Tumor-derived systems as novel biomedical tools—turning the enemy into an ally

Abstract

Cancer is a complex illness that presents significant challenges in its understanding and treatment. The classic definition, "a group of diseases characterized by the uncontrolled growth and spread of abnormal cells in the body," fails to convey the intricate interaction between the many entities involved in cancer. Recent advancements in the field of cancer research have shed light on the role played by individual cancer cells and the tumor microenvironment as a whole in tumor development and progression. This breakthrough enables the utilization of the tumor and its components as biological tools, opening new possibilities. This article delves deeply into the concept of "tumor-derived systems”, an umbrella term for tools sourced from the tumor that aid in combatting it. It includes cancer cell membrane-coated nanoparticles (for tumor theranostics), extracellular vesicles (for tumor diagnosis/therapy), tumor cell lysates (for cancer vaccine development), and engineered cancer cells/organoids (for cancer research). This review seeks to offer a complete overview of the tumor-derived materials that are utilized in cancer research, as well as their current stages of development and implementation. It is aimed primarily at researchers working at the interface of cancer biology and biomedical engineering, and it provides vital insights into this fast-growing topic.
Graphical Abstract

Full text

Desaietal. Biomaterials Research (2023) 27:113
https://doi.org/10.1186/s40824-023-00445-z
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Biomaterials Research
Tumor-derived systems asnovel biomedical
tools—turning theenemy intoanally
Nimeet Desai1, Pratik Katare1, Vaishali Makwana2, Sagar Salave3, Lalitkumar K. Vora4* and Jyotsnendu Giri1*
Abstract
Cancer is a complex illness that presents significant challenges in its understanding and treatment. The classic defini-
tion, "a group of diseases characterized by the uncontrolled growth and spread of abnormal cells in the body," fails
to convey the intricate interaction between the many entities involved in cancer. Recent advancements in the field
of cancer research have shed light on the role played by individual cancer cells and the tumor microenvironment
as a whole in tumor development and progression. This breakthrough enables the utilization of the tumor and its
components as biological tools, opening new possibilities. This article delves deeply into the concept of "tumor-
derived systems”, an umbrella term for tools sourced from the tumor that aid in combatting it. It includes cancer
cell membrane-coated nanoparticles (for tumor theranostics), extracellular vesicles (for tumor diagnosis/therapy),
tumor cell lysates (for cancer vaccine development), and engineered cancer cells/organoids (for cancer research). This
review seeks to offer a complete overview of the tumor-derived materials that are utilized in cancer research, as well
as their current stages of development and implementation. It is aimed primarily at researchers working at the inter-
face of cancer biology and biomedical engineering, and it provides vital insights into this fast-growing topic.
Keywords Cancer cell membrane, Extracellular vesicles, Tumor cell lysate, Tumor organoids, Cancer, Cancer research
*Correspondence:
Lalitkumar K. Vora
L.Vora@qub.ac.uk
Jyotsnendu Giri
jgiri@bme.iith.ac.in; enarm@bme.iith.ac.in
Full list of author information is available at the end of the article

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Graphical Abstract
Introduction
Global cancer burden andtreatment strategies
Cancer represents a heterogeneous group of dis-
eases characterized by the uncontrolled proliferation
of abnormal cells within the human body. Cancerous
cells do not adhere to the programmed growth, divi-
sion, and apoptosis cycle, unlike healthy cells. eir
origins are diverse, stemming from various cell types,
and the causative factors are typically multifaceted,
including genetic mutations, exposure to carcinogenic
agents, hereditary predisposition, and the aging pro-
cess [1]. ese rogue cells are notorious for subvert-
ing the body’s intrinsic mechanisms that regulate cell
growth and repair, ultimately leading to the forma-
tion of tumors. Furthermore, they can disseminate to
distant anatomical sites via either the bloodstream or
the lymphatic system, a phenomenon recognized as
metastasis. is invasive potential constitutes a cardi-
nal hallmark of cancer and is closely associated with
an unfavorable prognosis [2]. Cancer has emerged as a
global health crisis, substantially impacting public well-
being. A report published by the World Health Organi-
zation in 2019 revealed that cancer accounted for
three out of every ten premature deaths attributable to
noncommunicable diseases in 183 countries [3]. 2020
witnessed a significant global burden of cancer, with
a staggering 19.3 million new cases diagnosed and 10
million cancer-related fatalities recorded. Unfortu-
nately, this concerning trend is expected to persist, with
projections indicating a substantial surge in cancer
incidence in the coming years. By 2040, it is estimated
that an alarming 28.4 million new cases of cancer will
be reported worldwide [4]. e escalating incidence of
cancer underscores the urgent need to develop effective
strategies to comprehensively understand and combat
this malignancy.
Significant strides have been made in cancer research
over recent decades. Researchers across the globe
have been diligently dedicated to unraveling the intri-
cate facets of this disorder, and as our comprehension
deepens, therapeutic interventions consistently yield
promising clinical outcomes. For decades, the pillars of
cancer treatment have been surgical procedures, radia-
tion therapy, and chemotherapy, with their utilization
becoming increasingly refined through advancements
in technology and expanding knowledge. e progres-
sion of surgical techniques and the incorporation of
robotic systems have empowered surgeons to execute

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intricate procedures with heightened precision, mini-
mal invasiveness, and reduced recovery periods [5].
Radiation therapy has also undergone a transformative
journey, transitioning from conventional external beam
radiation to cutting-edge methodologies such as pro-
ton therapy, stereotactic body radiation therapy, and
intensity-modulated radiation therapy, enhancing the
precision of cancerous tissue targeting while minimiz-
ing exposure to healthy tissue [6]. Similarly, innovative
chemotherapeutic agents have been meticulously engi-
neered to selectively target distinct molecular pathways
and cellular mechanisms implicated in cancer prolifera-
tion and survival, rendering them more efficacious and
less deleterious than traditional chemotherapy agents
[7]. In conjunction with conventional therapies, these
pioneering treatment modalities have notably elevated
overall survival rates across various cancer types and
have opened avenues for managing malignancies that
were once deemed intractable.
Progress in diagnostic interventions has significantly
contributed to enhancing the depth of insight into a
patient’s cancer condition. e utilization of advanced
imaging techniques, such as positron emission tomog-
raphy, computed tomography, and magnetic resonance
imaging (MRI), has revolutionized our ability to access
real-time visual data about a tumor’s location, dimen-
sions, and staging [8]. Concurrently, biopsies have
evolved to furnish exceedingly precise and specific infor-
mation concerning the molecular attributes of tumors
and the presence of genetic mutations or variations [9].
ese strides in diagnostic interventions have substan-
tially elevated the precision of cancer diagnosis and
facilitated tailored treatment strategies, yielding superior
patient outcomes.
Overview oftumor‑derived systems
An exciting and evolving field within cancer research
involves the development of biomaterial-based platforms
tailored for precise and localized delivery of antican-
cer drugs, immunomodulatory biomolecules and diag-
nostic contrast agents. In most cases, these platforms
integrate targeting ligands, facilitating precise interac-
tions with diverse cellular elements within the intricate
context of the tumor microenvironment (TME) [10].
Predominantly, these constituents encompass cancer
cells, which can be selectively targeted through ligands
such as arginine-glycine-aspartic acid peptide [11],
folate [12], transferrin [13], or hyaluronic acid [14]. Fur-
thermore, the targeting approach can also be extended
to immune cells to directly modulate their anticancer
immune responses. Dendritic cells (DCs), for instance,
can be effectively modulated by exploiting surface recep-
tors such as mannose receptors [15], Fc receptors [16],
and Toll-like receptors [17] to enhance their functional
capabilities. Reprogramming tumor-associated mac-
rophages (TAMs) can be accomplished through selective
targeting strategies, leveraging the overexpression of IL-4
and galactose-type lectin receptors [18, 19]. In parallel,
the immunosuppressive functions of myeloid-derived
suppressor cells (MDSCs) can be attenuated by target-
ing specific myeloid cell markers, such as CD11b+ and
CD33+ [20]. Finally, nonimmune cells such as cancer-
associated fibroblasts (CAFs), which play a pivotal role in
fostering tumor growth by facilitating the remodeling of
the cancer extracellular matrix (ECM), can be effectively
targeted through surface markers such as fibroblast acti-
vation proteins [21].
While this approach offers flexibility in selecting the
optimal delivery site and has shown promising results,
as evident from the extensive academic literature on the
subject, its clinical translation remains limited [22]. e
primary obstacle lies in the complexity of the manufac-
turing process, which hampers scalability and results in
elevated production costs, rendering the final product
inaccessible to end users [23]. Consequently, despite the
availability of numerous naturally sourced and synthetic
biomaterials, the scientific community continues to
explore and develop superior alternatives.
Profound insights and practical solutions are often
achieved through a deep and comprehensive understand-
ing of the problem at hand. is principle holds true in
various aspects of life, including the realm of biomedical
science. Taking this into account, certain cellular charac-
teristics of tumors can be exploited for biomedical pur-
poses. Notably, tumor-derived extracellular vesicles (EVs)
play a pivotal role in mediating the exchange of molecu-
lar components and signaling events, collectively contrib-
uting to the progression of pathological conditions. ese
EVs possess remarkable attributes, such as high stability,
versatility in cargo loading, and excellent biocompatibil-
ity, rendering them valuable candidates for drug delivery
systems [24]. When sourced from tumors, EVs bear spe-
cific surface markers that function as intrinsic ligands,
facilitating targeted interactions with cancer cells and
specific immune cells within the TME. Furthermore, the
profiling of exosomal cargo, encompassing nucleic acids,
proteins, and lipids, is a vital diagnostic tool for assessing
and monitoring tumor progression [25].
In addition to EVs, the cancer cell membrane (CCM)
plays a critical role in tumor development, progression,
and metastasis. Altered membrane composition, struc-
ture, and functionality equip cancer cells with the ability
to thrive in hostile environments, evade immune surveil-
lance, and migrate to distant locations. When isolated
in a functionally viable state, CCM can be harnessed
as a fundamental component in the emerging class of

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nanocarriers termed CCM-coated nanoparticles. Com-
prising a synthetic nanoparticulate core loaded with
drugs or contrast agents concealed by a layer of cancer
cell-derived membrane, this approach imparts a "bio-
mimetic" character to conventional nanoparticles. is
strategy offers enhanced biocompatibility, immune eva-
sion, and homotypic tumor targeting [26, 27].
Beyond the therapeutic and diagnostic potential of EVs
and CCM, tumors themselves can be engineered as a
modality for comprehending and combating cancer. e
induction of an effective and specific immune response
against cancer cells is pivotal to successful immuno-
therapy. While multiple antigens can be targeted for this
purpose, the utilization of immunogenically dying tumor
cells or tumor cell lysate (TCL) offers a comprehen-
sive array of epitopes, promoting a multivalent immune
response [28]. Finally, cancerous tissues can be cultured
to establish cancer cell lines, which are the foundational
cornerstone of cancer research. Progress in biotechnol-
ogy and molecular science has empowered the genetic
manipulation of these cancer cell lines, further expanding
their utility in cancer research. Notably, they play a piv-
otal role in unraveling disease biology and advancing the
development of novel biomolecular interventions [29].
e aforementioned systems can be collectively
grouped as “tumor-derived biomedical tools,” as illus-
trated in Fig. 1. ese systems possess a multitude of
distinctive attributes that have attracted substantial
attention in the field of bioengineering for potential clini-
cal applications.
About this manuscript
is comprehensive review aims to thoroughly analyze
tumor-derived systems, highlighting their state-of-the-
art applications in various areas, such as drug delivery,
immunotherapy, cancer detection and diagnosis, vaccine
development, and fundamental cancer research.
rough an extensive survey of the latest literature,
several outstanding research studies showcasing these
systems’ immense potential in the fight against can-
cer were identified. e reference selection process was
conducted systematically to ensure the sources’ highest
quality and relevance. Specific criteria for inclusion were
established, encompassing factors such as relevance to
the topic, recency of publication, credibility of the source,
and the significance of the study. To retrieve the relevant
literature, comprehensive keyword searches were con-
ducted on PubMed. ese searches were performed from
2003 to 2023, and the search strategy included using
these keywords and filters as per PubMed’s capabilities.
Subsequently, the search results were meticulously
reviewed to ensure the utmost rigor in their use. e
retrieved reference was subjected to a comprehensive
evaluation, carefully considering how well they aligned
with predefined criteria for inclusion. e grounds on
which some studies were excluded included factors such
as a lack of direct relevance to the subject matter, out-
dated information, unreliable/questionable sources, or
studies that did not contribute substantively to the over-
arching theme of this review. Figure2 depicts the year-
wise distribution of publications (from 2003 to 2023)
sourced from our comprehensive searches on PubMed. It
provides a visual depiction of the evolving research land-
scape in the field over the past several years.
e subsequent sections of this review paper are struc-
tured to address distinct categories of tumor-derived
systems. Drawing from an extensive review of the litera-
ture, comprehensive insights encompassing fundamental
principles, the developmental or fabrication process, and
a case-based examination of their practical utility have
been presented. It is pertinent to mention that, for brev-
ity and reader engagement, our discussions pertaining
to the application aspects are primarily based on select
studies that furnish distinctive insights, effectively show-
casing the versatile utility of these tumor-derived sys-
tems. is strategic approach is implemented to enhance
the readability and overall enjoyment of the manuscript
for our readers while maintaining scientific rigor.
Cancer cell membrane
Understanding CCM
In recent years, the field of cancer treatment has under-
gone a revolutionary transformation with the introduc-
tion of novel anticancer drugs as integral components of
chemotherapy regimens. e lack of selectivity, systemic
cytotoxicity, and occurrence of multidrug resistance
have provided a significant obstacle to effectively utiliz-
ing these anticancer drugs [30]. In this context, strategies
that facilitate tumor-targeted delivery by encapsulating
these drugs into nanoparticles have demonstrated excel-
lent promise in improving treatment effectiveness. e
recent rise of nanoparticle-derived products receiving
regulatory approval is a testament to their clinical fea-
sibility [31]. However, it is worth mentioning that the
majority of these marketed nanoparticles rely solely on
the “enhanced permeability and retention” (EPR) effect
(a passive targeting approach that takes advantage of the
tumor’s leaky vessels and the poor lymphatic system) to
reach the tumor vicinity [32]. In the absence of an active
targeting approach, the exceedingly heterogeneous
nature of vessel fenestrations (for most types of tumors)
or the lack of leaky vasculature (as observed in slow-
growing tumors) acts as a biological barrier that restricts
the overall reach of nanoparticles, indirectly requiring a
higher concentration of nanoparticles to be administered
[33]. Furthermore, these nanoparticles may face immune

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Fig. 1 Schematic representing different categories of tumor-derived systems and their utilization as biomedical tools. A Potentiating tumor
targeting by coating nanoparticles with functional CCM. B Employing tumor-derived EVs as diagnostic tools and delivery vectors for anticancer
therapy. C Developing TCL as a potent cancer vaccine component. D Employing cancer cell lines and tumor organoids as multipurpose tools
in cancer research

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recognition challenges contingent upon their particle
dimensions and chemical composition. is recognition
can result in their premature elimination from the blood-
stream, preventing them from reaching the targeted
tumor site [34]. Biomimetic functionalization, especially
using CCM, has evolved as a lucrative approach to endow
nanoparticles with more prolonged circulation, effective
delivery, and active targeting [35].
e use of CCM endows nanoparticles with several
distinct advantages. First, if isolated from source can-
cer cells or patient‐derived xenografts, CCM provides a
unique opportunity to design personalized treatment for
addressing cancer heterogeneity due to the coexistence
of several cell types with varied phenotypes. Improved
patient specificity yields better clinical outcomes [36].
Second, the endogenous nature of CCM confers supe-
rior biocompatibility and safety. Finally, particles coated/
camouflaged with CCM demonstrate extended sys-
temic circulation without eliciting an immune response,
facilitating remarkable tumor site-specific localiza-
tion. is feat is achieved without the need to employ
complex surface modifications, as seen in other active
targeting strategies such as biological ligands, target-
ing peptides, or aptamers [37, 38]. Cancer cell adhesion
molecules (CCAMs) are the common underlying rea-
son for the abovementioned advantages associated with
CCM. CCAMs encompass various membrane receptors,
chiefly including (but not limited to) the immunoglobu-
lin superfamily (Ig-SF), selectins, integrins, and cadherins
[39–41]. CCAMs are pivotal in establishing cell‒cell and
cell-ECM interactions and are indirectly responsible for
cancer recurrence, invasion, and metastasis [42]. From a
functional perspective, cadherins (mainly cadherin-1, 2,
and 19; protocadherin) aid in cell‒cell adhesion within
tumors by regulating cell migration and gene regulation
(through catenin pathways) [43]. Integrins (mainly IT-β1,
β3, β4, and β5; IT-α1, α2, and α5) are essential for cell
proliferation, differentiation, and migration due to their
capacity to transmit ECM signals to the cell [44]. Selec-
tins and Ig-SF members (ALCAM, Contactin, ICAM,
MCAM, NCAM) activate signaling cascades that pro-
mote malignant behavior by negatively modulating the
immune cells within the TME [45, 46]. In addition to
CCAMs, the surface of a cancer cell is also enriched with
CD47. is ubiquitous transmembrane protein inter-
acts predominantly with signal regulatory protein-α
expressed by macrophages and DCs [47]. It primarily
functions as a "do not eat me" signal by activating protein
phosphatases and inhibiting immune phagocytosis [48].
By applying suitable CCM isolation techniques, these
membrane receptors can be retained in a functional state,
and their subsequent coating onto nanoparticles allows
them to engage in homotypic adhesive interactions (with
tumors) and evade systemic immune cells [49].
Fig. 2 Graphical plot illustrating the number of scientific papers published over the last two decades (from 2003 to 21 September 2023)
on biomedical systems that can be categorized as “tumor-derived.” These papers were systematically identified in the Pubmed database
by employing keywords and filters tailored to this research focus

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Leveraging CCM forbiomimetic functionalization
Fang et al. [50] were among the first groups to study
the homologous binding mechanisms of nanoparticles
coated with CCM. Compared to bare nanoparticles or
nanoparticles coated with erythrocyte membrane, the
authorsfound that poly(lactic-co-glycolic acid) (PLGA)
nanoparticles coated with cell membrane isolatedfrom
the MDA-MB-435 metastatic cancer cell line under-
went substantial cellular attachment to the source cells.
e cancer cell-specific affinity of the CCM coating was
further validated when the CCM-coated PLGA core dis-
played a similar uptake profile to the bare PLGA core
when incubated with human foreskin fibroblasts (as a
negative control). After this pioneering study, CCM iso-
lated from various cell lines has been widely reported
[51]. Regardless of the cancer cell type, CCM can be iso-
lated using a simple and scalable top-down approach.
e standard procedure for membrane isolation typi-
cally entails treating the source cells with a hypotonic
lysis buffer comprising various components, including
phenylmethyl sulfonyl fluoride, sodium bicarbonate, and
ethylenediaminetetraacetic acid. Following incubation
in lysis buffer, the cells were disrupted by gentle homog-
enization using mechanical pressure (preferably with
a Dounce homogenizer). Recently, many commercial
membrane protein extraction kits have become available
on the market that contain proprietary buffer solutions
that replace homogenization with sequential freeze‒thaw
cycles (by submerging the cells into liquid nitrogen) [52].
In both scenarios, the cell lysate obtained is subjected to
thorough differential ultracentrifugation, leading to the
isolation of a purified CCM pellet. e final CCM vesi-
cles are typically prepared by washing them once with
a solution containing Tris-hydrochloride and ethylen-
ediaminetetraacetic acid, followed by physical extrusion
through a 400-nm polycarbonate (PC) membrane [53].
Any nanoparticle can be coated with these isolated CCM
vesicles by coextrusion through a PC membrane of lower
pore size [54]. Other reported methods to coat CCM
include sonication (after prior incubation) and microflu-
idic-assisted nanoparticle coating. e presence of func-
tional membrane surface receptors can be validated using
western blotting, whereas the effective coating of CCM
on the nanoparticle can be assessed using transmission
electron microscopy (TEM) [55]. Measurement of the
hydrodynamic diameter and zeta potential using photon
correlation spectroscopy (also known as dynamic light
scattering) can provide semiquantitative confirmation of
nanoparticle coating [56].
While the premise of CCM-coated nanoparticles is
lucrative, it should be noted that the current gold-stand-
ard protocols to coat nanoparticles with CCM are not
fully efficient. In a recent study, Liu etal. [57] introduced
a fluorescence quenching assay to assess the integrity
of the cell membrane coating. By studying the coating
of CMM (isolated from diverse cell types such as CT26
cells and HeLa cells) with multiple nanoparticulate cores
(magnetite, gold, PLGA, and porous silicon), the authors
demonstrated that up to 90% of the nanoparticles are
only partially coated (with more than 60% of the sample
population having a coating degree < 20%). In in vitro
homologous targeting studies, it was observed that the
nanoparticles, despite being partially coated, were still
capable of being internalized by the target cells. Exten-
sive molecular simulations were conducted to gain
further insights into the endocytic entry mechanism.
Based on these simulations, the authors proposed that
nanoparticles with a high coating degree (≥ 50%) enter
the cells individually, whereas those with a low coating
degree (< 50%) require aggregation before internaliza-
tion (Fig.3). In a follow-up study, the authors explored
the addition of external phospholipids as “helpers” to
enhance CCM fluidity and promote the final fusion of
lipid patches. e nanoparticles coated with this method
showed a high ratio of complete coating (23%) and supe-
rior tumor-targeting capabilities compared to nanopar-
ticles coated conventionally [58]. In addition to external
phospholipids, designing “hybrid membranes” by com-
bining CCM with membrane vesicles isolated from other
cell sources, such as erythrocytes, platelets, immune cells
(macrophages, cytotoxic T lymphocytes (CTLs)), and
cellular TME components (MDSCs, CAFs), has been
explored [59, 60].
Applications intumor theranostics
e inherent ability of CCM-coated nanoparticles to
localize in close proximity to the TME has been lever-
aged to design several advanced cancer theranostics
platforms [61]. is approach benefits from diverse core
materials, ranging from natural biomaterials such as chi-
tosan, alginate, and silk fibroin to synthetic systems such
as polymeric, lipid-based, or metallic nanosystems [62,
63]. Depending on the intended application, the core can
be equipped with functional cargo such as biomolecules,
immune adjuvants, or contrast agents [64]. Some recent
applications are discussed below.
To tackle critical challenges in cancer treatment,
such as low drug loading, poor solubility of anticancer
drugs, and targeting specificity, Wu etal. [65] devised
a sophisticated system comprising paclitaxel (PTX)
nanocrystals coated with the SK-BR-3 cell membrane.
e coated membrane was modified with Herceptin (a
monoclonal antibody that selectively binds to HER2
receptors), making the final platform (HCNCs) an
excellent candidate for treating HER2-positive breast
cancer. Upon analysis via TEM, HCNCs displayed a

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characteristic cubic shape and measured approximately
220 nm in size. e authors utilized FITC-labeled
HCNCs to investigate cellular uptake and homo-
typic targeting. Findings from confocal laser scanning
microscopy (CLSM) indicated significantly elevated
uptake of HCNCs compared to uncoated nanocrys-
tals. Notably, this enhanced uptake was predominantly
observed in SK-BR-3 cells, underscoring the plat-
form’s selectivity. In a BALB/c nude mouse model, the
intravenous administration of HCNCs demonstrated
remarkable tumor localization with minimal unin-
tended distribution to vital organs. e inclusion of
Herceptin further potentiated the platform’s therapeu-
tic effects. HCNCs exhibited a proapoptotic capability,
as evidenced by the upregulation of proapoptotic pro-
teins, including caspase-3 and Bax, alongside a corre-
sponding decrease in the antiapoptotic protein Bcl-2.
HCNCs achieved an in vivo tumor inhibition rate of
83.1 ± 3.54% (Fig. 4A). e positive result highlights
CCM’s ability to potentiate conventional nanosystems
by imparting a selective targeting ability.
To enhance cancer immunotherapy, Li etal. [67] devel-
oped a biomimetic system (termed CFIN) by combining
triblock polymer-based nanomicelles with 4T1 CCM.
e nanomicelles were loaded with indocyanine green
(ICG, a photothermal agent) and NLG919 (an effec-
tive IDO-1 enzyme inhibitor). is system addresses
the challenge of poor tumor immunogenicity by com-
bining photothermal and immune therapies. CFIN are
approximately 220nm in size and have a negative sur-
face charge of -23 mV. When exposed to an 808 nm
laser for 10 min, they exhibited excellent photother-
mal properties, increasing their temperature by 34.5°C.
Under laser irradiation for just 1min, CFIN effectively
reduced cell viability to less than 4% in 4T1 cells. is
result highlighted the ability of CFIN to ablate cancer
cells through efficient photothermal energy conversion.
Moreover, CFIN induced immunogenic cell death (ICD),
Fig. 3 Assessment of cell membrane coating integrity and its impact on the internalization mechanism. In subfigure (i), TEM images are presented
for various nanoparticle cores (Fe3O4, ZIF-8, Au, PLGA, and porous silicon) before and after cell membrane coating, accompanied by quantification
of the ratio of complete cell membrane coating (Scale bar: 100 nm). Subfigure (ii) displays the distribution of cell membrane coating degrees
on a SiO2 core, determined from TEM images (n = 325). The inset provides information on the proportion of SiO2 cores with a low cell membrane
coating degree, specifically below 50%. In subfigure (iii), a schematic illustration is presented, elucidating potential endocytic entry mechanisms
for partially coated cores. Adapted with permission from [57] (Copyright Springer Nature, 2021)

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Desaietal. Biomaterials Research (2023) 27:113
promoting the expression of calreticulin and stimulating
DC phagocytosis. In a murine 4T1 tumor model, CFIN
treatment combined with laser irradiation led to nearly
complete inhibition of primary tumor growth (93.5%
inhibition rate) and delayed tumor progression at distant
sites. Immunofluorescence staining revealed enhanced
DC maturation and increased T lymphocyte infiltration
within the tumor. CFIN-mediated delivery of NLG919
also inhibited IDO-1, reducing the immunosuppressive
TME and improving proinflammatory cytokine expres-
sion. While the previous example focused on targeted
drug delivery, CFIN effectively harnesses photother-
mal properties to induce ICD and promote an immune
response against cancer cells, exemplifying the versatility
of CCM-coated nanoparticles.
In a remarkable advancement in breast cancer treat-
ment, Pan et al. [66] combined a CCM coating with a
nanoscale metal–organic framework (MOF) core loaded
Fig. 4 A Herceptin-functionalized CCM-coated PTX nanocrystals for targeted therapy of HER2-positive breast cancer. Subfigure (i) displays TEM
images comparing uncoated NCs and HCNCs, revealing successful coating (scale bar: 200 nm). Subfigure (ii) exhibits CLSM images illustrating
the cellular uptake of FITC-labeled NCs and HCNCs in SK-BR-3 cells, demonstrating enhanced uptake by HCNCs (Scale bars: 50 µm). Subfigure (iii)
demonstrates the biodistribution patterns of HCNCs in tumors and main organs. Figure (iv) shows the western blot analysis of β-actin, caspase-3,
Bax, and Bcl-2 proteins, enabling the assessment of the apoptotic capability of HCNCs. Finally, subfigure (v) depicts the in vivo antitumor effect
of HCNCs. Adapted with permission from [65] Copyright Elsevier, 2022). B CCM-coated MOF for multimodal tumor therapy. Subfigure (i) provides
TEM images of PFTT@CM, showing the structure of the MOF coated with CCM. Subfigure (ii) demonstrates the cumulative release of Fe3+
from PFTT@CM at different pH levels, indicating its acid-dependent dissociation. Subfigure (iii) presents the ability of PFTT@CM, mediated by Fe.3+,
to deplete GSH. Subfigure (iv) reveals the continuous catalysis of H2O2 by PFTT@CM, monitored through a 3,3′,5,5′-tetramethylbenzidine assay
over a 30-min duration. Adapted with permission from [66], (Copyright Elsevier, 2022)

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with photosensitizers. is innovative strategy com-
bined photodynamic therapy (PDT) with chemotherapy
for enhanced efficacy. e MOF core, containing Fe-
tetrakis (4-carboxyphenyl) porphyrin and loaded with
tirapazamine (TPZ), exhibited acid-responsive prop-
erties. e platform, termed PFTT@CM, featured a
hydrodynamic diameter of 201nm and a zeta potential
of − 44.22 mV. e TPZ loading efficiency was deter-
mined to be 27.1 ± 7.4%. e CCM coating facilitated
immune evasion and tumor retention. Upon reach-
ing cancer cells through endocytosis, the nanoparticles
decompose within lysosomes, releasing Fe3 + ions that
catalyze the conversion of endogenous hydrogen perox-
ide (H2O2) into highly reactive hydroxyl radicals (•OH)
while depleting glutathione in the TME. is modula-
tion induced ferroptosis, a specific form of cell death, and
enhanced PDT by increasing oxygen levels in the TME,
leading to cancer cell apoptosis. PFTT@CM exhibited
therapeutic effects of approximately 19.7% (ferroptosis),
43.8% (PDT), and 22.9% (TPZ-based chemotherapy)
against MDA-MB-231 breast cancer cells at a concentra-
tion of 100μg/mL (Fig.4B). Hypoxia activation of TPZ
within cancer cells was confirmed. e combined thera-
peutic approach demonstrated superior anticancer
efficacy compared to individual modalities. Invivo exper-
iments in tumor-bearing nude mice showed that PFTT@
CM preferentially accumulated at the tumor site, with
significant fluorescence intensity observed even 96 h
after injection. is synergistic treatment led to com-
plete tumor suppression for 18days following a single
administration.
To tackle the formidable challenge posed by multiple
myeloma (MM), a hematological malignancy known for
its aggressive nature and impact on patients’ lives, Qu
et al. [68] developed a novel approach by utilizing the
phenomenon of "bone marrow homing," wherein MM
cells migrate and return to the bone marrow for survival
and proliferation. ey fabricated bortezomib (BTZ)-
loaded polymeric nanoparticles and further coated them
with the MM cell membrane through physical extrusion,
forming MPCEC@BTZ nanoparticles. BTZ is a protea-
some inhibitor and an established first-line treatment for
MM. is platform exhibited remarkable antitumor effi-
cacy in a systemic orthotopic transplantation MM model.
e success of this approach can be attributed to the
bone marrow localization facilitated by the presence of
bone marrow-specific protein markers, including CD44,
CD147, CXCR4, and CD138, on the MM cell membrane.
e MPCEC@BTZ nanoparticles effectively delayed
tumor progression within the BM, improved overall sur-
vival rates, and reduced systemic side effects without
causing histological toxicity in major organs (Fig. 5A).
is innovative platform holds great promise for
enhancing the treatment outcomes of MM and improv-
ing patient well-being.
In an interesting study, Li et al. [69] engineered an
antibody-anchored membrane (AAM) nanovaccine by
incorporating anti-CD40 single-chain variable fragments
(scFv) into tumor cell membranes. is process involved
recombinant gene expression, leading to the overexpres-
sion of anti-CD40 scFv on the tumor cell surface. e
resulting membrane, enriched with anti-CD40 scFv, was
then coated onto a polymeric core using water-bath soni-
cation and serial extrusion, yielding nano-AAM/CD40
particles with a mean size of 130.3nm. CD40, a critical
tumor necrosis factor receptor superfamily member, is
known for its elevated expression on antigen-presenting
cells (APCs) such as DCs. e presence of anti-CD40 scFv
on nano-AAM/CD40 enabled specific binding to CD40
receptors on APCs, facilitating efficient delivery of the
nanovaccine to lymph nodes. is platform resulted in
the maturation of DCs, characterized by the upregulation
of costimulatory molecules, effective cross-presentation
of antigens, and cytokine production. ese responses
culminated in the activation and differentiation of T
cells. In a prophylactic invivo study, CD40-humanized
transgenic mice vaccinated with nano-AAM/CD40 dem-
onstrated complete prevention of tumor growth when
challenged with MC38 tumor cells in 50% of the mice for
up to 80days. To broaden the spectrum of tumor anti-
gens targeted, the researchers loaded the polymeric core
with tumor lysate, resulting in nano-AAM/CD40/lysate.
is modification significantly enhanced the expression
of costimulatory molecules and the production of IL-12
while increasing the density of CD8 + tumor-infiltrating
T cells. Treatment with a low nano-AAM/CD40/lysate
dose extended median survival compared to low-dose
nano-AAM/CD40 alone (Fig.5B). is innovative nano-
vaccine platform demonstrates versatility in combination
therapy for cancer treatment and is characterized by its
precise targeting and immunostimulatory properties.
In the realm of anticancer therapy, the utilization of
CCM nanoparticles extends beyond their therapeutic
potential. It offers opportunities for early cancer detec-
tion and investigation of cancer metastasis through the
development of highly sensitive and specific tumor imag-
ing platforms [70, 71]. Rao etal. [54] reported the devel-
opment of CC-UCNPs, a platform that combines CCM
with upconversion nanoparticles (UCNPs). UCNPs stand
out among fluorescence agents due to their unique opti-
cal and chemical properties, including exceptional light
penetration depth, narrow emission peaks, high pho-
tostability, large Stokes shifts, low toxicity, and mini-
mal background fluorescence. Nevertheless, UCNPs
have limitations, such as the lack of targeting ability and
susceptibility to the systemic immune system. ese

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challenges were addressed through the application of
CCM coating. CCM, obtained via hypotonic lysis, was
efficiently coated onto UCNPs using extrusion, result-
ing in a core–shell-like structure with a hydrodynamic
diameter of approximately 100nm, including a cancer
membrane layer of approximately 10nm (Fig.6A). e
antiphagocytosis properties of CC-UCNPs were assessed
invitro by incubating them with RAW 264.7 cells. Uptake
was quantified by measuring the yttrium ion content over
various time intervals. CC-UCNPs exhibited minimal
uptake compared to their uncoated counterparts. Fur-
thermore, when subjected to a 980nm NIR laser, these
particles displayed exceptional luminescence. In vivo
assessments involved using MDA-MB-435 human breast
cancer cells, DU145 human prostate cancer cells, CAL27
human squamous cancer cells, and HCT116 human colo-
rectal cancer cells to prepare CC-UCNPs. Using a murine
tumor model, the study demonstrated highly efficient
Fig. 5 A Targeted therapy of multiple myeloma based on bone marrow homing. Subfigure (i) shows a schematic of cell membrane-coated
polymeric nanoparticles for the treatment of multiple myeloma. Subfigure (ii) shows the ex vivo fluorescence imaging of the femur tissues at 48 h
postinjection of DiR-loaded MPCEC nanoparticles in a 5 T multiple myeloma murine model. Adapted with permission from [68], (Copyright Wiley–
VCH, 2022). B Genetically engineered antibody-anchored tumor cell membrane as nanovaccines. Subfigure (i) shows a schematic illustration
of the design of the nano-AAM/CD40 and nano-AAM/CD40/lysate. Subfigure (ii) shows the flow cytometry analysis of in vitro DC maturation
following different treatments. Subfigure (iii) represents ELISA analysis of IL-12p70 production by DCs without and with treatments. Figure (iv)
represents the mean tumor growth curve of MC38 tumors in mice after different treatments. Here, p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Adapted with permission from [69], (Copyright Wiley–VCH, 2023)

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Desaietal. Biomaterials Research (2023) 27:113
Fig. 6 A CC-UCNPs for highly specific tumor imaging. Subfigure (i) shows a schematic diagram highlighting the preparation, function,
and application of CC-UCNPs. Subfigure (ii) shows TEM analysis of uncoated UCNPs and CC-UCNPs. The cancer membrane was negatively
stained with uranyl acetate. Adapted with permission from [54], (Copyright Wiley–VCH, 2016). B In vivo tumor visualization using CCM-coated
nanoconjugates as MRI contrast agents. Here, subfigure (i) presents the results of T1-weighted magnetic resonance imaging (MRI)
along with corresponding pseudocolor images of tumor-bearing mice. The images were captured at different time points following the intravenous
injection of MSNPs equivalent to 2.5 µmol of Gd.3+. Subfigure (ii) demonstrates the tumor-to-background (T/B) and tumor-to-muscle
(T/M) contrast ratios. Adapted with permission from [72], (Copyright Wiley–VCH, 2019). C Imaging and surgical navigation of glioma using
CCM-coated lanthanide-doped nanoparticles. Subfigure (i) shows brightfield and near-infrared II (NIR-II) fluorescence images of mice
with glioma before and after surgery. Additionally, subfigure (ii) displays an H&E-stained image of the whole brain containing the tumor,
alongside a corresponding fluorescence microscope image of the tumor using DiO-labeled CC-LnNPs in green. Adapted with permission from [73],
(Copyright Wiley–VCH, 2022)

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targeting when the CCM source cell matched the host
tumor. Comprehensive biosafety evaluations through
blood biochemistry, hematology tests, and histological
analyzes revealed no significant alterations or organ dam-
age, confirming the platform’s excellent biocompatibility.
Yi et al. [72] introduced MSNPs, a biocompatible
nanostructure platform for high signal-to-noise ratio
MRI imaging. ese MSNPs consist of self-assembled
NaGdF4 and CaCO3 nanoconjugates encased within a
HeLa cell membrane. In MRI, T1 and T2 relaxation times
are crucial for image quality. T1 agents enhance signal
intensity in T1-weighted images, while T2 agents do so
in T2-weighted images. MSNPs are unique because their
T1 source, Gd3+ ions, is spatially confined, initially result-
ing in an "OFF" MRI signal. However, when exposed to
a slightly acidic TME, embedded CaCO3 nanoparticles
generate CO2 bubbles, creating an "ON" MRI signal.
Invivo experiments on mice with tumors compared the
performance of MSNPs with that of the commercial MRI
contrast agent Magnevist® (gadopentetic acid). Before
MSNP injection, the tumor site appeared dark, but
30min after injection, it began to illuminate. Approxi-
mately 195min postinjection, the tumor site exhibited
significant contrast enhancement, achieving a tumor-to-
background ratio of approximately 48, effectively illumi-
nating the entire tumor. Notably, MSNPs outperformed
Magnevist®, primarily due to their pronounced tendency
to accumulate within tumors. Detailed analysis of specific
regions of interest revealed that the tumor-to-muscle
signal ratios were approximately 61.6 times higher for
MSNPs than for Magnevist® (Fig.6B).
Liu et al. [74] developed a CCM-coated nanosystem
called ZGM to enhance metabolic glycan labeling for
tumor diagnostic imaging. By incorporating a MOF-
azidosugar complex (ZIF-8-Ac4GalNAz), this plat-
form selectively targets homotypic cancer cells through
receptors for cell-specific glycan labeling. ZGM cellular
internalization occurs through cholesterol-dependent
endocytosis, with efficient release from lysosomes due
to the "proton-sponge" effect. Notably, ZGM achieved
significant metabolic glycan labeling within 12 h with-
out preincubation. e CCM coating protected against
macrophage phagocytosis, extending blood circulation
and enhancing labeling. In vivo experiments demon-
strated ZGM’s capability to visualize multiple tumor
cell-selective glycans in homotypic tumors, particularly
distinguishing between breast cancer subtypes, including
the triple-negative and luminal A subtypes. is selec-
tivity holds clinical promise for precise cancer subtype
diagnosis.
In a recent study, Wang et al. [73] leveraged mem-
brane fragments derived from brain tumors to improve
brain tumor resection accuracy using lanthanide-doped
nanoparticles (LnNPs). ese coated nanoparticles,
termed CC-LnNPs, exhibited fluorescence in the near-
infrared-IIb window (NIR-IIb, 1500–1700 nm). is
study aimed to address the challenges associated with low
spatial resolution and limited permeability of the blood‒
brain barrier (BBB). Homotypic interaction between the
coated nanoparticles and brain tumor cells assisted in
crossing the BBB. CC-LnNPs exhibited several advan-
tages, including higher temporal and spatial resolution,
improved stability, and lower background signals than
the clinically approved imaging agent indocyanine green.
Consequently, the boundaries of brain tumors could be
visualized more clearly. By leveraging NIR-IIb fluores-
cence as a guide, the researchers successfully visualized
and precisely resected glioma tissue located approxi-
mately 2.3mm within the brain (Fig.6C).
Extracellular vesicles
Understanding CCM
EVs are nanosized vesicles enclosed by a lipid bilayer
that are actively released into the extracellular milieu by
various eukaryotic cells, including cancer cells and other
pathological cell types [75]. ey can be detected in vari-
ous somatic fluids, including blood, urine, bile, saliva,
breast milk, and cerebrospinal fluid [76, 77]. When ini-
tially discovered in 1967, EVs only function in eliminat-
ing cell debris. However, as research has progressed, their
importance as imperative bidirectional mediators of cell-
to-cell/cell-to-microenvironment signaling in various
biological processes has been established [78]. EVs are
usually produced in response to intracellular and extra-
cellular stress, such as platelet activation, pH changes,
hypoxia, complement protein exposure, irradiation,
chemotherapy, and necrosis [79]. e International Soci-
ety for Extracellular Vesicles emphasizes that EVsshould
be categorized according to their physical character-
istics, surface protein markers, and/or cell source. e
most widely applied nomenclature (based on biogenesis
and size distribution) classifies EVs into three subtypes:
exosomes (30–150 nm), microvesicles (100 nm-1 μm),
and apoptotic bodies (1–5μm) [80]. e following sec-
tion will exclusively focus on exosomes and microvesi-
cles, as the applicability of apoptotic bodies is limited.
Exosome formation begins when the endosome mem-
brane folds inward, creating a multivesicular endosome.
is endosome combines with the cell membrane and
releases fully formed exosomes into the extracellular
space through exocytosis [81]. In contrast, microvesi-
cles are produced by the outward budding of the
plasma membrane. Both vesicles lack functional nuclei,
rendering them unable to replicate independently [82].
e ‘endosomal sorting complex required for trans-
port’ protein plays a crucial role in their formation.

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Based on their biogenesis method, it is anticipated that
these EVs contain the same membrane proteins and
lipids as the source cell and thus can accurately rep-
resent the cell’s condition without direct access to it
[83]. EVs are enrichedwith diverse biomolecules, such
as nucleic acids, proteins, lipids, and metabolites, that
are derived from source cells. To ensure the reliability
of using EVs in biomedicine, a validation approach for
EVs that involves the verification of specific protein
markers is recommended. In the validation process, it
is suggested to include at least one membrane protein
marker, one cytosolic protein marker, and one non-
EV protein marker [84]. To establish the specificity of
EV-associated proteins, excluding proteins originating
from the nucleus, mitochondria, endoplasmic reticu-
lum, and Golgi complex is essential. ese intracellular
proteins can serve as negative control markers during
the validation process [85]. By adhering to this recom-
mended approach, researchers can ensure the accu-
rate characterization and identification of EV proteins
[86]. Most research has identified small noncoding
RNAs (specifically microRNAs) as the predominant EV
cargo. Isolated EVs may typically comprise hundreds
of microRNA species in varying concentrations, all of
which play crucial roles in intercellular communica-
tion. e standard RNA profiles for EVs collected from
different fluids/tissues are available in databases such as
ExoRBase [87], exRNA atlas [88], and miRanda [89].
Role incancer development
e intercellular communication facilitated by EVs is
paramount for tumorigenesis and metastasis. Multiple
studies have demonstrated that the cargo transported
by tumor-derived EVs accurately mirrors the dynamic
changes occurring within tumor cells throughout vari-
ous stages, including initiation, progression, invasion,
metastasis, and possible relapse [90]. Alteration of tumor
neovasculature (enhanced angiogenesis and vascular
leakage), formation of premetastatic niches, and modu-
lation of the immune response and drug resistance are
some of the critical areas where EVs play a significant role
[91]. Figure7 provides a schematic overview of the role
played by EVs (and their cargo) in cancer biology.
Tumor-derived EVs play a crucial role in cancer biol-
ogy by transferring cargo molecules that contribute to
tumor progression and alter the phenotype of recipi-
ent cells. One key aspect influenced by EVs is vascu-
lar growth, facilitated by activating endothelial cells
(ECs). is activation is primarily attributed to vascular
endothelial growth factor (VEGF) [92]. Additionally, sev-
eral EV proteins have been identified to promote angio-
genesis, including carbonic anhydrase 9 [93], annexin II
[94], myoferlin [95], and wnt4 [96]. In addition, extensive
research has been conducted on EV miRNAs, specifically
miR-130a and miR-92a, to elucidate their involvement in
tumor angiogenesis [97].
e development of premetastatic niches can be facili-
tated by EVs, which may alter cellular signaling, weaken
Fig. 7 Overview of the role played by tumor-derived EVs (and their cargo) in cancer biology

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Desaietal. Biomaterials Research (2023) 27:113
interendothelial junctions, and increase vascular per-
meability [98]. Tumor-derived soluble factors such as
Angptl4, SDF-1, and CCL2 are carried by EVs and pro-
mote vascular leakage, facilitating metastasis. Notably,
hypoxic tumor cells release more EVs than their nor-
moxic counterparts, which are regulated by hypoxia-
inducible factors (HIFs) [99]. Among various HIF
isomers, HIF-1α and HIF-2α are the most well studied
[100, 101]. ey bind to hypoxia response elements in
the promoters of target genes and regulate the expres-
sion of genes involved in vesicle biogenesis, trafficking,
and release [102]. EVs released under hypoxic conditions
contain various constituents, including VEGF [103], the
long noncoding RNA CCAT2 [104], and some noncod-
ing RNA miRNAs (miR-25-3p and miR-9) [105], which
directly influence the proliferation and migration of ECs.
Additionally, miR-23a, miR-92-3p, miR-103, miR-181c,
and miR-105 are enriched in these EVs and collectively
contribute to the suppression of genes involved in main-
taining vascular integrity, ultimately leading to vascular
leakage [106]. Furthermore, cancer cells secrete interleu-
kin 3 (IL-3), stimulating ECs to secrete EVs that further
promote neovessel formation [107]. e development of
premetastatic niches is also aided by tumor-derived EV
stimulation of angiogenesis, upregulation of inflamma-
tory chemicals, and suppression of immune responses
[108]. ese EVs also influence the migration of myeloid
cells and contribute to the formation of premetastatic
sites by controlling the levels of proinflammatory mol-
ecules. Factors and proteins such as TNF-α, transforming
growth factor-β (TGF-β), IL-6, IL-10, VEGF, S100, and
integrins play essential roles in this complex process [109,
110]. Moreover, EVs exert regulatory effects on DCs,
stimulating CD8 + T cells, altering pH levels, and impair-
ing antigen cross-presentation abilities [111]. Remark-
ably, EVs have the potential to stimulate the immune
system and induce an antitumor immune response
through the presentation of tumor-associated antigens
(EA, HER2, mesothelin, CD24, EpCAM, etc.) to immune
cells, particularly cytotoxic T cells [112–114].
miRNAs such as the miR-200 family, miR-23a, and
miR-92a-3p are highly concentrated in EVs produced
by cancer cells and play a role in triggering epithelial-
mesenchymal transition (EMT) in epithelial cells. [115].
EMT is closely connected to several proteins linked to
EV-mediated EMT, including HRAS, flTF III, and TGF-β
[116–118]. EMT prominently generates CAFs that fur-
ther secrete factors synergizing with tumor metastasis,
such as IL-1β, IL-6, IL-8, and matrix metalloproteinases
[119]. In this context, generating CAFs through EMT
contributes to establishing a premetastatic niche by can-
cer cell-derived EVs, as they interact with and prime
cells from distant organs. Furthermore, CAFs release
proinflammatory cytokines that suppress the normal
function of immune cells, maintaining a protumorigenic
environment.
Moving forward, the impact of EVs on drug resistance
is a critical aspect to consider. Tumor-derived EVs have
been identified as key contributors to the development
of drug resistance, operating through two distinct path-
ways: the activation of calcium/calmodulin-dependent
protein kinases and the Raf/MEK/ERK kinase cascade
[120]. Multiple cancers have been reported to exhibit
multidrug resistance due to EV-mediated transfer of
P-glycoprotein [121], multidrug resistance-1 [122], and
ATP-binding cassette subfamily B member 1. ese EVs
further contribute to drug resistance by sequestering
cytotoxic drugs, effectively reducing their concentration
at target sites. Moreover, they serve as decoys by trans-
porting membrane proteins and capturing monoclonal
antibodies designed to target cancer cell surface recep-
tors. Finally, it is worth noting that EVs derived from
resistant cancer cells can transmit messenger proteins
that induce drug resistance in sensitive cells [123, 124].
is interconnected network of EV-mediated processes
underscores the multifaceted role of EVs in cancer pro-
gression and drug resistance.
Isolation techniques
Based on the abovementioned explanation that highlights
the crosstalk between cancer cell EVs and components of
the TME, it is evident that EVs hold tremendous poten-
tial for manipulation as tools against cancer itself. Before
planning any biomedical applications, an optimal and
consistent method to isolate EVs from cancer cells must
be employed. Rapid isolation time (preferably under 1h),
high retrieval efficiency with purity (maximum EV yield
with minimal protein/free nucleic acid contaminants),
and affordability are some of the desirable characteristics
of isolation methods [125]. For viable clinical translation,
the method should additionally be able to handle high
sample volumes and be compatible with automation. An
overview of some prominent EV isolation techniques and
their respective advantages/disadvantages is discussed in
Table1.
After the successful isolation of EVs, it becomes
imperative to comprehensively characterize their clini-
cally relevant attributes. Initial assessments encompass
the determination of size and morphology, which can be
accomplished through scanning electron microscopy and
atomic force microscopy. However, for precise quantifi-
cation of EV concentrations, specialized methodologies
such as scanning ion occlusion sensing or nanoparticle
tracking analysis are essential [147, 148]. To gain insight
into the composition of EVs, including their associ-
ated proteins and nucleic acids, targeted proteomic and

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Table 1 Techniques for EV isolation
Technique (sample volume and
isolation time) Schematic Overview Advantages Limitations Ref
Differential ultracentrifugation
(1.5 mL-25 mL, 3 h-9 h)
• Minimal reagents,
consumables, exper-
tise needed
• Suitable for large
volumes
• Chemical-free, EV-
friendly
• Reliable reproduc-
ibility
• Costly equipment
• Low throughput
• Elevated protein
contamination
• Cross-contamina-
tion risk
• Sterility concerns
[126–128]
Density gradient centrifugation
(1.5 mL-25 mL, 2 h-40 h)
• High purity (gold
standard)
• Enables specific
EV subpopulation
isolation
• Affordable reagents
• High equipment
cost
• Time/labor intensive
• Possibility of viral
particle contamina-
tion (from sucrose)
• Significant sample
loss
[129–131]
Ultrafiltration (10 μL-150 mL, < 2 h) • Simple, highly
versatile
• Rapid, purity-
enhancing
• Suitable for large
samples
• Limited filter lifespan
(clogging risk)
• Non-EV protein
contamination
• EV distortion
[132–134]
Size exclusion chromatography
(200 μl-20 ml, 1 min/mL)
• Rapid isolation
• Preserves EV
integrity
• Prevents aggrega-
tion
• High purity, repro-
ducible
• Low yield
• Low sample volume
(not more than 2–5%
of the column
volume)
• Requires EV con-
centration for down-
stream applications
[135–137]
Solubility precipitation (50
μL-10 mL, 30–120 min)
• Quick process
• Kit use reduces
labor/equipment
needs
• Maintains physi-
ological pH
• Costly reagents
• Non-EV protein
contamination
• Poor purity (pres-
ence of undifferenti-
ated EV subtypes)
[138–140]
Immunoaffinity-based capture
(10 μL-10 mL, < 2 h)
• High purity, repro-
ducibility
• High specificity
to EV subpopulations
• Short processing
time
• High reagent costs
• Require prior knowl-
edge of exosome
tags
• Functional loss
without antibody
detachment
[141–143]
Microfluidic devices (Variable,
1–15 μL/min)
• Low sample volume
and minimal con-
sumables
• Rapid isolation
with high purity
• Real-time control,
automation option
• Requires use-
specific design
that increases cost
• Low throughput
[144–146]

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transcriptomic analyzes are indispensable [149]. e fol-
lowing subsections will focus on exploiting EVs as tools
against cancer.
Therapeutic applications
Bioengineered EVs are excellent templates for develop-
ing advanced cancer nanomedicines. eir intrinsic ori-
gin and structural attributes endow them with distinct
tumor-targeting characteristics, including augmented
systemic circulation and improved tumor penetration,
owing to their favorable immunogenic profile and EPR
effect, respectively. Furthermore, their capacity for deep
tumor penetration arises from their deformability and
mechanical flexibility. In addition, they exhibit selective
cellular internalization due to the presence of glycans and
membrane-soluble ligands that specifically interact with
cancer cells [150]. Isolated/purified EVs can be directly
used after loading with appropriate exogenous cargo.
Small molecule anticancer or drug-loaded nanoparticles
can be incorporated into EVs by coincubating them with
donor cancer cell lines. While this active loading method
is simple, the final loading efficiency is heavily influenced
by the physiochemical properties of the drug/drug-nano-
carrier system and protocol parameters, which demands
extensive optimization [151]. EVs can be passively loaded
using saponin-based permeabilization, electroporation
(ideal for siRNA or miRNA loading), and mechanical
methods, such as freeze‒thawing, sonication, and extru-
sion [152].
Proper membrane modification strategies can expo-
nentially increase the therapeutic potential of cancer
cell-derived EVs. Smyth etal. [153] reported the first suc-
cessful conjugation of ligands to the surface of exosomes
(derived from mouse 4T1 cells) using copper-catalyzed
azide-alkyne cycloaddition (click chemistry). is study
laid the foundation for exploring click chemistry in EV
research due to its rapid reaction times, high specificity,
and compatibility in aqueous buffers. Subsequently, Jia
etal. [154] reported the click chemistry-based conjuga-
tion of neuropilin-1-targeted peptide to the membrane
of exosomes loaded with superparamagnetic iron oxide
nanoparticles and curcumin. With the aid of surface-
conjugated peptides, the exosomes smoothly crossed the
BBB and provided lucrative results for targeted imaging
and therapy of glioma. In a different study, azido-modi-
fied exosomes obtained from MDA-MB-231 cells were
labeled with azadibenzylcyclooctyne fluorescent dyes
and applied to examine the invivo pharmacokinetics of
exosomes in MDA-MB-231 tumor-bearing mice [155].
In addition to click chemistry, various noncova-
lent membrane modifications have been reported with
interesting applications. Tamura et al. [156] reported
positively charged exosomes with enhanced targeting
efficiency prepared by electrostatic interactions between
negatively charged exosome membranes and cationized
pullulan. Sato et al. [157] developed an exosome-lipo-
some hybrid by fusing exosomes with antibody- or pep-
tide-functionalized liposomes via a freeze‒thaw method.
e hybrid demonstrated active targeting ability while
preserving exosome functionality. Recently, artificial
“chimeric” exosomes prepared by combining integrated
cell membrane proteins (isolated from cancer cell EVs)
with synthetic phospholipid bilayers have been exten-
sively reported as novel platforms with high drug-loading
capacity and EV-specific functionality [158]. e follow-
ing section discusses some recent applications of tumor-
derived EVs in cancer therapy.
Jiao etal. [159] reported the targeted delivery of exog-
enous recombinant P53, an apoptosis-inducing protein,
using EVs derived from breast cancer cells. is approach
involved conjugating P53 proteins with a mitochondria-
targeting triphenylphosphonium (TPP) derivative. e
TPP/P53 conjugate was loaded into EVs through elec-
troporation, resulting in the creation of TPP/P53@EVs.
TEM imaging revealed that unaltered EVs had a homo-
geneous and bright white interior, indicative of inherent
exosomal proteins and RNAs. Electroporation removed
the original cargo of EVs and replaced it with TPP/P53
conjugates, resulting in 2–8 TPP/P53 orbicular particles
within each EV. Cellular targeting was assessed using
MCF-7 and SK-BR-3 cells incubated with Cy5-labeled
TPP/P53@EVs, showing TPP/P53 distribution through-
out the cells, with notable concentration at the cell
center and periphery, indicating mitochondrial targeting.
Invivo biodistribution studies in a 4T1 mammary cancer
mouse model demonstrated preferential accumulation of
TPP/P53@EVs within tumors, with minimal exposure in
the liver, lungs, and kidneys. Once fused with the tumor
cell membrane, TPP/P53 proteins are released into the
cytosol and subsequently transferred to mitochondria.
e presence of p53 led to the downregulation of antia-
poptotic proteins (Bcl-2), upregulation of proapoptotic
proteins (Bax), and increased calcium levels, promoting
mitochondrial outer membrane permeabilization and
caspase-dependent apoptosis. Notably, no observable
toxicity or side effects were detected during the treat-
ment duration in the animal models, underscoring the
system’s potential safety and efficacy (Fig.8A).
Huang etal. [160] combined Hiltonol (a TLR3 agonist)
and the ICD inducer human neutrophil elastase (ELANE)
within exosomes derived from MDA-MB-231 breast can-
cer cells to promote the activation of type one conven-
tional DCs (cDC1s) within the TME. ese exosomes
were further modified with α-lactalbumin (α-LA), a
breast-specific immunodominant protein, to enhance
targeting and immunogenicity. TEM analysis revealed

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Fig. 8 Tumor-derived EVs in cancer therapy. A TPP/P53@EVs for treating breast cancer. Subfigure (i) displays the morphological features of the EVs
in both unloaded and loaded states. Subfigure (ii) demonstrates the tumor cell targeting ability and mitochondrial localization capacity of TPP/
P53@EVs using Cy5 labeling and immunofluorescence imaging. Subfigure (iii) reveals the in vivo biodistribution of TPP/P53@EVs through ex vivo
fluorescence imaging. Subfigure (iv) depicts the in vivo toxicity assessment of TPP/P53@EVs through the expression analysis of specific antigens,
namely, Bax, Bcl-2, Caspase-3, and Ki-67. Adapted with permission from [159] Copyright Elsevier, 2022). B HELA-Exos as an in situ DC-primed
vaccine for breast cancer. Subfigure (i) presents a schematic detailing the preparation process. Subfigure (ii) presents TEM images of the HELA-Exos,
allowing for observation of their morphological characteristics. Scale bar: 100 nm. Subfigure (iii) displays the results of calcein-AM fluorescence
assays used to evaluate the HELA-Exo in vitro killing capabilities against MCF7, MDA-MB-435S, and SKBR3 cells. Figure (iv) illustrates the intratumoral
accumulation of cDC1s/CD8 + T cells resulting from HELA-Exo treatment. Subfigure (v) shows the results of a flow cytometry-based analysis
of CD11c + DCs. Adapted with permission from [160], (Copyright Springer Nature, 2022)

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Desaietal. Biomaterials Research (2023) 27:113
that the resulting HELA-Exos had an average diameter
of approximately 113nm. Invitro assessments using the
calcein-AM fluorescence test demonstrated that HELA-
Exos effectively induced cell death in MCF7, MDA-MB-
435S, and SKBR-3 breast cancer cell lines, resulting in
a 90% reduction in cancer cell viability compared to a
control group treated with tumor-derived exosomes
(Texs) alone. Invivo evaluation of HELA-Exos focused
on assessing the accumulation of cDC1s and CD8 + T
cells in the TME. Immunofluorescence analysis revealed
significantly increased infiltration of DCs, identified by
markers CD11c + and CD103 + , in both the TME and
draining lymph nodes compared to the group treated
with Hiltonol alone. Flow cytometry further confirmed
the accumulation of the cDC1 subset within the TME,
providing strong evidence of the immunogenic activity of
HELA-Exos (Fig.8B).
Extensive global research is being conducted to
improve the antigen recognition site and enhance the
immunosuppressive TME. However, the goal of achiev-
ing these improvements remains elusive. Addressing
this challenge, Taghikhani etal. [161] reported a study
in which they utilized miRNA-modified tumor-derived
extracellular vesicles (mt-EVs) to selectively enhance
the maturation and antigen presentation capabilities of
DCs. rough careful investigation, specific miRNAs
(Let-7i, miR-142, and miR-155) that facilitate the desired
therapeutic effects of DC maturation and antigen pres-
entation were selectively loaded into mt-EVs. Various
combinations and permutations of miRNAs were tested,
and it was discovered that miR-142 exhibited the highest
expression levels when analyzed using real-time PCR. To
assess DC maturation, flow cytometry analysis was per-
formed. e expression levels of surface molecules such
as CD11c, MHCII, CD86, and CD40 on DCs treated with
mt-EVs were significantly higher than those observed
in DCs treated with tumor-derived EVs (t-EVs) alone.
Notably, CD11c MHCII expression reached 80% in the
mt-EV-treated group and only 65.3% in the t-EV group.
e targeted delivery capability of mt-EVs was also evalu-
ated. e expression of miRNAs was solely observed in
tumor tissue and nearby lymph nodes, with no detection
in other tissues.
Nguyen etal. [162] developed a nanosystem using EVs
derived from the CT26 murine colorectal cancer (CRC)
cell line loaded with doxorubicin (DOX) to create CT26-
EV-DOX nanoparticles. TEM images confirmed that
the CT26-EV-DOX nanoparticles had a particle size
of approximately 217.9 nm. e researchers evaluated
the cytotoxicity of these nanoparticles against various
types of cancer cells and observed that they exhibited
toxicity specifically toward CT26 cells. In vitro experi-
ments were conducted using both 2D and 3D cell culture
models to investigate cellular uptake. In the 2D setup,
cells were stained with DAPI, while DOX was visual-
ized by its red color. Confocal laser scanning microscopy
(CLSM) images confirmed that CT26-EV-DOX nano-
particles were taken up more readily by their parent cells
than other cell types. In the 3D tumor setup, CT26 cell
spheroids were used, and CT26-EV-DOX nanoparticles
demonstrated efficient penetration into the parent cells
within the spheroids.
Conventional nanoparticles undergo rapid clearance
by macrophages, particularly Kupffer cells in the liver. To
address this challenge, Qiu etal. [163] developed a novel
strategy utilizing exosome-like nanovesicles (ENVs)
derived from exosomes of metastatic breast cancer 4T1
cells. ENVs demonstrated the ability to modify the distri-
bution of nucleolin on the cell surface, thereby suppress-
ing phagocytosis by Kupffer cells. Pretreatment with 4T1
ENVs protected therapeutic drugs against elimination
by Kupffer cells, thus enhancing the therapeutic efficacy
and minimizing potential adverse effects associated with
chemotherapeutic drugs. e underlying mechanism
behind this phenomenon was attributed to the translo-
cation of membrane nucleolin from the inner face of the
plasma membrane to the cell surface, facilitated by ENVs,
along with intercellular calcium fluxes. ese events
resulted in the modulation of gene expression involved
in macrophage phagocytosis. Notably, the research-
ers observed that mice preadministered ENVs exhibited
reduced uptake of DOTAP:DOPE liposomes (DDL) in
the liver. Consequently, doxorubicin-loaded DDL was
redirected to the lungs instead of the liver, effectively
impeding breast cancer lung metastasis.
Diagnostic applications
In addition to anticancer therapy, the major application
of EVs is as biomarkers for cancer diagnosis due to the
stability and diversity of their biomolecular cargo (such
as proteins, lipids, and nucleic acids). ese molecules
can reflect the presence and staging of tumors, making
EVs valuable diagnostic tools. All cell types in the body
shed EVs, and their molecular contents are dependent on
the cells or tissues of origin. An ideal diagnostic approach
should preferentially identify tumor-specific biomark-
ers at premetastatic phases via a noninvasive method. In
this context, body fluid samples that include circulating
exosomes loaded with preserved tumor-associated miR-
NAs can be a novel biomarker source [164]. However,
the specific cargo of EVs is not always correlated with the
overexpression of molecules in the cells of origin. It can
be affected by microenvironmental conditions such as
inflammation and metabolic balance [165].
EVs containing biomarkers such as glypican-1 [166],
DEL-1 [167], and survivin [168] help distinguish between

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Desaietal. Biomaterials Research (2023) 27:113
benign and malignant diseases in breast cancer. EV-CD47
has also been proposed as a possible biomarker for breast
cancer [169]. Alterations in the levels of exosomal pro-
tein markers (compared to healthy controls), such as
gamma-glutamyltransferase in prostate cancer [93], car-
cinoembryonic antigen in CRC [170], and NY-ESO-1 in
non-small cell lung cancer [171], are some prominent
examples for cancer diagnosis. Table2 discusses several
cancer-specific exosomal markers and their application
in cancer diagnosis. ese exosomes can aid in medical
decision-making by providing contextual information
during treatment. Overall, EVs’ stable and diverse cargo
makes them a promising avenue for cancer diagnosis and
monitoring.
Liquid biopsy is a minimally invasive process that uses
biological fluids such as blood, urine, or saliva and pro-
vides real-time information and simple sample storage
[190]. It examines severalelements, including EVs, cells,
and circulating tumor DNA [191]. EV liquid biopsy, in
particular, has become a potential technique for cancer
diagnosis, prognosis, and treatment monitoring because
of its stability in circulation. e process includes isola-
tion, purification, and detection of EVs from the liquid
biopsy sample. Numerous methodologies are available
for comprehensively examining biomolecular constitu-
ents within cancer cells. ese methods encompass DNA
sequencing, which furnishes insights into the genetic
attributes of cancer; RNA sequencing, which elucidates
the gene expression profiles of cancer cells; and prot-
eomics analysis, which offers a glimpse into the protein
expression profiles associated with cancer [192]. Liquid
biopsy of EVs offers several advantages over traditional
tissue biopsy, including its noninvasiveness, the ability
to monitor cancer over time, and the potential for early
detection. However, the technique is still in the early
stages of development, and further research is needed
to validate its clinical utility [193]. e following section
highlights some interesting applications of tumor-derived
EVs in cancer diagnosis.
Wang etal. [194] introduced an innovative microflu-
idics-based device, the EV Click Chip, coupled with a
specialized nanosurface, designed to efficiently isolate
tumor-derived EVs. ese vesicles were explicitly tar-
geted to identify disease-relevant mRNAs, primarily
in the context of prostate cancer (PCa). e EV Click
Chip facilitated the isolation of pure populations of
tumor-derived EVs, overcoming contamination issues
by nontumor-derived EVs. Using reverse transcriptase-
droplet digital polymerase chain reaction (RT-ddPCR),
the researchers developed the EV Digital Scoring Assay
(DSA), tailored for rapidly detecting mRNA contents
originating from PCa-derived EVs. e assay employed
a panel of 11 carefully selected mRNA markers derived
from PCa tissue and blood samples, as illustrated in
Fig.9A. e assay’s outcome, the Met score, effectively
classified PCa in patients as either metastatic or local-
ized. Receiver operating characteristic (ROC) analysis
validated the Met score, demonstrating a higher sensitiv-
ity (approximately 85%) compared to the commonly used
prostate-specific antigen (PSA), with a sensitivity of only
approximately 65%. Compared to conventional methods
such as ultracentrifugation and the ExoQuick Assay, the
EV Click Chip showed superior purification capabilities,
even with smaller plasma samples. However, the study
acknowledged limitations, such as the need for more
appropriate gene candidates specific to EV-based stud-
ies. As research into EV cargo continues, the assay holds
potential for further optimization by targeting new or
evolving disease biology.
Dong et al. [195] introduced the nano-Villi chip,
inspired by the efficient surface area of intestinal villi, as
an innovative method for capturing tumor-derived EVs
from limited blood plasma samples. is chip comprises
two key components: a silicon nanowire with anti-epithe-
lial cell adhesion molecule (anti-EPCAM) grafting and a
polydimethylsiloxane-based chaotic mixer equipped with
a microchannel. e herringbone arrangement within
the mixer facilitates contact between the anti-EPCAM-
grafted nanowire and tumor-derived EVs, enhancing
capture efficiency. RNA content from captured EVs was
assessed using a Qubit 3.0 Fluorometer and Qubit RNA
HS assay, followed by reverse transcription droplet digi-
tal PCR. Longer silicon wires significantly improved RNA
recovery rates (82 ± 8%) compared to shorter silicon wires
(60 ± 6%) and flat silicon substrates (31 ± 1%). e pres-
ence of anti-EPCAM was critical for efficient EV capture,
primarily targeting EVs with diameters between 30 and
300 nm. e clinically applicable nano-Villi chip dem-
onstrated its utility for quantitatively detecting genetic
alterations in non-small cell lung cancer (NSCLC), high-
lighting its potential in clinical settings (Fig.9B).
Kang et al. [196] introduced an innovative approach
termed the extracellular vesicle on demand (EVOD) chip,
designed to detect and diagnose lung cancer by isolat-
ing cancer-associated exosomes derived from lung can-
cer cells. is method relies on inverse electron-demand
Diels–Alder click chemistry and involves several tech-
nical steps. First, a cross-linking agent is employed to
immobilize trans-cyclooctene prelinked with primary
amines onto a capture surface, creating a three-dimen-
sional isolation structure. Subsequently, tetrazine mole-
cules are attached to the surfaces of the exosomes, either
through direct bonding with N-hydroxysuccinimide ester
or by conjugation with specific antibodies. e EVOD
chip capitalizes on the rapid membrane-bound reac-
tion between trans-cyclooctene and tetrazine within the

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Desaietal. Biomaterials Research (2023) 27:113
Table 2 Reported exosome protein biomarkers for cancer diagnostic applications
Cancer type Exosome Protein Markers Function Application Ref
Prognosis Diagnosis Therapeutic
target Disease
monitoring
Breast cancer EpCAM Involved in tumor progression
and metastasis
✓ ✓ -✓[172]
HER2 Overexpression leads to more
aggressive tumor growth
and a poorer prognosis
-✓-✓[173]
CA 15–3 Involved in tumor growth
and metastasis
✓[174]
PKG1 (Protein Kinase G1) Plays a role in cell prolifera-
tion and migration. It also regu-
lates estrogen receptor signaling
-✓- - [175]
Colorectal cancer CD 147 (Basigin) It activates PI3K/AKT, MAPK/ERK,
and JAK/STAT signaling pathways
to promote tumor development,
invasion, and metastasis
-✓- - [176]
CEA (Carcinoembryonic Antigen) Activate recipient cell signaling
pathways, especially the EGFR
pathway, to promote tumor
growth, invasion, and angio-
genesis
✓ ✓ - - [177]
CD 166/ALCAM (Activated Leuko-
cyte Cell Adhesion Molecule) Promote tumor growth
and metastasis
✓ ✓ - - [178]
CD 9 CD9-positive exosomes play
a role in the dissemination of CRC
✓-✓ ✓ [179]
Ovarian cancer HE4 (Human Epididymis Protein
4) Modulates EGFR-MAPK signaling
pathway to influence can-
cer cell adhesion, migration,
and the growth of tumors
-✓- - [180]
Mesothelin Promote tumor development
and metastasis
✓ ✓ ✓ - [181]
Lung cancer EGFR (Epidermal Growth Factor
Receptor) Exosomal EGFR activates
downstream signaling path-
ways to promote tumor growth
and metastasis
-✓-✓[182]
KRAS Lung cancer often has KRAS
protein mutations, which are
implicated in numerous cellular
signaling pathways
✓ ✓ - - [183]
Rab3D It activates AKT/GSK3β
and induces cancer cell EMT,
promoting invasion
- - ✓- [184]
PSMA (Prostate-Specific Mem-
brane Antigen) It plays a role in the degrada-
tion of folate and is abundantly
expressed during various phases
of prostate cancer, particularly
following a relapse in therapy
-✓-✓[185]
Prostate cancer PCA 3(Prostate Cancer Antigen 3) Regulates cancer cell prolifera-
tion, invasion, and metastasis -✓- - [186]
CA 19–9 It is produced due to abnormal
glycosylation, a process com-
monly seen in cancer progres-
sion, resulting in the formation
of various glycosylated residues
- - - ✓[187]

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Desaietal. Biomaterials Research (2023) 27:113
device, ensuring instantaneous and efficient isolation of
exosomes. Following isolation, the exosomes are liberated
from the chip by cleaving the disulfide bond with dithio-
threitol (DTT). e authors systematically fine-tuned the
concentration and flow rate of DTT release to optimize
the recovered exosome yield and purity. is precision
enhancement in the isolation process holds immense
potential for the in-depth study of cancer-associated
exosomes. It may usher in new clinical applications in
cancer diagnosis and treatment. By offering a rapid and
effective EV isolation technique, the EVOD chip opens
up new avenues for investigating the role of EVs in cancer
and advancing early cancer detection strategies.
In a similar study by Notarangelo etal. [197], a novel
technique known as nickel-based isolation (NBI) was
introduced. NBI utilizes a nickel-functionalized matrix
to capture EVs via electrostatic interactions, ensuring
EV stability and integrity preservation. Chelating agents
release EVs while maintaining their structural integ-
rity for subsequent analysis. NBI demonstrates efficient
selective enrichment of heterogeneous EVs within the
50–700 nm size range, providing a time-efficient and
cost-effective method. It addresses the common issues
of protein contaminants and surface charge fluctuations
associated with traditional isolation methods. To over-
come challenges related to correlating vesicle size with
their cell of origin, the study introduces the concept of
EV lineages. ese are mixed vesicle populations posi-
tive for a cell type-specific marker, indicating a common
parental origin. e approach allows for unbiased recov-
ery of different EV lineages, resulting in a homogeneous
suspension of polydisperse EV lineages. Additionally, the
study presents a new droplet digital polymerase chain
reaction assay that eliminates the need for RNA extrac-
tion. e platform identified fractions of secreted EVs
carrying tumor biomarkers with enhanced sensitivity and
accuracy, potentially reevaluating the mutational status
in at least 10% of the analyzed patients. is suggests that
NBI-ddPCR could be used to infer the presence of spe-
cific cell subpopulations that are difficult to detect in tis-
sue biopsies, providing a deeper understanding of tumor
heterogeneity and its evolution during therapy.
Whole cells andtumor lysates
Understanding cancer vaccines
Vaccines, which prevent disease by training the body’s
immune system to rapidly and specifically terminate
harmful pathogens, are widely regarded as one of the
greatest medical inventions. ey have single-handedly
contributed to saving countless lives by facilitating the
elimination of smallpox and the near eradication of other
life-threatening diseases, such as polio and diphtheria
[198]. For the past 50years, the lucrative proposition to
develop therapeutic cancer vaccines (TCVs) has been a
research hotspot, but most of these efforts have met with
unsatisfactory outcomes. While vaccines against human
papillomavirus and hepatitis B (cervical and liver cancer,
respectively) have received Food and Drug Administra-
tion (FDA) approval, the poor clinical translatability of
other TCVs can be attributed to the immunosuppressive
context of their utilization (suboptimal antigens, lack of
immunostimulatory adjuvants, inadequate tumor locali-
zation of cytotoxic T-cell lymphocytes) [199].
e use of personalized antigen sources (especially
modified whole cancer cells and TCLs), alone or in com-
bination with appropriate adjuvants, has opened new
Table 2 (continued)
Cancer type Exosome Protein Markers Function Application Ref
Prognosis Diagnosis Therapeutic
target Disease
monitoring
Pancreatic cancer MUC1 It suppresses the immune
response and promotes tumor
growth. MUC1-expressing
exosomes increase cancer cell
migration, invasion, and angio-
genesis
-✓-✓[188]
AFP (alpha-fetoprotein) AFP exosomes transfer onco-
genic chemicals to promote
tumor growth and invasion
and suppress T cells and natural
killer cells, which kill cancer cells
-✓-✓[188]
Liver cancer ANGPT2 (Angiopoietin 2) It promotes tumor growth
and angiogenesis and is associ-
ated with increased aggres-
siveness, invasion, and tumor
metastasis
✓- - ✓[189]

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Desaietal. Biomaterials Research (2023) 27:113
avenues to develop robust TCVs [200]. Before deep-
diving into these novel TCV opportunities, it is vital to
comprehend the key elements and immune mechanisms
that cause tumor immunity. e main goal of any TCV
is to induce a robust antitumor T-cell response. TCVs
engage and leverage several facets of cancer immunity,
such as the presentation of cancer antigens, priming and
activation of T cells, and cancer cell recognition and sub-
sequent elimination [201]. By coadministering adjuvants,
both innate and adaptive responses can be triggered
simultaneously. Pattern recognition receptors (PRRs),
e.g., TLRs, identify and retort pathogen-associated
molecular patterns, thereby triggering nonspecific innate
immune responses. e transcription factor nuclear fac-
tor κB (NF-κB) is then stimulated, leading to enhanced
synthesis of cytokines/chemokines and activation of lym-
phocytes [202]. Finally, TCVs induce an adaptive cyto-
toxic T-cell lymphocyte-mediated antitumor response by
serving as a chemotactic platform to commence immune
crosstalk with APCs. TCVs present immunogenic tumor
antigens to APCs, leading to recruitment and matura-
tion. ese mature APCs, under the influence of key
Fig. 9 Tumor-derived EVs as a diagnostic agent. A Metastasis detection and monitoring of prostate cancer progression using the PCa EV Digital
Scoring Assay. Adapted with permission from [194], (Copyright Elsevier, 2023). B Schematic illustration depicting the design of the NanoVilli
Chip, which takes inspiration from natural biological structures. The chip incorporates densely packed arrays of silicon nanowires that have been
grafted with anti-EpCAM antibodies. This design enables the chip to achieve remarkably efficient and reproducible immunoaffinity capture
of tumor-derived EVs. Adapted with permission from [195], (Copyright American Chemical Society, 2019)

Page 24 of 52
Desaietal. Biomaterials Research (2023) 27:113
stimulatory signals and cytokines, activate CD8 + T cells.
ese individual steps generate an enduring immuno-
logical memory proficient in constraining tumor growth
and preventing relapse/metastasis [203]. A schematic
overview of TCV-mediated tumor immunity is depicted
in Fig.10.
Cell/lysate‑derived tumor antigens
Tumor antigen selection is a critical aspect of TCV devel-
opment. An ideal antigen should be exclusively produced
by cancer cells, present on all cancer cells, and be an inte-
gral part of cancer cell survival. Utilization of such anti-
gens will decrease the probability of “immune escape”
by means of antigen downregulation (thus yielding high
immunogenicity) [204, 205]. A single antigen will rarely
possess all these attributes. Regardless, tumor antigens
are classified into two broad categories: tumor-specific
antigens (TSAs) and tumor-associated antigens (TAAs).
Different tumor antigen sources and their attributes are
highlighted in Fig. 11. TAAs are abnormally expressed
“self-proteins” of cancerous cells. Examples of TAAs that
have been identified and utilized in TCVs include cancer/
germline antigens (e.g., MAGE-A1, MAGE-A3, and NY-
ESO-1) [206]; cell lineage differentiation antigens (e.g.,
tyrosinase, MART-1, prostate-specific antigen) [207]; and
overexpressed cancer antigens (e.g., hTERT, HER2, meso-
thelin, and MUC-1) [208]. While TAAs produce specific
cellular and humoral immune responses, their “self-anti-
gen” nature makes them susceptible to central immune
tolerance, requiring costimulators or repeated vaccina-
tion to amplify the immune response [209]. Since TAAs
are also expressed by normal cells, significant efforts are
necessary to address any potential risk to normal cells. In
this sense, TSAs are superior antigens, as they are absent
in normal cells. Oncogenic viral antigens and neoanti-
gens are examples of TSAs. Neoantigens are a subtype
of major histocompatibility complex (MHC)-binding
peptide originating from cancer-specific mutations
[210]. “Shared” neoantigens are common mutations that
can be largely identified in a wider range of patients (for
particular cancers), making them excellent targets for
off-the-shelf TCVs. “Private” neoantigens are rare muta-
tions that are highly patient-specific and appropriate for
more personalized vaccine development [211]. It should
be noted that neoantigen identification is an expensive
and laborious process.
TAAs are self-antigens expressed abnormally in tumor
cells, while TSAs are tumor-specific and expressed by
oncoviruses or cancer mutations. TSAs are more effec-
tive because high-affinity T cells may already be present,
while TAAs require a potent vaccine to “break tolerance.”
Some TSAs are shared among patients, while others
are unique and require personalized therapy. Employ-
ing whole cancer cells or TCLs as TCV components is
a viable proposition based on the available knowledge.
ey can supply all prospective antigens, removing the
obligation to target the best antigen in a specific tumor.
Multiple tumor antigens can be targeted simultaneously,
provoking an assorted immune response that avoids anti-
gen loss [212]. “Cellular” vaccines can be prepared using
modified whole tumor cells or TCLs generated by irra-
diation or repetitive freeze‒thawing. Cancer cells can be
sourced from the patient (autologous) or an appropri-
ate donor (allogeneic) for use as antigens. Nevertheless,
autologous cancer cells impart stronger tumor-specific
immunity, as they carry the entire antigenic profile of
the patient’s tumor. In theory, tumor cell lines can also
be used, but patient-derived primary cells are favored, as
their preexposure to the immune system provides addi-
tional tumor antigens [204].
Applications incancer immunization
Various strategies have been devised to enhance the
immunogenicity of these cancer cell-derived compo-
nents. Using retroviral or adenoviral transduction, can-
cer cells can be modified to express molecules relevant
for immune activation, such as cytokines (especially
granulocyte–macrophage colony-stimulating factor),
chemokines, or costimulatory molecules that function
as adjuvants [213]. By inducing ICD in cancer cells
before using them (whole or in lysate form), various
damage-associated molecular patterns (DAMPs), such
Fig. 10 Mechanisms responsible for promoting tumor immunity and generating effective T-cell responses against tumors within the human
body. TCVs activate the immune response by presenting cancer antigens to DCs, activating CTLs that provide long-term immunity. A Developing
immunity against cancer is cyclic and involves both immunostimulatory and inhibitory factors. The cycle consists of seven main steps, starting
with the release of antigens from cancer cells and ending with their eradication. TCVs and combination immunotherapy affect particular phases
of the cancer immune cycle. In addition, blocking PD-L1 or PD-1 can eliminate the suppression of T-cell-mediated cancer cell death. Vaccines boost
the presentation of the cancer antigen. Anti-CTLA4 promotes the priming and activation of antigen-specific T cells. B DCs in the TME or peripheral
blood interact with tumor antigens, which triggers the immune system to produce antitumor T-cell responses. These antigens are delivered to T
cells by activated DCs, which causes effector and memory T cells to be activated and differentiate. Once cancer cells are targeted and destroyed,
memory T cells can produce an effective secondary response when subjected to subsequent exposure to the same tumor antigen. Adapted
with permission from [199] (Copyright Elsevier, 2021)
(See figure on next page.)

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Desaietal. Biomaterials Research (2023) 27:113
Fig. 10 (See legend on previous page.)

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Desaietal. Biomaterials Research (2023) 27:113
as calreticulin, heat shock proteins (HSP 70/90), pen-
traxin-3, and uric acid, are released, which synergize with
dendritic cell maturation and T-cell responses against the
tumor [214]. eoretically, immunization with antigens
derived from cancer cells will undoubtedly expand their
therapeutic uses. Nevertheless, their clinical efficacy may
be constrained by uncertainty regarding their optimum
dose and route of administration [215]. is drawback
can be circumvented by using considerately bioengi-
neered delivery platforms, some of which are discussed
below.
Noh etal. [216] introduced an innovative immunomod-
ulatory nanoliposome system named Tumosome, which
combines tumor cell membranes from whole tumor
lysates with two lipid-based adjuvants: 3-O-desacyl-4′-
monophosphoryl lipid A (MPLA) and dimethyldiocta-
decylammonium bromide (DDA). MPLA, derived from
Salmonella Minnesota, was chosen as a TLR ligand to
stimulate key cytokines involved in immune activation,
such as IL-6, IL-1β, IL-12, IFN-β, and TNF-α, via the
TLR4 receptor. As MPLA and DDA are hydrophobic,
they require delivery in the form of an emulsion or lipo-
some. DDA, known for its cationic properties, aids in the
uptake of tumor antigens by APCs. Tumosome prepara-
tion involves tumor cells obtained from the patient or an
allogenic tumor cancer cell line. e study utilized fluo-
rescence techniques to demonstrate enhanced cellular
uptake of Tumosomes. Furthermore, the secretion of the
proinflammatory cytokines IL-6 and TNF-α was meas-
ured via ELISA to assess the potential of Tumosomes to
activate and mature immune cells, specifically bone mar-
row-derived dendritic cells (BMDCs) and bone marrow-
derived macrophages. e secretion of cytokines was
concentration-dependent (Fig.12A). Overall, this study
underscores Tumosomes’ capacity for simultaneous
tumor antigen delivery and immune cell activation, sug-
gesting their potential in cancer immunotherapy.
Lin Ma etal. [217] reported the development of a can-
cer vaccine by solubilizing the entire TCL fraction using
8M urea. is solubilized lysate was then incorporated
into a vaccine formulation comprising PLGA, creating
a versatile vaccine capable of carrying various tumor
antigens. e size of the resulting nanovaccine was
meticulously controlled at 300nm to facilitate efficient
delivery into APCs. e tumor was initially fragmented
into microsized pieces, followed by centrifugation. e
water-soluble component in the supernatant was col-
lected, while the insoluble component in the precipitate
was solubilized using 8 M urea. ese water-soluble
and insoluble components were then loaded into PLGA
nanoparticles, leading to the development of nanovac-
cines A and B, respectively. Using the double-emulsion
method and mixing/coating techniques, all essential anti-
gens were loaded inside and on the surface of the PLGA
nanoparticles. is nanovaccine exhibited the ability to
activate both adaptive and innate immune responses.
e study revealed a significant increase in the abun-
dance of various immune cells at the tumor site in the
nanovaccine-treated group. Notably, the levels of DCs,
macrophages, B cells, and T cells were notably elevated,
reflecting the activation of adaptive immunity against
cancer. B cells and tertiary lymphoid structures were also
observed, suggesting their role in enhancing immuno-
therapy. Furthermore, nanovaccine treatment resulted
in increased levels of natural killer cells and enhanced
CD8 + and CD4 + T-cell activity, characterized by ele-
vated cytotoxic interferon-gamma (IFN-γ) secretion.
Most macrophages at the tumor site following nanovac-
cine treatment exhibited the type 1 macrophage (M1)
phenotype, known for its effectiveness in eliminating
Fig. 11 Schematic representation of different tumor antigen sources and their attributes. Recreated with permission from [202], (Copyright
Springer Nature, 2019)

Page 27 of 52
Desaietal. Biomaterials Research (2023) 27:113
cancer cells. Flow cytometry studies further supported
these findings, showing an increase in cytotoxic T cells
after nanovaccine treatment (Fig.12B).
Wang etal. [218] employed polydopamine nanopar-
ticles loaded with tumor lysate (TCL@PDA NPs) for
cancer immunotherapy. e TCL@PDA NPs demon-
strated excellent storage stability and minimal cytotoxic-
ity. Upon uptake by BMDCs, TCL@PDA NPs promoted
antigen assimilation and BMDC maturation, leading to
enhanced surface molecule expression and the secretion
of 1-related cytokines. In vivo administration of
TCL@PDA NPs to mice resulted in significant tumor
growth suppression in treatment and prevention models.
is was accompanied by increased subpopulations of
CD4 + and C D8 + T cells in the spleen and lymph nodes
and an increase in the production of memory T cells,
providing long-term protection against malignancies.
e increased number of CTLs and M1-type TAMs and
decreased subpopulation of immunosuppressive cells in
the tumor tissues demonstrated the antitumor activity
Fig. 12 Using cancer cell tumor lysate for immunomodulatory effects. A Tumosomes for enhancing antitumor immunity. Here, subfigure (i)
illustrates the synthesis of multifaceted Tumosomes with immunostimulant adjuvants and helper lipids. Subfigure (ii) demonstrates immune
cell maturation by measuring the levels of proinflammatory cytokines such as TNF-α and IL-6 with ELISA. Adapted with permission from [216],
(Copyright Wiley–VCH, 2017). B Nanovaccine preparation using whole TCL. Here, subfigure (i) depicts a brief graphic representation of the synthesis
of the nanovaccine. Subfigure (ii) shows the activation of adaptive immunity by the activation of DCs, macrophages, B cells, and T cells. Subfigure
(iii) shows the activation of innate immunity. Increased levels of CD8 + and CD4 + T cells increase the IFN-γ responsible for tumor death. Figure (iv)
shows enhanced macrophage type I expression by investigating F4/80, CD163, CD80, PD-1, and PD-L1 through immunohistochemistry. Adapted
with permission from [217], (Copyright Wiley–VCH, 2021)

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Desaietal. Biomaterials Research (2023) 27:113
of TCL@PDA NPs. Furthermore, empty PDA NPs dem-
onstrated the ability to modulate DC maturation by
enhancing their capacity to express MHC II and secrete
1-related cytokines, inhibiting tumor development by
promoting the production of activated T cells and reduc-
ing the subpopulation of MDSCs in tumors.
In a similar study, Wang etal. [219] developed a tumor
microparticle vaccine. It involved coating live tumor
cells with a specialized layer containing epigallocat-
echin-3-gallate (EGCG) and aluminum (III) (Al(III)).
EGCG, a well-known polyphenol with anticancer prop-
erties, stimulated macrophage activation by enhancing
1 cytokines such as TNF-α and INF-γ while reducing
immunotolerance-related cytokines such as IL-10. Al(III)
is an effective aluminum adjuvant known for eliciting a
robust humoral immune response. EGCG and Al(III)
were coordinated to form an EGCG/Al(III) complex,
which was then coated with inactivated B16 tumor cells,
resulting in the TCL@EGCG/Al(III) complex. is coat-
ing process was successfully applied to four different
types of cancer cells, demonstrating compatibility. Cell
viability studies indicated that the process had no adverse
effects on cell viability. Furthermore, the study investi-
gated antigen uptake by DCs using microparticles as a
delivery system. e results showed that DCs efficiently
phagocytosed these microparticles, displaying dendritic
structures indicative of their maturation. Flow cytometry
analysis quantified antigen uptake and revealed signifi-
cantly increased uptake by DCs when using the micro-
particle delivery system compared to soluble antigens.
After antigen uptake, BMDCs matured and expressed
activation markers, enhancing their capacity to present
antigens and prime T cells. Notably, the microparticle
delivery system upregulated the expression of CD40,
CD80, MHC I, and MHC II on the surface of BMDCs,
indicating its role in BMDC maturation. Additionally, it
induced the production of 1-related cytokines such
as IL-12p70, TNF-α, IL-6, and interferon-γ, which are
crucial for promoting the differentiation of T cells into
CD8 + cytolytic T lymphocytes, highlighting its signifi-
cant potential as a personalized cancer immunotherapy
adaptable to different cancer types.
Numerous studies have investigated the role of adju-
vants in conjunction with TCL and their synergistic
effects in targeting and eliminating tumor cells. Ashrafi
et al. [220] conducted similar research to explore the
impact of propranolol, an adjuvant, when combined
with a tumor lysate vaccine in a mouse model of breast
cancer. eir study focused on evaluating immune
responses and tumor growth. e findings revealed that
administering propranolol alongside the vaccine con-
taining TCL derived from the 4T1 breast cancer cell
line significantly enhanced lymphocyte proliferation and
cytokine production within the TME. Notably, cytokines
such as IL-2, IL-10, IL-12, IFN-, and IL-17 were notably
increased. Moreover, the propranolol/vaccine combina-
tion effectively suppressed tumor growth compared to
the group immunized solely with tumor lysate. ese
results underscore the potential of propranolol as an
adjuvant in cancer immunotherapy. e study also sug-
gests that the propranolol/vaccine mixture may induce
1 cytokine responses, which are recognized for their
involvement in antitumor immune reactions. Addition-
ally, the combination of propranolol and tumor lysate
vaccine holds promise for expanding IL-17-based thera-
pies in breast cancer treatment.
Shi etal. [221] synthesized chitosan nanoparticles dec-
orated with mannose to target DCs. ese nanoparticles
were loaded with TCL derived from B16 melanoma cells
(Man-CTS-TCL NPs). When tested in BMDCs, the sys-
tem demonstrated significantly higher antigen uptake
than other groups, as assessed by flow cytometry. More-
over, the Man-CTS-TCL NPs promoted the maturation
of DCs, as evidenced by the enhanced expression of sur-
face markers such as CD80, CD86, and CD40. A cyto-
toxic T lymphocyte assay was performed to assess T-cell
efficacy in tumor-mediated specificity. Splenocytes,
as effector cells, were cocultured with B16 melanoma
cells, and lysis of the target cells was evaluated at differ-
ent effector:target (E:T) cell ratios. e results showed
that effector T cells obtained from mice immunized
with Man-CTS-TCL NPs exhibited a higher efficiency in
inducing the CTL response against B16 melanoma target
cells, with a lysis rate of approximately 35% compared to
the control group’s rate of approximately 12%. C57BL/6
mice with B16 melanoma vaccinated with Man-CTS-
TCL NPs displayed increased CD8 + T cells in the spleen
and elevated expression of IFN-γ.
Engineered cancer cells andtumor organoids
Understanding engineered cancer cell lines
Cancer cell lines are cells obtained from cancerous tis-
sues and can proliferate continuously in laboratory con-
ditions, offering a continuous supply of cancer cells for
scientific investigations. Cancer cell lines are typically
generated by immortalizing cancer cells, which involves
introducing genetic changes that hinder or bypass the
mechanisms that regulate cell growth and division. Com-
mon methods of immortalization include inducing muta-
tions in cell cycle control genes such as TP53, RB1, or
PTEN, which usually inhibit uncontrolled cell growth
and division [222]. Once immortalized, cancer cell lines
can be propagated and maintained in culture indefinitely,
making them an invaluable resource in cancer research.
ey are extensively utilized to scrutinize the biology of
cancer, test new cancer treatments, evaluate drug toxicity

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Desaietal. Biomaterials Research (2023) 27:113
and effectiveness, and pinpoint prospective targets for
cancer therapies [223]. Cancer cell lines offer numerous
benefits for scientific research. ey can be grown in sub-
stantial quantities, enabling experiments and testing on a
large scale. ey can also be cryopreserved and preserved
for long periods, allowing researchers to work with the
same cell lines for years or even decades. Furthermore,
cancer cell lines are often easier to manipulate than
whole animals, which enhances experimental control and
reproducibility [224].
During the early 1990s, the National Cancer Institute
in Bethesda, MD, introduced a new "disease-oriented"
approach for evaluating new anticancer drugs. is
involved using a panel of 60 human cancer cell lines
derived from nine different types of cancer (brain, colon,
leukemia, lung, melanoma, ovarian, renal, breast, and
prostate) to perform high-throughput screening of poten-
tially marketable drug candidates [225]. e primary
focus was to narrow down the most likely candidates
subjected to further preclinical assessment in xenograft
models. However, this approach had poor robustness in
identifying promising candidate drugs, leading to the
adoption of the hollow fiber assay (which involves the
implantation of hollow fibers, which are small, semiper-
meable tubes containing cancer cells into a mouse/rat).
e fibers are then exposed to various anticancer drugs
to assess their cytotoxicity [226]. e NCI uses only 12
human cell lines for regular preliminary screening before
moving on to time-consuming and labor-intensive xeno-
graft experiments for the most promising therapeutic
candidates.
Despite widespread efforts to reduce and eliminate
animal testing for anticancer drug screening, cancer cell
line-based in vitro methods have some critical draw-
backs that make them unreliable. ese drawbacks can
be narrowed down to the extensive genetic/epigenetic
changes of cells in culture, lack of tumor heterogene-
ity (as in primary cancer), and total absence of relevant
components constituting the complex TME [227, 228].
Several strategies have been employed to mitigate these
limitations. One approach involves utilizing primary can-
cer cells derived directly from tumor biopsies or surgical
specimens obtained from cancer patients. ese primary
cells closely mirror the biological characteristics of the
original tumor, offering a high degree of fidelity to the
source malignancy. Another strategy involves the use of
genetically modified cancer cell lines. ese cell lines are
engineered to carry specific genetic alterations, such as
the overexpression or knockdown of genes implicated
in cancer development and progression. Additionally,
they can be modified to express specific cytokines and
growth factors known to be present in the TME [229].
Primary tumor cells provide a superior representation of
the genetic and phenotypic diversity present in clinical
tumor samples. However, the degree of heterogeneity is
often not comprehensively defined, rendering the inter-
pretation of experimental outcomes challenging. Even
well-defined cancer lines can pose a challenge in screen-
ing therapies targeted toward specific oncogenic mecha-
nisms due to the complex network of mechanisms that
drive tumor growth [230]. Conversely, models created by
modifying cell lines to overexpress specific cancer bio-
markers provide a clear understanding of the oncogenic
mechanism. However, artificially elevating the expression
of an oncogene to nonphysiological levels fails to model
the intricate cascade of events that lead to tumor forma-
tion invivo [231]. ese trade-offs are significant factors
contributing to the low success rates of clinical trials of
targeted cancer therapies.
One way to tackle this problem is by using the grow-
ing range of CRISPR/Cas9 genome-editing tools to create
tailor-made biomarker-specific cancer models from the
existing library of human cancer cell lines. Emmanuelle
Charpentier and Jennifer Doudna were awarded the 2020
Nobel Prize in Chemistry for their groundbreaking work
in developing CRISPR/Cas9 gene editing technology,
which allows precise modifications to DNA sequences
in living organisms [232]. e technology consists of
two main components: the Cas9 protein and a guide
RNA (gRNA). e Cas9 protein is a nuclease enzyme
that can cut DNA at specific locations. e gRNA is a
short RNA molecule complementary to a specific DNA
sequence, guiding the Cas9 protein to the target site.
Delivering the Cas9 protein and gRNA to a cell makes it
possible to change the DNA sequence at the target site.
e mechanism of CRISPR/Cas9 involves a process that
occurs naturally in certain bacteria as a defense against
viral infections. Small RNA molecules called CRISPRs
are a memory of past viral infections in these bacteria.
When the bacteria are infected again by the same virus,
the CRISPR RNA molecules guide the Cas9 protein to
the viral DNA, which is then cut and destroyed [233].
e laboratory facilitates precise genetic mutations by
introducing a nonfunctional version of a gene to create
a knockout or loss-of-function mutation or by intro-
ducing a modified gene to create a specific point muta-
tion. Insertion or deletion of specific DNA sequences is
also possible [234]. It is possible to use CRISPR-based
genome engineering to make accurate modifications to
the genome of a particular cell line. is can be done to
create cell lines that accurately mimic the natural devel-
opment of cancer in healthy tissue or to induce specific
cancer genotypes found in clinical patient samples [235].
In recent years, a number of studies utilizing CRISPR/
Cas9 to create cancer cell lines for drug development and

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Desaietal. Biomaterials Research (2023) 27:113
cancer biology research have emerged. e following sec-
tion will delve into a few significant studies.
Engineered cancer cell lines incancer research
Zhang etal. [236] used the CRISPR/Cas9 system to spe-
cifically knock out the Mediator complex subunit 12
(MED12) gene to develop cancer cell line-based inherit-
able drug-resistant models. MED12 encodes a mediator
complex subunit that can confer transcriptional resist-
ance to chemotherapy. e authors created a MED12KO
A375 cell model (melanoma) resistant to B-Raf proto-
oncogene, serine/threonine kinase (BRAF). ey
reported that this cell model could be established in three
weeks, much faster than the traditional method (gradi-
ent-dosage induction), which takes several months. Addi-
tionally, the induced mutations were genomically stable
during passaging. A small-scale drug screening study
was performed to find new combinations of drugs that
could effectively treat multidrug-resistant melanoma.
ey focused on MED12, which blocks the glycosyla-
tion of immature forms of the TGF-β receptor, a protein
that activates TGF-β signaling. Loss of MED12 leads to
activation of TGF-βR signaling, which confers resistance
to BRAF inhibitors. e authors showed that inhibiting
TGF-βR signaling can restore drug sensitivity in cells
that lack MED12. ey also identified several new com-
binations of TGF-β inhibitors and BRAF inhibitors that
showed strong synergy in suppressing drug resistance.
e A375 cell line, normally sensitive to BRAF inhibitors
such as vemurafenib, became resistant to these drugs by
activating the TGF-βR signaling pathway. Nevertheless,
the bioengineered cells regained drug sensitivity through
TGF-βR inhibition (Fig.13A).
Gonçalves et al. [238] employed a multifaceted
approach that involved analyzing a vast dataset com-
prising over 199,000 drug sensitivity measurements for
397 anticancer drugs across 484 cancer cell lines. ese
data were integrated with genome-wide CRISPR loss-of-
function screens to assess gene fitness. e study yielded
valuable insights into drug targets, specificity, isoform
selectivity, and drug potency. Researchers have identi-
fied robust pharmacogenomic biomarkers by scrutinizing
the correlation between drug response and gene fitness
data. ese biomarkers hold the potential for predict-
ing drug responses and shed light on alternative tar-
gets that could be leveraged in combination therapies.
However, it is worth noting that nearly half of the drugs
tested did not exhibit a significant association with gene
fitness effects. Several factors could contribute to this
outcome, including drug polypharmacology, distinctions
between protein inhibition and knockout, incomplete
target inhibition, functional redundancy among pro-
tein isoforms, and inherent limitations of CRISPR‒Cas9
screens. e authors proposed that this approach could
be integrated into drug development, particularly dur-
ing the hit-to-lead or lead optimization stages. Moreo-
ver, they suggested that combining this approach with
other experimental and computational methods could be
instrumental in investigating the mechanisms of action
for novel and uncharacterized compounds. As additional
data from CRISPR knockout screening and CRISPR
activation and inhibition studies become available, this
approach is expected to become even more valuable in
unraveling cellular drug mechanisms and enhancing drug
development processes.
Similarly, Behan et al. [237] conducted a study to
identify key genes that are selectively required for the
fitness of cancer cells, which could be exploited as
therapeutic targets. ey used CRISPR/Cas9 to disrupt
genes in over 300 cancer models from 30 cancer types
across 19 different cancerous tissues. By combining this
information with patient genomic data, the research-
ers created a data-driven framework that generated a
ranked list of potential new targets for various cancer
types. ese critical genes represent vulnerabilities in
cancer cells and could contribute to the initial stages of
drug development by offering a more varied and effec-
tive portfolio of cancer drug targets. e principles
outlined in this study have the potential to enhance
the success of cancer drug development by provid-
ing a data-driven approach to identifying new targets
(Fig.13B).
In their research on cancer metastasis and EMT, Shu
etal. [239] developed a novel cell model using CRISPR/
Cas9 technology. e aim was to create a more accurate
and physiologically relevant model for studying EMT in
breast cancer. To achieve this, the researchers introduced
a modified gene, termed ECAD-EmGFP, into MCF10A
breast epithelial cells. is modified gene allowed them
to monitor the progression of EMT in real time. e
key modification involved tagging EmGFP at the ECAD
gene’s C-terminus, a critical EMT marker. e success-
ful knock-in of the ECAD-EmGFP gene was confirmed
at multiple levels, including DNA, mRNA, and pro-
tein. Subsequently, when these engineered cells were
treated with TGF-β, a well-known inducer of EMT, they
underwent the EMT process. is was evident through
a decrease in ECAD-GFP expression and a concurrent
increase in vimentin and fibronectin expression, char-
acteristic changes associated with EMT. Additionally,
the researchers observed that the cells undergoing EMT
displayed enhanced migration capabilities, a hallmark
feature of EMT in cancer cells. is physiologically rele-
vant cell model provides insights into the biology of EMT
in cancer and holds promise for drug discovery efforts

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Desaietal. Biomaterials Research (2023) 27:113
Fig. 13 A Leveraging CRISPR/Cas9 to create drug-resistant cancer cell lines for use as advanced drug screening tools. Here, subfigure (i) illustrates
the structure and working process of the CRISPR/hCas9 system. Subfigure (ii) depicts immunofluorescence analysis of MED12 protein levels
in MED12KO cells using different stains. Cells were stained with Alexa 488-phalloidin, rabbit anti-MED12 antibody/rhodamine red-labeled goat
anti-rabbit IgG, and DAPI. Subfigure (iii) shows western blot analysis confirming MED12 protein levels. Subfigure (iv) compares the response of BRAF
inhibitor-resistant clones to various inhibitor combinations [Colors indicate different drug efficiencies: no significant inhibitory effect (white,
cell coverage ≥ 80%), mild inhibitory effect (yellow, 60% ≤ cell coverage < 80%), moderate effect (orange, 30% ≤ cell coverage < 60%), and strong
inhibitory effect (red, cell coverage < 30%)]. Adapted with permission from [236] (Copyright Elsevier, 2018). B A schematic overview of a novel
strategy to prioritize targets in multiple cancer types by incorporating CRISPR–Cas9 gene fitness effects, genomic biomarkers and target tractability
for drug development. Adapted with permission from [237] (Copyright Springer Nature, 2019)

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Desaietal. Biomaterials Research (2023) 27:113
targeting EMT-related processes in cancer progression
and metastasis.
Table3 summarizes several studies utilizing CRISPR to
generate bioengineered cancer cell lines and their appli-
cation in cancer research.
Need fortumor organoids
Despite their enormous contributions to accelerating
research, cancer cell lines are held back by their inability
to replicate the intricate biology and pathophysiology of
their original tumors. As an alternative, tumor organoids
offer a superior and independent invitro means of study-
ing cancer, providing a more personalized and accurate
model for studying cancer biology and testing new can-
cer therapies [249]. Tumor organoids are a groundbreak-
ing development in cancer research that provides a more
personalized and accurate model for studying cancer
biology and testing new cancer therapies. ey are 3D
structures developed by embedding cancer cells in a gel-
like substance that mimics the TME. ey are useful in
cancer research because they provide a more realistic
representation of the TME than traditional 2D cell cul-
tures, which lack the complexity and heterogeneity of real
tumors [250]. ey allow for the incorporation of other
cell types that are present in the TME, such as immune
cells, fibroblasts, and ECs. ese cell types play impor-
tant roles in tumor growth and response to therapy, and
their inclusion in tumor organoids allows for a more real-
istic representation of the TME [251]. ese organoids
can be derived directly from patient biopsies and offer a
unique opportunity to study the effects of different drugs
on individual patients based on their genetic and molecu-
lar characteristics. is is particularly important for test-
ing the effectiveness of targeted therapies, which rely on
identifying specific genetic mutations or biomarkers pre-
sent in the tumor [252–254].
e mechanisms of cancer therapy resistance can be
investigated using tumor organoids. By growing orga-
noids from tumors resistant to a particular therapy,
researchers can identify the genetic and molecular
changes that lead to resistance and develop new strate-
gies to overcome them [255]. Recent research using
advanced tumor organoids and engineering approaches
has enhanced our understanding of the role of immune
cells in the TME. is can help researchers understand
how immune cells contribute to tumor growth and
identify new targets for immunotherapy [256]. Tumor
organoids offer a valuable platform for investigating the
impact of various microenvironmental factors on tumor
development and therapeutic outcomes. For example,
researchers can study the effects of hypoxia or acidosis
(high acidity) on tumor growth and identify new targets
for therapy [257]. By sourcing cancer cells from patients,
tumor organoids can also serve as powerful models for
studying tumor heterogeneity. Patient-specific tumor
organoids accurately replicate the diverse genetic, phe-
notypic, and spatial heterogeneity of tumors [258]. ese
3D models capture the genetic and epigenetic diversity of
distinct neoplastic subclones and enable studying their
responses to treatments. Additionally, organoids incor-
porate nonneoplastic TME cells, allowing the investiga-
tion of complex cellular interactions and niche-specific
signaling [259]. By faithfully preserving tumor heteroge-
neity, tumor organoids provide a valuable tool for person-
alized treatment strategies and a deeper understanding of
tumor biology (Fig.14).
Overall, the reconstruction of the TME using engi-
neered tumor organoids has enabled the discovery of
novel targets and the development of more effective
therapies to improve patient response rates. e follow-
ing section highlights compelling instances that illustrate
the distinctive applications of tumor organoids in cancer
research, providing valuable insights into their utility.
Organoids incancer research
Dominijanni etal. [260] designed a 3D organoid model
for investigating the intricate interactions within the
TME during liver metastasis of CRC. is model was
engineered to emulate the transformation of hepatic
stellate cells (HSCs) into myofibroblasts, a phenomenon
linked to tumor growth and resistance to chemotherapy
due to excessive ECM production. e model’s core con-
sists of a collagen-based 3D hydrogel enveloping a CRC
spheroid. It offers precise control over the structural and
cellular aspects of the TME. Following exposure to TGF-
β, a cytokine known to induce HSC activation, the TME
exhibited increased stiffness attributed to heightened
ECM synthesis and bundling orchestrated by LX-2 cells,
akin to the behavior of CAFs in the liver. is enhanced
rigidity in the TME was correlated with chemoresist-
ance. Moreover, the organoid model revealed that colla-
gen fibers within the TME, when influenced by TGF-β,
displayed enhanced alignment, length, and width char-
acteristics indicative of HSC activation and fibrotic tis-
sue formation. Immunohistochemical staining revealed
that cancer cells embedded within a denser ECM began
expressing epithelial markers. ese findings underscore
the critical role of the TME in influencing cancer pro-
gression and chemoresistance. e 3D organoid model
serves as a valuable tool for gaining deeper insights into
TME-mediated effects on cancer cells.
Tsai etal. [261] addressed the need for comprehen-
sive in vitro models of pancreatic adenocarcinoma
microenvironments, which are vital for studying
stromal and immune interactions within pancreatic
tumors. ey developed patient-matched, organotypic

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Desaietal. Biomaterials Research (2023) 27:113
Table 3 Bioengineered cancer cell lines using CRISPR technology
Cancer Cell Line Type CRISPR Target Gene and Function Key Findings/Potential Therapeutic Application Ref
Murine melanoma B16F10 cell line • CD47: It acts as a signal on tumor cells to prevent their
phagocytosis by macrophages
• IL-12: Promotes immune response against tumors by inhibit-
ing angiogenesis, boosting T-cell growth, and inducing TAM
polarization
• Tumor cells were genetically engineered to produce IL-12
while simultaneously knocking out the CD47 gene
• This dual modification enhanced the immune response
by promoting increased macrophage activity
[240]
Human melanoma cell lines WM115, CM150-Post, and NZM40 • Early B-cell factor 3 (EBF-3): It acts as a putative epigenetic
driver of melanoma metastasis • DNA methylation editing was conducted, with particular
emphasis on the EBF-3 promoter region
• Study highlights the promising capabilities of CRISPR for con-
ducting epigenetic editing in disease-related investigations
[241]
The human NSCLC cell line A549 and the cisplatin-resistant
A549 cell line (A549/DDP) • NNT: It encodes for an enzyme known as nicotinamide
nucleotide transhydrogenase, which plays a crucial role
in mitochondrial energy metabolism
• CRISPR-based DNA hypermethylation leads to the silencing
of NNT in cisplatin-resistant lung cancer cells
• Restoring NNT expression reduces resistance to cisplatin
and suppresses autophagy
[242]
ID8 cells (murine ovarian surface epithelial cells) • Trp53/p53: A crucial tumor suppressor gene that is vital
in controlling the cell cycle
• Breast cancer susceptibility gene 2: Functions as a tumor sup-
pressor gene, aiding in the prevention of cancer cell formation
• Novel ID8 derivatives were generated by introducing single
or double deletions of suppressor genes
• This approach enabled the modeling of high-grade serous
carcinoma for further study
[243]
NPC (Nasopharyngeal carcinoma) cell line CNE2 • KLHDC4: It interacts with tumor suppressor protein p53,
thereby augmenting its capacity to initiate apoptosis in cancer
cells
• Gene editing of KLHDC4 led to the inhibition of cell prolifera-
tion, reduced colony formation, and suppressed tumor growth
in mice
• Findings suggest KLHDC4 holds promise as a potential thera-
peutic target and a prognostic biomarker
[244]
SH-Y5Y neuroblastoma cell line • Chromosome 6q region: Loss of 6q genetic material, includ-
ing tumor suppressor CDKN1A, affects cell growth and prolif-
eration and is implicated in neuroblastoma progression
• Chromosome 11q region: 11q genetic material loss, includ-
ing tumor suppressors ATM and CBL, is linked to aggressive
neuroblastoma
• CRISPR‒Cas9 mediated deletions in chromosome 11q and 6q
revealed oncogenic advantages, suggesting therapeutic
potential in targeting these deletions
[245]
Human cell lines Panc-1 and SUIT-2 and the murine cell line
TB32047 • KrasG12D: It is postulated to be a key driver in tumor initiation
and progression • Three Kras heterozygous cell lines were investigated,
and the resulting knockout clones displayed characteristics
akin to those of wild-type cells during standard growth condi-
tions
[246]
Adriamycin-resistant (A2780/ADR) ovarian cancer cell line • ABCB1: It encodes P-glycoprotein (a membrane protein
that aids in the transport of molecules across cell membranes).
ABCB1 mutations are associated with anticancer drug resist-
ance
• Downregulation of the ABCB1 results in heightened sen-
sitivity of drug-resistant ovarian cancer cells to doxorubicin
treatment
[247]
PC-9 (lung adenocarcinoma cell line) • Epidermal growth factor receptor (EGFR): It encodes a protein
that governs cell growth, survival, and migration. Abnormali-
ties in this gene can drive cancer growth
• CRISPR/Cas9 introduced EGFR T790M mutation into PC9 lung
cancer cells, creating clones resistant to gefitinib but sensitive
to AZD9291
[248]

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Desaietal. Biomaterials Research (2023) 27:113
models encompassing human pancreatic cancer orga-
noids, CAFs, and T cells. eir research unveiled the
significant influence of fibroblasts, which secrete par-
acrine cytokines such as IL-6, on tumor survival and
growth. ese intricate in vitro models are valuable
for investigating the stromal and immune components
of the TME and hold promise for personalized drug
testing, as organoids can be cultured from fine-needle
aspiration biopsy specimens. e models offer clinical
applications such as evaluating genetic and phenotypic
markers, guiding individualized therapies, and aiding
target identification. Moreover, they facilitate the study
of immunotherapeutic strategies and immune check-
point inhibition by incorporating lymphocytes into
pancreatic cancer organotypic cultures.
Fig. 14 Patient-specific tumor heterogeneity reproduction in tumor organoids. Tumor organoids accurately mimic the unique characteristics
of individual patients’ tumors, reflecting the diverse cellular and environmental factors that contribute to tumor heterogeneity. These organoid
models, derived directly from patients, effectively reproduce the phenotypic, epigenetic, and spatial diversity observed within and between
tumors. Moreover, tumor organoids provide a means to study the heterogeneous TME, including the presence and functions of noncancerous TME
cells, signaling through specific factors within their respective niches, and the altered composition of the ECM. Consequently, tumor organoids
hold significant promise for modeling personalized responses to anticancer treatments in clinical settings. Adapted with permission from [250],
(Copyright Springer Nature, 2022)

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In a similar study, Lim et al. [262] constructed an
in vitro coculture model using hyaluronic acid hydro-
gels to explore angiocrine interactions between ECs and
hepatocellular carcinoma (HCC). is model provided
insights into the potential roles of ECs in driving tumor
progression independently of perfusion. e coculture
induced the upregulation of MCP-1, IL-8, and CXCL16,
aligning with known angiocrine signaling patterns. RNA
sequencing analysis highlighted the activation of tumor
necrosis factor signaling, indicating that ECs stimulate
HCC cells to establish an inflammatory microenviron-
ment that recruits immune cells. Furthermore, the model
demonstrated that angiocrine crosstalk influenced mac-
rophage polarization toward a proinflammatory and
proangiogenic phenotype, resembling tumor-associated
macrophages observed in HCC. is platform serves as a
valuable tool for exploring the intricate interplay between
angiogenesis and the immune microenvironment and
assessing the clinical potential of antiangiogenic therapy
in HCC (Fig.15A).
Tumor organoids offer the potential for revolution-
ary high-throughput drug testing, using automation
and robotics to assess multiple drugs simultaneously,
expediting drug development [264]. On this note, Phan
etal. [265] reported a high-throughput tumor organoid
drug screening platform for personalized medicine.
ey introduced a mini-ring approach that simplifies the
geometry for seeding cells around the well rims, enabling
compatibility with automation and high-throughput
screening. is method was tested with four patient-
derived tumor organoids from ovarian and perito-
neal carcinomas. ey exposed these organoids to 240
kinase inhibitors and assessed viability, number, and
size changes, identifying personalized responses tailored
to each tumor. Impressively, the results were available
within a week of surgery, making this approach rapid and
efficient for informing treatment decisions. is method
offers notable advantages, such as using a small number
of cells, negating the need for extensive invitro or invivo
expansion that can lead to divergence from the tumor’s
characteristics. It is particularly advantageous for sam-
ples that struggle to grow invivo, reducing the time and
cost of generating patient-derived xenografts (PDXs). e
authors identified several effective molecules for treating
a rare ovarian carcinosarcoma, which lacks a standard,
optimized first-line drug regimen. is emphasizes the
promise of this platform, highlighting its speed, versatil-
ity across various systems and drug screening protocols,
potential for full automation, adaptability to different
support materials (beyond Matrigel), and scalability to
384-well plates.
In a related investigation, Schuster etal. [263] devel-
oped a microfluidic 3D organoid culture and analysis
system that can provide hundreds of organoid cultures
with combinatorial and dynamic drug treatments, ena-
bling real-time organoid analysis. Platform validation
encompassed individual, combinatorial, and sequen-
tial drug screens on human-derived pancreatic tumor
organoids. e findings demonstrated that tempo-
rally modified drug treatments exhibited superior effi-
cacy in vitro compared to constant-dose monotherapy
or combination therapy. is platform holds signifi-
cant potential for advancing organoid models, enabling
screening approaches closely mimicking real patient
treatment courses. Consequently, it can contribute to
personalized therapy decision-making. e microfluidic
platform utilized in this study exhibited high reproduc-
ibility and robustness, making it well suited for accom-
modating complex treatment combinations and temporal
sequences of culture conditions. e microfluidic archi-
tecture offers precise addition of reagents at specific
times, effectively eliminating the major errors that may
arise from manual approaches. Furthermore, the plat-
form incorporates numerous repeated conditions and
controls through identical well units exposed to identical
conditions, enhancing accuracy. One notable advantage
of the platform lies in its temporal capabilities, which
facilitate testing thousands of drug combinations to rep-
licate real-life patient treatments in a procedural manner.
is feature provides valuable insights into the efficacy
and potential synergistic effects of various drug combina-
tions, aiding in identifying optimal treatment strategies
(Fig.15B).
Alternately, Maloney etal. [266] developed a new tech-
nique for generating high-throughput tumor organoids
using an immersion printing method that employs extru-
sion-based bioprinting. Tumor cells were mixed with
hyaluronic acid and collagen hydrogels and printed into
a viscous gelatin bath. e bath provided the necessary
structural support to form spheroids in 96-well plates.
e study demonstrated that this technique can fabri-
cate tumor organoids from glioblastoma and sarcoma
patient biopsies and tumor cell lines, making it a promis-
ing method for generating organoids for drug screening
purposes.
Organoids inimmunotherapy
Recent research has made significant advances in our
understanding of the role of immune cells in the tumor
TME using advanced tumor organoids and engineering
approaches. e TME can impede the immune system’s
ability to effectively eradicate tumor cells, but immuno-
therapy has successfully reversed this effect. However,
the variability in response among patients indicates that
a deeper understanding of the mechanical interactions
between immune and tumor cells is required to improve

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Desaietal. Biomaterials Research (2023) 27:113
Fig. 15 A Hepatocellular carcinoma organoid cocultures to understand the interplay between angiogenesis and the immune milieu. Here,
subfigure (i) shows confocal microscopy images of live cocultures comprising fluorescently labeled HCC cell line-derived spheroids (Huh7)
or PDX-derived organoids and ECs (HUVECs). Subfigure (ii) depicts the assessment of angiocrine signaling using the “Proteome Profiler Human
Angiogenesis” antibody array. Subfigure (iii) shows the upregulation in protein levels of angiocrine factors (MCP-1, IL-8, and CXCL16) in HCC cells
directly cocultured with ECs. Adapted with permission from [262] (Copyright Elsevier, 2022). B Automated microfluidic 3D cellular/organoid
culture platform for dynamic and combinatorial drug screening. Here, subfigure (i) shows a programmable membrane-valve-based microfluidic
chip that can provide automated stimulation profiles. Subfigure (ii) shows a 3D culture platform (which can be controlled by the microfluidic
chip) that can produce many parallel/dynamical culture experiments. Subfigure (iii) shows a cross-section of the two-layer multichambered 3D
culture chamber device (containing 200 individual chambers that are compatible with Matrigel). Subfigure (iv) depicts chemical inputs that can be
preprogrammed to provide combinatorial and time-varying stimulations to the 3D culture chamber device. Subfigures (v) and (vi) show organoids
or 3D cellular structures that can be continuously observed through time-lapse imaging and representative images of quantitative cellular assays,
respectively. Adapted with permission from [263] (Copyright Springer Nature, 2020)

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Desaietal. Biomaterials Research (2023) 27:113
response rates and develop novel therapeutics. Immu-
notherapy has revolutionized cancer treatment with two
key approaches: adoptive T-cell therapies and immune
checkpoint blockers (ICBs). ACT introduces activated T
cells to specifically target tumor cells, while ICBs boost
the immune system’s response by blocking inhibitory sig-
nals used by cancer cells to evade the immune system.
ese approaches have shown great promise in improv-
ing patient outcomes [267]. In a study by Michie etal.
[268], the efficacy of tumor-specific cytotoxicity of T
cells, specifically CAR T cells and TCR T cells, was evalu-
ated using patient-derived organoids (PDOs) as a plat-
form. is study investigated the impact of combining
CAR T cells with birinapant, an inhibitor of apoptosis, on
the growth of PDOs. e findings demonstrated that the
combination therapy significantly reduced the growth of
PDOs in a TNF-dependent manner, whereas CAR T cells
alone showed limited efficacy. ese results highlight the
potential of PDOs as a valuable tool for evaluating the
efficacy of combination therapies involving T cells and
other targeted agents. is approach could ultimately aid
in the development of more effective treatment strategies
for cancer patients.
In another relevant study, Schnalzger etal. [221] devel-
oped a preclinical model using 3D PDOs to assess the
cytotoxicity of chimeric antigen receptor (CAR) in a
native tumor immune microenvironment mimicking
model. ey also established a live-cell imaging protocol
to monitor cytotoxic activity at the single organoid level.
e study demonstrated a stable effector-target cell inter-
action in the coculture of natural killer cells with CRC
or normal organoids on an ECM layer. e authors also
used CRC organoids to evaluate the tumor antigen-spe-
cific cytotoxicity of CAR-engineered NK-92 cells target-
ing EGFRvIII or FRIZZLED receptors. is platform can
be used to assess CAR efficacy and tumor specificity in
a personalized manner. Epithelial-only PDOs, which do
not contain stromal or immune elements, can be used
to select T cells that react to tumors. is coculture
approach can be utilized to concentrate, activate, and
determine the effectiveness of tumor-reactive lympho-
cytes [269].
Neal et al. [270] developed ALI (air–liquid interface)
organoids by expanding and serially passaging physically
processed cancer fragments using WENR (WNT3A,
EGF, NOGGIN, and RSPO1) base medium. e authors
demonstrated that these ALI PDOs retained stromal and
immune cells and successfully reproduced the expansion,
activation, and tumor cytotoxicity of TILs responding to
PD-1/PD-L1 ICB, similar to the microfluidic approach.
Notably, CD8 + TIL expansion, activation, and tumor cell
killing were observed after just one week of anti-PD-1
treatment in ALI PDOs derived from various human
tumor biopsies, including RCC, NSCLC, and melanoma.
It is essential to consider the material and composition
of the organoid culture devices used in immunotherapy
studies, including those involving ICB. e results sug-
gest that ALI PDOs could be an effective platform for
assessing the efficacy of ICB treatments for different
types of cancer.
In a study conducted by Jenkins etal. [271], the authors
demonstrated the utility of exvivo systems incorporat-
ing features of the TME for investigating the response
to ICBs. ey employed murine-derived and patient-
derived organotypic tumor spheroids (MDOTS/PDOTS),
which retained autologous lymphoid and myeloid cell
populations and exhibited short-term responsiveness
to ICB in a three-dimensional microfluidic culture. e
study findings revealed that MDOTS derived from estab-
lished immunocompetent mouse tumor models effec-
tively recapitulated the response and resistance to ICB.
Furthermore, the authors identified that inhibition of
TBK1/IKKϵ could enhance the response to PD-1 block-
ade, demonstrating the predictive value of this ex vivo
model in assessing tumor response invivo. e authors
propose that profiling MDOTS/PDOTS represents an
innovative platform for evaluating ICB, utilizing estab-
lished murine models as well as clinically relevant patient
specimens. is approach holds the potential to facilitate
advancements in precision immuno-oncology and the
development of effective combination therapies.
Translational considerations
Translating tumor-derived systems from laboratory
research to clinical applications involves a range of com-
plex considerations vital for their successful implementa-
tion. With their unique properties and capabilities, these
systems offer exciting prospects for advancing healthcare
and transforming our approach to combating cancer. By
enhancing our understanding of the disease, enabling
precise and early diagnosis, and facilitating personalized
treatment strategies, tumor-derived systems have the
potential to revolutionize cancer care. However, achiev-
ing effective translation requires careful attention to sev-
eral key factors that must be considered (Fig.16).
Manufacturing consistency andquality control
Manufacturing consistency refers to the ability to repro-
duce these systems reliably and consistently across differ-
ent batches and production runs. It involves establishing
robust and standardized manufacturing processes that
yield products with consistent properties and perfor-
mance characteristics. Manufacturing consistency is
important because it ensures that the tumor-derived
systems used in preclinical studies accurately represent
those that will be tested in clinical trials and ultimately

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Desaietal. Biomaterials Research (2023) 27:113
used in patient care [272]. Achieving manufacturing
consistency requires careful optimization and control of
various production parameters, such as the sourcing of
cancer cells/tumors, selection of raw materials, fabrica-
tion/preparation methods, manufacturing equipment,
and system-specific process parameters [273]. By estab-
lishing well-defined manufacturing protocols and quality
control procedures, it can be ensured that the final prod-
ucts meet the desired specifications consistently [274].
Quality control is another crucial aspect of manufac-
turing tumor-derived systems for clinical translation.
It involves a set of processes and procedures designed
to assess the quality, purity, and safety of the prod-
ucts. Quality control measures are implemented at dif-
ferent stages of the manufacturing process, from raw
material selection to final product testing. It includes
various analytical techniques and tests to evaluate the
physicochemical properties, stability, and performance
of tumor-derived systems [275]. ese tests may involve
assessing particle size and distribution, surface charge,
drug loading and release characteristics, biocompatibility,
and stability under various storage conditions. e use
of validated analytical methods and strict adherence to
quality control standards ensures that the manufactured
tumor-derived systems consistently meet the required
specifications [276].
Implementing robust quality control measures helps
identify any manufacturing deviations or potential batch-
to-batch variations that may impact the safety and effi-
cacy of tumor-derived systems. It allows for the detection
of impurities, contaminants, or any other factors that
could compromise the quality of the final product. By
ensuring high-quality manufacturing processes and prod-
ucts, manufacturers can have confidence in the reliability
Fig. 16 Key considerations for clinical translation of tumor-derived systems. The initial steps involve optimizing the manufacturing process
and implementing stringent quality control protocols to ensure the reproducibility, scalability, and consistency of the systems. Prior to clinical
translation, rigorous evaluation of safety and toxicological profiles must be conducted in preclinical stages, addressing any potential risks associated
with their use. Subsequently, the efficacy of the tumor-derived systems needs to be demonstrated through well-designed clinical trials, providing
robust evidence of their therapeutic potential. Obtaining regulatory approval from relevant authorities is essential for ensuring compliance
with safety and efficacy standards. Additionally, addressing any intellectual property and patent-related concerns is critical to establishing
ownership, encouraging innovation, and supporting commercialization efforts

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and reproducibility of tumor-derived systems, which
is crucial for their successful translation into clinical
applications [277]. Manufacturing consistency and qual-
ity control are essential not only for meeting regulatory
requirements and obtaining approvals but also for build-
ing trust among clinicians, researchers, and patients.
Consistent and high-quality tumor-derived systems
instill confidence in the medical community and facilitate
their integration into routine clinical practice. Moreo-
ver, robust manufacturing processes and quality control
measures contribute to the scalability and commercial
viability of these systems, making them more accessible
and cost-effective for broader clinical use [278].
Safety andtoxicity considerations
Safety and toxicity considerations play a pivotal role in
the successful clinical translation of any medical inter-
vention, including tumor-derived systems. Ensuring the
safety of patients is of utmost importance, and a thor-
ough evaluation of the potential risks and adverse effects
associated with these systems is critical. In the context
of tumor-derived systems, safety considerations involve
assessing their potential toxicity and the impact they
may have on the overall health of patients. is involves
examining both short-term and long-term effects, as well
as evaluating the potential for systemic toxicity or dam-
age to vital organs or tissues [279]. Preclinical studies
are conducted to investigate the safety profile of tumor-
derived systems before they are introduced into clinical
trials. ese studies involve testing the systems in labo-
ratory models, such as animal models or in vitro cell
culture models, to assess their biocompatibility and any
potential harmful effects. Key parameters that are evalu-
ated include cytotoxicity, immunogenicity, genotoxicity,
and organ-specific toxicity [280].
Toxicity studies aim to identify any adverse effects
caused by tumor-derived systems. is involves assess-
ing the potential for inflammation, organ dysfunction, or
other systemic responses. Various techniques and meth-
odologies are employed to analyze the biophysical and
biochemical interactions of the systems within biological
systems, shedding light on their safety profile. Moreover,
the potential for off-target effects must be considered.
Tumor-derived systems, particularly those used for tar-
geted therapy, should have minimal impact on healthy
cells or tissues. Evaluating the selectivity of these sys-
tems and their ability to discriminate between cancerous
and noncancerous cells is crucial to minimize the risk of
unintended harm [281].
Safety considerations also extend to the manufactur-
ing and storage processes of tumor-derived systems.
Proper storage conditions and stability studies are con-
ducted to ensure the integrity and efficacy of the systems
throughout their shelf life. Addressing safety and toxic-
ity concerns in the early stages of development allows
researchers and clinicians to identify and mitigate poten-
tial risks. It helps refine the design and formulation
of tumor-derived systems to minimize adverse effects
and enhance patient safety. By comprehensively under-
standing the safety profile and toxicity considerations,
researchers can develop robust safety guidelines and
protocols for the clinical application of tumor-derived
systems, thus facilitating their successful translation into
clinical practice [282].
Clinical trials andecacy studies
Clinical trials are structured research studies conducted
on human subjects to assess the safety and efficacy of
new treatments, diagnostic methods, or medical devices.
In the context of tumor-derived systems, clinical trials
provide an opportunity to gather critical data on their
performance, tolerability, and clinical outcomes. ese
trials are typically conducted in multiple phases, starting
with small-scale studies in a limited number of patients
and progressing to larger trials involving diverse patient
populations [283]. Efficacy studies, on the other hand,
specifically focus on evaluating the effectiveness of a par-
ticular intervention or treatment approach. In the case
of tumor-derived systems, efficacy studies aim to deter-
mine the extent to which these systems can effectively
diagnose, treat, or monitor cancer. ese studies involve
rigorous data collection and analysis to measure specific
clinical endpoints, such as tumor response rates, pro-
gression-free survival, overall survival, or quality of life
improvements [284].
Both clinical trials and efficacy studies are essential for
establishing the safety and efficacy of tumor-derived sys-
tems in a real-world clinical setting. ey provide crucial
evidence that informs regulatory decisions, treatment
guidelines, and clinical practice. e data generated from
these studies not only support the approval and regula-
tory clearance of tumor-derived systems but also guide
healthcare professionals in making informed decisions
regarding their adoption and use [285, 286]. Moreover,
clinical trials and efficacy studies help identify the patient
populations that are most likely to benefit from tumor-
derived systems. ey contribute to the development
of personalized treatment strategies by elucidating the
predictive factors, biomarkers, or patient characteristics
associated with positive responses to these interventions.
is knowledge allows for the tailoring of treatment plans
and the identification of patients who are most likely to
derive significant clinical benefits [287].
Additionally, clinical trials and efficacy studies pro-
vide an opportunity to compare tumor-derived systems
against existing standards of care or alternative treatment

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approaches. Comparative studies can demonstrate the
superiority, noninferiority, or added value of tumor-
derived systems in terms of safety, efficacy, or patient
outcomes [288]. ese head-to-head comparisons are
crucial for making informed decisions about the clini-
cal adoption and integration of these systems into rou-
tine practice [289]. e results generated from clinical
trials and efficacy studies contribute to the overall body
of evidence supporting the translation of tumor-derived
systems into clinical practice. ey provide the scientific
basis for regulatory approvals, reimbursement decisions,
and healthcare providers’ adoption of these systems.
Additionally, these studies contribute to the ongoing
refinement and optimization of tumor-derived systems,
helping to improve their performance, safety profiles,
and patient outcomes. Table4 provides an overview of
selected clinical trials involving tumor-derived systems.
e clinical trial data presented in this table were dili-
gently sourced from ClinicalTrials.gov (https:// clini caltr
ials. gov/), a reputable and comprehensive clinical trials
registry. e unique NCT numbers associated with each
clinical trial have been included in the table to ensure the
traceability and accuracy of the referenced studies.
Regulatory challenges
Regulatory challenges encompass the complex regulatory
framework and requirements set by regulatory agencies
to ensure the safety, efficacy, and quality of medical prod-
ucts before they can be introduced into clinical practice.
One of the primary regulatory challenges is obtain-
ing clearance for the use of tumor-derived systems in
clinical settings. Regulatory agencies, such as the FDA
in the United States, require extensive preclinical and
clinical data to support the safety and effectiveness of
new medical technologies. is process involves rigor-
ous evaluation of the scientific evidence, including data
from invitro studies, animal models, and clinical trials
[290, 291]. Meeting these regulatory requirements can be
time-consuming, costly, and challenging, requiring sub-
stantial resources and expertise. Additionally, challenges
arise from the need to comply with various regulations
and guidelines specific to different regions or countries.
Different regulatory frameworks may exist in different
jurisdictions, each with its own specific requirements
and approval processes [292]. Companies and research-
ers must navigate these diverse regulatory landscapes to
ensure compliance and obtain the necessary approvals
to advance their tumor-derived systems toward clinical
translation.
Another significant regulatory challenge is the evolving
nature of regulatory guidelines for novel technologies. As
tumor-derived systems represent innovative approaches,
they may not fit neatly into existing regulatory
frameworks. is can lead to uncertainties and ambigui-
ties in determining the appropriate regulatory pathway
for these systems [293]. It is crucial to engage in proac-
tive communication with regulatory agencies to seek
guidance and clarification on the regulatory require-
ments specific to tumor-derived systems. Collaboration
between researchers, industry stakeholders, and regula-
tory authorities is essential to address these challenges
and establish clear regulatory pathways for the clinical
translation of tumor-derived systems [294]. Moreover,
ensuring postmarket surveillance and monitoring is
another important regulatory challenge. Once tumor-
derived systems are approved for clinical use, ongoing
monitoring of their safety and efficacy is necessary to
identify any potential adverse effects or long-term risks.
Postmarket surveillance involves collecting and analyz-
ing real-world data from patients and healthcare pro-
viders to assess the performance and safety profile of
these systems. Compliance with postmarket surveillance
requirements is crucial to maintain regulatory approval
and ensure the continued safe and effective use of tumor-
derived systems in clinical practice [295].
Intellectual property andpatents
Intellectual property (IP) and patents play a significant
role in the clinical translation of innovative technologies,
including tumor-derived systems. ese factors are cru-
cial considerations due to their potential impact on the
successful commercialization and adoption of these sys-
tems in clinical practice. One of the primary concerns
related to IP is the need for researchers and developers
to protect their novel inventions and discoveries. Obtain-
ing patents provides legal protection and exclusive rights
over intellectual property, preventing others from using,
manufacturing, or selling the same technology without
permission [296]. By securing patents for tumor-derived
systems, researchers and companies can establish owner-
ship and control over their innovations, which is essential
for attracting investment, establishing partnerships, and
commercializing the technology.
e presence of intellectual property protection
fosters a competitive environment by incentivizing
innovation and research and development activities.
Companies and investors are more willing to invest
resources and capital into translating tumor-derived
systems into clinical applications when they have a
strong IP portfolio [297]. e existence of patents can
provide a competitive advantage, enabling the devel-
opment of market exclusivity and generating rev-
enue through licensing agreements or product sales.
In the context of clinical translation, IPs and patents
also contribute to technology transfer and collabora-
tion between academia and industry. Researchers and

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Table 4 Selected ongoing/completed clinical trials involving tumor-derived systems (Source: https:// clini caltr ials. gov/)
System NCT Number Status Overview of Study
EVs NCT05270174 Not yet recruiting Evaluation and validation of exosomal long noncoding RNA ELNAT1 as an independent
predictor of lymph node metastasis in bladder cancer
NCT04556916 Recruiting Investigation of circulating blood-based biomarkers (including tumor-derived exosomes)
for early detection of prostate cancer
NCT04394572 Completed Investigation of protein markers transported by tumor exosomes for noninvasive colorectal
cancer diagnostic
NCT02507583 Completed The study explored immunotherapy of malignant glioma using exosomes derived
from patient-derived cancer cells after treatment with an investigational antisense molecule
NCT05286684 Recruiting Investigation of cerebrospinal fluid microvesicles using a high-throughput clinical proteomic
approach for improved profiling of metastatic tumor meningitis
NCT01344109 Withdrawn The study assessed the use of tumor-derived exosomes as a marker for response to therapy
in women receiving neoadjuvant chemotherapy for newly diagnosed breast cancer
NCT05397548 Recruiting Investigation of circulating exosomal long noncoding RNA GC1 as a potential biomarker
for the detection of gastric cancer
NCT05463107 Not yet recruiting Investigation of a potential correlation between exosomal protein biomarkers and pathologi-
cal manifestation in thyroid follicular neoplasm
NCT03334708 Recruiting Development of a minimally invasive test for early diagnosis and treatment response
monitoring in pancreatic cancer using blood-based biomarkers (including tumor-associated
exosomes)
NCT03236675 Active, not recruiting Investigated the potential of detecting patient-specific gene rearrangement/mutation
from circulating exosomes in patients of non-small cell lung cancer
TCL NCT01635283 Completed Evaluation of safety and efficacy on survival of patients with low-grade glioma, treated
with autologous DCs pulsed with autologous TCL
NCT02215837 Active, not recruiting Evaluation of safety and efficacy of chemotherapy combined with autologous TCL-pulsed DCs
for gastric cancer
NCT00405327 Completed Study of TCL as a vaccine for high-risk solid tumor patients following stem cell transplantation
NCT03114631 Completed Evaluation of safety and efficacy of novel peptide combined with TCL-pulsed DCs as immuno-
therapy in pancreatic cancer
NCT01204684 Active, not recruiting Evaluation of safety and efficacy of immunotherapy using autologous tumor lysate-pulsed
DCs in patients with an intracranial brain tumor
NCT02678741 Completed Safety and tumor response assessment of autologous TCL, yeast cell wall particles, and DCs
vaccine with checkpoint inhibitors in stage IV melanoma
NCT01678352 Completed Evaluation of a novel vaccination regime comprised of TCL and imiquimod (an FDA-approved
immune response modifier) in glioma patients
NCT03360708 Active, not recruiting Evaluation of safety and feasibility of malignant glioma TCL-pulsed autologous DCs vaccine
in glioblastoma patients at first or second recurrence
NCT03395587 Recruiting Evaluating the efficacy of integrating vaccination with TCL-loaded mature DCs into standard
radio/chemotherapy for newly diagnosed glioblastoma patients

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academic institutions may collaborate with industry
partners to further develop and commercialize tumor-
derived systems. IP rights and licensing agreements
facilitate the transfer of technology from academic
settings to the private sector, enabling the necessary
resources, expertise, and infrastructure for clinical
translation [298].
Moreover, IP considerations also influence regula-
tory pathways and market entry strategies. Patents can
impact the ability of other companies or organizations to
develop similar technologies, creating barriers to entry
for potential competitors. is exclusivity can offer a
certain level of market protection and enable the patent
holder to gain a foothold in the market [299]. However,
it is crucial to balance IP protection with considerations
of accessibility and affordability to ensure that innovative
technologies, such as tumor-derived systems, are acces-
sible to patients in need. Additionally, IP and patents can
influence pricing and reimbursement considerations.
e existence of patent protection can allow companies
to establish pricing strategies that recoup investment
costs and drive profit. However, it is important to strike
a balance between recouping investment and ensuring
affordability for patients and healthcare systems [300].
e cost-effectiveness and potential health benefits of
tumor-derived systems must be carefully evaluated to
determine their value proposition in relation to existing
treatment options.
Conclusion andfuture outlook
e growing body of scientific literature on tumor-
derived systems underscores their pivotal role in can-
cer research and therapy. is suggests that this field
will remain a focal point of research in the years ahead.
Tumor-derived systems hold immense promise due to
their inherent biological relevance, making them valuable
tools for unraveling cancer biology and forging new ther-
apeutic avenues. In this comprehensive review, we aim
to explore tumor-derived systems in depth, tracing their
scientific roots as potential biomedical instruments for
cutting-edge applications. By delving into these systems’
technical intricacies and scientific rationale, we aim to
inspire the research community to propel these platforms
to the forefront of our battle against cancer.
Among the array of systems discussed earlier, cancer
cell-derived EVs have already made substantial strides
in clinical diagnostics. EVs serve as a noninvasive source
of cancer-specific biomarkers, enabling early detec-
tion, diagnosis, and disease progression monitoring.
Analyzing EVs allows for the characterization of tumor
Table 4 (continued)
System NCT Number Status Overview of Study
Tumor Organoids NCT05842187 Recruiting Evaluation of the consistency between in vitro tumor organoid drug sensitivity and the thera-
peutic efficacy of in vivo drug treatment in patients with metastatic pancreatic or gastric
cancer
NCT04777604 Not yet recruiting Evaluation of patient-derived organoids as predictive platforms to select appropriate neoad-
juvant chemotherapy before surgery, based on unique genomic mutations
NCT05203549 Recruiting Assessment of the consistency between treatment responses in patient-derived organoids
and actual clinical outcomes in 250 gastric cancer patients
NCT05304741 Recruiting The study aims to establish organoid-based platforms that represent different types
(advanced/recurrent/metastatic) of colorectal cancer patients and apply them to drug screen-
ing
NCT05007379 Not yet recruiting Development and evaluation of patient-derived breast cancer organoids to test the antitu-
mor activity of novel chimeric antigen receptor-macrophages
NCT04342286 Completed Development of a reproducible organoid culture model using human kidney cells and their
evaluation for developing personalized/targeted therapy
NCT05669586 Recruiting Evaluation of the consistency and accuracy of patient-derived lung cancer organoids,
to select personalized treatment regiments for patients with resistance to multiline standard
therapies
NCT04865315 Active, not recruiting Development of patient-derived organoids of high-grade and low-grade gliomas and their
utilization in identifying underlying mechanisms that contribute to malignancy and treat-
ment resistance
NCT04826913 Not yet recruiting Evaluation of a high throughput device based on 3D nanomatrices and 3D tumors with func-
tional vascularization for personalized drug screening
NCT05196334 Recruiting Investigation of pancreatic ductal adenocarcinoma organoids cocultured with CAFs for phar-
macotyping using relevant chemotherapeutic agents used in the clinic

Page 43 of 52
Desaietal. Biomaterials Research (2023) 27:113
heterogeneity, with different subsets of cancer EVs poten-
tially bearing unique molecular signatures reflective of
distinct tumor subpopulations. is valuable information
can guide personalized treatment strategies and monitor
treatment responses. Initially, EV-based cancer diagnosis
faced challenges, including EV destruction due to factors
such as unsuitable temperatures, tensile forces, chemi-
cals, and prolonged storage durations [301]. Recent tech-
nological advancements have allowed the analysis of EVs
using rapid yet robust techniques. Notably, commercially
available and FDA-approved EV-based liquid biopsy kits,
such as the ExoDx™ Lung Test (blood-based) and the
ExoDx™ Prostate Test (urine-based), employ proprietary
EV separation devices in conjunction with quantitative
polymerase chain reaction techniques for swift diagnosis
of lung and prostate cancer, respectively [302, 303]. Addi-
tionally, Exosome Diagnostics, USA, has introduced the
"MedOncAlyzer 170," a pancancer liquid biopsy system
that simultaneously analyzes exosomal RNA and circu-
lating tumor DNA in a single assay to uncover function-
ally significant mutations across multiple cancer types
[304]. While cost remains a current hurdle, several other
EV-based cancer diagnosis systems, such as ExoView™
by NanoView Biosciences and ExoSearchTM by Nor-
gen Biotek Corp., are under development and poised to
reduce costs as they gain widespread usage.
While diagnostic applications of EVs are being rapidly
utilized, the translation of these approaches for drug
delivery has not progressed as swiftly. A similar trend
is evident for cancer membrane-coated nanoparticles,
which have only been applied in preclinical models for
therapeutic purposes despite being conceptualized a dec-
ade ago. A potential reason for this lag is the absence of
regulatory guidelines for the in vivo utilization of such
systems. Given their tumor-derived origin, attributes
such as pharmacokinetic profiles, long-term therapeutic
safety, and toxicology require extensive study and stand-
ardization [305]. Moreover, scaling up manufacturing
processes poses a significant challenge for these systems.
Cancer cells are typically cultured in laboratory settings
using the conventional two-dimensional flask culture
method. e secreted EVs are isolated after a specific
incubation period in EV-enriched media. At the same
time, the CCM is collected using suitable downstream
processing techniques once the cell culture flask reaches
confluency.
For efficient and reproducible commercial produc-
tion, it is vital to implement scalable large-scale pro-
duction techniques that overcome the limitations of
conventional methods. In this context, bioreactors pro-
vide controlled production environments and scalability,
with the choice of bioreactor type (such as hollow fiber,
membrane, or microcarrier bioreactor) and optimization
of process parameters (nutrient composition, pH, tem-
perature, dissolved oxygen levels, and agitation speed)
crucial for achieving optimal productivity [306]. Optimal
productivity can be achieved by selecting the appropri-
ate bioreactor type (hollow fiber, membrane, or micro-
carrier bioreactor) depending on the desired output and
optimizing process parameters (such as nutrient com-
position, pH, temperature, dissolved oxygen levels, and
agitation speed). Scaling up production can also lead
to reduced operating costs, lowering the final product’s
overall cost [307]. Both bioengineered EVs and CCM-
coated nanoparticles offer unique applications, particu-
larly those that excel in homotypic targeting, enabling
them to reach challenging sites such as the bone mar-
row and cross the BBB [308]. By selecting the appropri-
ate combination of nanoparticle core and delivery cargo,
CCM-coated nanoparticles have been at the focal point
of interesting cancer theranostic applications. With the
resolution of these challenges, both tumor-derived deliv-
ery systems are poised to transition toward clinical thera-
peutic applications.
From an immunotherapeutic and cancer vaccine per-
spective, TCL has emerged as a promising tool. Research-
ers have demonstrated significant interest in this area,
with numerous preclinical studies reporting promising
results [309]. e TCL serves as a rich source of TAAs,
stimulating an immune response without requiring
specific antigen targeting or synthesis. is approach
effectively prevents tumor evasion from immune surveil-
lance and can be enhanced by incorporating TCL into
delivery systems alongside other immunostimulatory
biomolecules, generating durable immune memory to
inhibit tumor relapse and metastasis. While autologous
tumor cells theoretically provide an ideal source for cell
lysate-based vaccines due to their unique array of tumor
antigens, challenges related to limited availability and
difficulty in generating large quantities of autologous
tumor cells hinder widespread clinical use. In such cases,
enhancing the antigenicity of allogeneic tumor cell-
derived lysate remains an area for improvement [310].
Finally, engineered cell lines and tumor organoids have
made significant contributions to our understanding of
cancer development and drug discovery, complement-
ing insights from traditional two-dimensional cell lines.
Tumor organoids, in particular, offer distinct advantages
due to their intricate cell‒cell interactions, cell–matrix
interactions, and potential for cellular differentiation.
ese characteristics have allowed us to overcome the
limitations of conventional cell lines and gain deeper
insights into the complexity of cancer biology [311]. By
harnessing the power of engineered cell lines and tumor
organoids, we have expanded our understanding of can-
cer and accelerated the search for effective therapies.

Page 44 of 52
Desaietal. Biomaterials Research (2023) 27:113
Tumor organoids, with their diverse cell subtypes and
treatment responses more closely resembling invivo con-
ditions, present a promising alternative to animal-based
drug testing. Increasing advocacy by global regulatory
agencies for alternatives to animal testing in drug devel-
opment is driven by ethical concerns and the recognition
that animal models may not always accurately predict
human responses. Bioengineered cell lines and tumor
organoids can effectively support this effort [312].
Moving forward, stakeholders in the cell line supply
industry can seize the emerging demand for organoid
models by focusing on user-friendly and readily avail-
able organoid platforms. By investing in the creation of
standardized organoid systems, these companies can
meet the needs of researchers seeking more sophisticated
and physiologically relevant invitro models. is strate-
gic move not only presents a lucrative business oppor-
tunity but also extends its advantages to laboratories in
resource-limited countries, where establishing organoid
systems from scratch can be challenging [313]. Notably,
Hubrecht Organoid Technology, a nonprofit organiza-
tion, is dedicated to developing a biobank of thousands
of cancer-based models that closely mirror the hallmarks
and diversity of human cancer. ey obtain and generate
organoids from patient tumor tissues, which are exten-
sively analyzed through genome sequencing and expres-
sion profiling. e organization has created a thoroughly
characterized collection of cultures along with accompa-
nying clinical data. is resource is valuable for advanc-
ing fundamental research, identifying potential leads, and
investigating innovative therapeutic approaches, offering
advantages to both the industrial and academic sectors
[314]. Standardized organoid assays developed through
these platforms can serve as the gold standard for cancer
research in the future.
Tumor-derived systems, although promising, are beset
with intricate challenges. Tumors inherently manifest
heterogeneity rooted in a multitude of cell types and
genetic mutations. is heterogeneity, coupled with
temporal biological variability within tumors, gives rise
to formidable obstacles. ese complexities markedly
impact the precision of research outcomes in the realms
of disease modeling, drug testing, and therapeutic appli-
cations. Addressing these issues necessitates rigorous
standardization efforts and the development of innova-
tive methodologies, often guided by advanced analytical
techniques, to offset the influence of these inherent vari-
ations. is diligence is crucial to uphold the reliability of
tumor-derived systems, ensuring their suitability for both
research and clinical utility. Furthermore, the production
and analysis of tumor-derived systems can be financially
burdensome, curtailing their accessibility, especially
within resource-constrained healthcare settings. To
enhance affordability, there is an imperative need for the
development of cost-effective production techniques.
Collaborative endeavors, including public‒private part-
nerships and targeted funding initiatives, are instrumen-
tal in the pursuit of cost reduction.
While tumor-derived systems offer the potential for
personalized medicine, operationalizing patient-spe-
cific treatments presents logistical complexities. Timely
acquisition and processing of individual tumor samples
pose practical challenges. Streamlining these procedures
necessitates the integration of automation and the adop-
tion of personalized medicine approaches to expedite the
generation of patient-specific tumor-derived systems.
Last, the complexity inherent in the data generated by
tumor-derived systems, encompassing diverse omics data
types (genomics, proteomics, etc.), poses substantial hur-
dles in data interpretation and analysis. Handling these
intricate datasets is resource intensive and demands
advanced computational methodologies. Notably, the
fields of bioinformatics and machine learning hold the
potential to significantly facilitate the analysis and inter-
pretation of the multifaceted data outputs originating
from tumor-derived systems, thereby enhancing their
utility in cancer research and therapy.
In conclusion, tumor-derived systems, encompassing
a spectrum of innovative approaches, stand as powerful
allies in our ongoing battle against cancer. As we navigate
the intricacies of these systems, it is evident that they
offer invaluable insights into cancer biology and treat-
ment strategies. e landscape of tumor-derived systems
is poised for further exploration and development as
potent biomedical tools in the fight against cancer. eir
versatility, ranging from diagnostic applications such as
EV-based liquid biopsies to therapeutic potentials such
as membrane-coated nanoparticles, holds great promise.
Overcoming challenges related to standardization, cost,
patient-specific treatments, and data complexity is essen-
tial to harness the full potential of these systems. As we
venture forward, it is imperative that stakeholders in the
field, including researchers, clinicians, and industry part-
ners, collaborate to address these challenges. Continued
advancements in the understanding and utilization of
tumor-derived systems will undoubtedly shape the future
of cancer management, offering hope for improved
patient outcomes and innovative approaches to cancer
diagnosis and therapy.
Abbreviations
APCs Antigen-presenting cells
BBB Blood‒brain barrier
BMDCs Bone marrow-derived dendritic cells
BTZ Bortezomib
CAFS Cancer-associated fibroblasts
CCAMS Cancer cell adhesion molecules

Page 45 of 52
Desaietal. Biomaterials Research (2023) 27:113
CCM Cancer cell membrane
CLSM Confocal laser scanning microscopy
CRC Colorectal cancer
CTLs Cytotoxic T lymphocytes
DAMPs Damage-associated molecular patterns
DCs Dendritic cells
ECM Extracellular membrane
ECs Endothelial cells
EMT Epithelial-mesenchymal transition
EPR Enhanced Permeability and Retention
EVs Extracellular vesicles
FDA Food and Drug Administration
FITC Fluorescein isothiocyanate
gRNA Guide RNA
H2O2 Hydrogen peroxide
HIFs Hypoxia-inducible factors
ICBs Immune checkpoint blockers
ICD Immunogenic cell death
IP Intellectual property
Ig-SF Immunoglobulin superfamily
MDSCs Myeloid-derived suppressor cells
MM Multiple myeloma
MOF Metal–organic framework
MRI Magnetic resonance imaging
PC Polycarbonate
PDT Photodynamic therapy
PLGA Poly(lactic-co-glycolic acid)
PTX Paclitaxel
ROS Reactive oxygen species
TAAs Tumor-associated antigens
TAMs Tumor-associated macrophages
TCL Tumor cell lysate
TCVs Therapeutic cancer vaccines
TEM Transmission electron microscopy
TGF-β Transforming growth factor-β
TLR Toll-like receptor
TME Tumor microenvironment
TSAs Tumor-specific antigens
VEGF Vascular endothelial growth factor
Acknowledgements
J.G. would like to acknowledge the financial support provided by the Depart-
ment of Science and Technology, Ministry of Science and Technology, Science
and Engineering Board, India, through project numbers CRG/2020/005244
and IMPRINT (DST/IMP/2018/000687), as well as the Abdul Kalam Technology
Innovation National Fellowship granted by the Indian National Academy of
Engineering, India (INAE/121/AKF/37). All original figures were created with
Biorender.com.
Authors’ contributions
ND: Conceptualization, Writing-original draft, Figures; PK: Writing-original draft;
VM: Writing-original draft; SS: Writing-original draft; LV: Reviewing and edit-
ing, Resources, Funding Acquisition, Project Administration; JG: Supervision,
Reviewing and editing, Resources, Funding Acquisition, Project Administra-
tion. All authors read and approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
Not applicable.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests
Author details
1 Department of Biomedical Engineering, Indian Institute of Technology
Hyderabad, Kandi, Telangana, India. 2 Center for Interdisciplinary Programs,
Indian Institute of Technology Hyderabad, Kandi, Telangana, India. 3 Depart-
ment of Pharmaceutics, National Institute of Pharmaceutical Education
and Research-Ahmedabad (NIPER-A), Gujarat, India. 4 School of Pharmacy,
Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK.
Received: 21 June 2023 Accepted: 11 October 2023
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