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Investigating Metastasis Using In Vitro Platforms
Abstract
T he complexity of the metastatic cascade has led to the development of a plethora of 2‑ and 3‑dimensional in vitro assays to model the various steps of metastasis under a more controlled environment. These assays have been invaluable in cancer research not only as tools to delineate the molecular events that underpin metastasis but also to enable drug screens and validation of therapeutic targets. Here we review the advantages and limitations of current in vitro platforms used to investigate metastasis. In light of the overwhelming evidence showing that 3‑dimentional culture systems better mimic the tumor microenvironment and therapeutic response in vivo, recent advances made toward the development of 3‑dimensional culture systems and their applications are discussed in more detail. Relevant information on protocols and resources available to support scientists with an interest in metastasis research is also provided.
... The most commonly used model to investigate cancer extravasation is the transendothelial assay built on the Boyden chamber/Transwell platform (Fig. 1b) [24][25][26]. In this assay, endothelial cells are seeded on the inserted porous membrane (diameter~3-12 μm) and cultured in the upper chamber, and condition medium containing specific chemokines or cytokines to attract cancer cells is placed in the lower chamber. ...
... Similar to the cancer-endothelium adhesion and invasion assay, after the EC form a confluent monolayer, cancer cells are seeded on the top of the monolayer. The transmigrated cancer cells in the lower chamber within the following 48 h will be collected and quantified for further analysis [24][25][26][27]. More recently, a 3D extracellular matrix (ECM) such as Matrigel is often incorporated beneath the endothelial monolayer in this assay, which offers more physiological relevance by recapitulating the invasion of cancer cells into ECM following the extravasation [28]. ...
... Unlike the in vivo models in which the local biochemical environment is often hard to modulate, the high adjustability of this transendothelial migration assay allows us to determine the roles of specific cell types [32][33][34] and non-cell factors [35,36] under various biochemical and genetic settings in the cancer transendothelial migration. Moreover, as this assay can be easily scaled up, it is often preferred when conducting high-throughput drug screening for metastasis inhibitors [24]. ...
Metastasis is a multistep process that accounts for the majority of cancer-related death. By the end of metastasize dissemination, circulating tumor cells (CTC) need to extravasate the blood vessels at metastatic sites to form new colonization. Although cancer cell extravasation is a crucial step in cancer metastasis, it has not been successfully targeted by current anti-metastasis strategies due to the lack of a thorough understanding of the molecular mechanisms that regulate this process. This review focuses on recent progress in cancer extravasation visualization techniques, including the development of both in vitro and in vivo cancer extravasation models, that shed light on the underlying mechanisms. Specifically, multiple cancer extravasation stages, such as the adhesion to the endothelium and transendothelial migration, are successfully probed using these technologies. Moreover, the roles of different cell adhesive molecules, chemokines, and growth factors, as well as the mechanical factors in these stages are well illustrated. Deeper understandings of cancer extravasation mechanisms offer us new opportunities to escalate the discovery of anti-extravasation drugs and therapies and improve the prognosis of cancer patients.
... Common methods for assessing ovarian cancer cell proliferation/migration/invasion have included 2D culture growth studies, "scratch" wound healing assays, and penetration through transwell inserts. Scratch wound assays are relatively easy to set up, and very cheap to run and there are now many options for tracking and quantitating cell growth and migration, including the MetaMorph™ and Incucyte™ real-time Imaging systems (31). Migration assays through transwell inserts are more expensive and do not allow for real-time monitoring. ...
... Migration assays through transwell inserts are more expensive and do not allow for real-time monitoring. Microfluidic assays have the advantage that cells can be grown in controlled chemotactic gradients (31). However, these systems have not to date been utilized widely for ovarian cancer cell culture studies. ...
... For example, only a partial understanding of the involvement of proteases/MMPs in the spread and invasion of ovarian cancer cells can be drawn from experiments using matrices that lack structural properties of a relevant ECM. For example, matrigel is substantially less cross-linked and differs in overall composition compared to many tissues (29,31,33). ...
High grade serous epithelial ovarian cancer (HG-SOC) is one of the most devastating gynecological cancers affecting women worldwide, with a poor survival rate despite clinical treatment advances. HG-SOC commonly metastasizes within the peritoneal cavity, primarily to the mesothelial cells of the omentum, which regulate an extracellular matrix rich in collagens type I, III, and IV along with laminin, vitronectin, and fibronectin. Cancer cells depend on their ability to penetrate and invade secondary tissue sites to spread, however a detailed understanding of the molecular mechanisms underlying these processes remain largely unknown. Given the high metastatic potential of HG-SOC and the associated poor clinical outcome, it is extremely important to identify the pathways and the components of which that are responsible for the progression of this disease. In vitro methods of recapitulating human disease processes are the critical first step in such investigations. In this context, establishment of an in vitro "tumor-like" micro-environment, such as 3D culture, to study early disease and metastasis of human HG-SOC is an important and highly insightful method. In recent years, many such methods have been established to investigate the adhesion and invasion of human ovarian cancer cell lines. The aim of this review is to summarize recent developments in ovarian cancer culture systems and their use to investigate clinically relevant findings concerning the key players in driving human HG-SOC.
... This wound healing assay serves as a preliminary screening assay. It is also a cost-friendly and reliable method [20,21]. A wound is introduced in the middle of the monolayer cells and the ability of the cells to migrate towards the wound was assessed [20,21]. ...
... It is also a cost-friendly and reliable method [20,21]. A wound is introduced in the middle of the monolayer cells and the ability of the cells to migrate towards the wound was assessed [20,21]. According to Figure 4B, after 24 h, cells filled up the wounded area completely in the control sample. ...
... Another assay was attempted to further screen the anti-migration effects of BHAQ. This assay was designed to observe whether or not the cells could migrate to the other end of a membrane when induced with a chemoattractant [21]. The chart in Figure 5 represents the percentage of MDA-MB231 cells migrating through a Boyden chamber/membrane with a chemoattractant at the bottom of the chamber [22]. ...
Breast cancer is becoming more prominent in women today. As of now, there are no effective treatments in treating metastatic breast cancer. We have tested the cytotoxic and anti-migration effects of BHAQ, a synthesized anthraquinone, on two breast cancer cell lines, MCF-7 and MDA-MB231. Anthraquinones are an interesting class of molecules that display a wide spectrum of biological applications, including anticancer properties. Cellular inhibition was tested through a MTT assay, double acridine orange/propidium iodide staining and FACS cell cycle analysis. Inhibition of migration was tested by the wound healing method, and migration through a Boyden chamber. BHAQ was cytotoxic towards both cell lines in a dose dependent and possibly cell-dependent manner. Additionally, BHAQ also inhibited the migration of the highly metastatic MDA-MB231 cell line.
... In three independent experiments, each compound was tested in triplicate for each cell line. 56 The IC 50 of the most active compounds was calculated by nonlinear regression analysis using GraphPad Prism 9.3.1 software. ...
... The final compounds (19−34) and (35−38) were tested for their anticancer activity against MCF-7 and A549 cancer cell lines using an MTT assay. 56 Gedatolisib, a potent PI3Kα triazine inhibitor, and doxorubicin, one of the most effective and commonly used chemotherapeutic anticancer drugs, were used as positive standards for the MTT assay study. As shown in Table 2 and Figures 3 and 4, the most active compounds against MCF-7 and A549 cancer cell lines were compounds 27, 28, 29, 31, and 35. ...
The biological benefits of trisubstituted 1,3,5-triazine derivatives include their ability to reduce inflammation and fight cancer. A unique series of sulfonamide− triazine hybrid molecules were produced chemically by synthesizing triazine derivatives utilizing the usual nucleophilic aromatic substitution of cyanuric chloride via the solvent-free/neat fusion method. Fourier-transform infrared spectroscopy (FTIR), 1 H NMR, and 13 C NMR spectroscopic analyses were used to identify novel trisubstituted synthetic compounds. The synthesized compounds have a moderate inhibition percentage when tested at 100 μM against the phosphoinositol 3-kinases (PI3Kα) enzyme; compounds 20 and 34 showed 46 and 68% anti-PI3Kα activity, respectively. To comprehend the anticipated interactions, the most successful compounds were subsequently docked into a PI3Kα protein's binding site (PDB code: 6OAC, resolution: 3.15 Å). The final synthetic compounds' anticancer activity was tested on the breast (MCF-7) and lung (A549) cancer cell lines at doses of 100 and 50 μM for additional evaluation of anticancer characteristics. The IC 50 values for the sulfaguanidine−triazine derivatives 27, 28, 29, 31, and 35 ranged from 14.8 to 33.2 μM, showing that compounds containing sulfaguanidine and diethylamine in their structures significantly inhibited the activity. Compound 34 could be a promising lead compound for developing new target-selected anticancer compounds with low toxicity and high selectivity.
... Due to the clinical relevance of bone metastases in cancer, the ability to study this process in vitro is of high importance. However, due to the extensive complexity of the molecular pathways involved in the metastatic cascade, in addition to difficulties in recapitulating patient physiology, models are often limited and lack translational efficacy (92). ...
... For example, increasing evidence has indicated that the stromal microenvironment plays an integral role in the metastatic cascade (96,97). Therefore, 2-dimensional (2D) models which aim to allow the investigation of tumour cell interactions with the surrounding ECM have been generated, including scratch or wound healing assays and transwell cell migration assays (92). Although these assays provide improved translational efficacy in comparison to human cell line-based models, they fail to fully recapitulate the architectural and cellular complexity of metastatic tumour formation in vivo. ...
Prostate cancer (PCa) is one of the most prominent causes of cancer-related mortality in the male population. A highly impactful prognostic factor for patients diagnosed with PCa is the presence or absence of bone metastases. The formation of secondary tumours at the bone is the most commonly observed site for the establishment of PCa metastases and is associated with reduced survival of patients in addition to a cohort of life-debilitating symptoms, including mobility issues and chronic pain. Despite the prevalence of this disease presentation and the high medical relevance of bone metastases, the mechanisms underlying the formation of metastases to the bone and the understanding of what drives the osteotropism exhibited by prostate tumours remain to be fully elucidated. This lack of in-depth understanding manifests in limited effective treatment options for patients with advanced metastatic PCa and culminates in the low rate of survival observed for this sub-set of patients. The present review aims to summarise the most recent promising advances in the understanding of how and why prostate tumours metastasise to the bone, with the ultimate aim of highlighting novel treatment and prognostic targets, which may provide the opportunity to improve the diagnosis and treatment of patients with PCa with bone metastases.
... To date, a variety of in vitro platforms has been established to recapitulate cancer metastasis [40]. Given the simplicity and cost advantage of in vitro models over ex vivo and animal models, such models are preferred option especially in highthroughput drug screening [40]. ...
... To date, a variety of in vitro platforms has been established to recapitulate cancer metastasis [40]. Given the simplicity and cost advantage of in vitro models over ex vivo and animal models, such models are preferred option especially in highthroughput drug screening [40]. In this part, we aim to outline the strengths and weaknesses of current in vitro models which have been used to investigate cancer metastasis. ...
Purpose of review
Modelomics approach allows modelling and understanding of the mechanisms regulating living complex systems using computational and experimental models. Here, our aim was to highlight recent cancer models with the focus on bioengineering tool box to study bone metastasis cascade.
Recent findings
Bone is a common site for cancer metastasis. In the last few decades, various in vitro and in vivo models have been used to provide insights into the treatment of human cancer bone metastasis. Despite significant advances into the understanding of molecular and cellular mechanisms of cancer bone metastasis using traditional models, recent studies reported the functional failure of the new drugs when tested in humans and demonstrated the inability of current bone metastasis models to mimic corresponding human disease. The failure of cancer treatments highlights the importance of reviewing the efficiency and efficacy of current modelomics to accurately investigate bone metastasis.
Summary
Development of innovative cancer bone metastasis models is an essential step to develop new therapies or investigate the feasibility of current treatments. The emergence of bone on chip, tissue engineering of humanized mouse models has the potential to overcome the limitation of current platforms by better recapitulation of the cancer bone metastasis.
... Animal models are commonly used for studies on cancer biology and genetics as well as the preclinical investigation of cancer-therapy and the efficacy and safety of novel drugs [34]. Animal models represent the in vivo counterpart to cell-lines and suspension culture, while being superior in terms of physiological relevance offering imitation of parental tumors Murine models [37] Genetically engineered mouse model [36] Zebra fish model [42] Drosophila model [41] Chick embryo model [43,44] In vitro General 2D/3D in vitro models [46,53] Transwell model [48] Spheroid system [49] Microfluidic system [50] Tissue-engineered microvessel model [51] In silico Sequence analysis [63,69,74] General pathway analysis and network inference [132,133,135] Pan-cancer [62,82,134,139] Chemical perturbation mapping [64,66,68] Pharmacogenomic mapping [99,102,117,136] Genome-phenotype mapping [81] Clinical data integration [106] Structure mapping [102,103] Structure and activity [100,101] Framework for key events and mode of action [97,98] Image classification [85,87] Growth prediction [91][92][93] and a heterogeneous microenvironment as part of an interacting complex biochemical system. In general, animal models primarily based on murine or rodent models can be subdivided into the following groups of (I) xenograft models, which refer to the heterotopic, subcutaneous intraperitoneal or orthotopic implantation into SCID (Severe Combined Immune Deficiency) or nude mice, (II) syngenic models involving the implantation of cells from the same strain into non-immunocompromised mice, and (III) genetically engineered models, which allow for RNA interference, multigenic mutation, inducible or reversible gene expression [35,36]. ...
... Generally, such cell culture models focus on key aspects of metabolism, absorption, distribution, excretion of chemicals or other aspects of cell signaling pathways, such as aspects of metastasis under a controlled environment [53]. Scale-up systems attempt to emulate the physiological variability in order to extrapolate from in vitro to in vivo [54]. ...
Background:
Improving our understanding of cancer and other complex diseases requires integrating diverse data sets and algorithms. Intertwining in vivo and in vitro data and in silico models are paramount to overcome intrinsic difficulties given by data complexity. Importantly, this approach also helps to uncover underlying molecular mechanisms. Over the years, research has introduced multiple biochemical and computational methods to study the disease, many of which require animal experiments. However, modeling systems and the comparison of cellular processes in both eukaryotes and prokaryotes help to understand specific aspects of uncontrolled cell growth, eventually leading to improved planning of future experiments. According to the principles for humane techniques milestones in alternative animal testing involve in vitro methods such as cell-based models and microfluidic chips, as well as clinical tests of microdosing and imaging. Up-to-date, the range of alternative methods has expanded towards computational approaches, based on the use of information from past in vitro and in vivo experiments. In fact, in silico techniques are often underrated but can be vital to understanding fundamental processes in cancer. They can rival accuracy of biological assays, and they can provide essential focus and direction to reduce experimental cost.
Main body:
We give an overview on in vivo, in vitro and in silico methods used in cancer research. Common models as cell-lines, xenografts, or genetically modified rodents reflect relevant pathological processes to a different degree, but can not replicate the full spectrum of human disease. There is an increasing importance of computational biology, advancing from the task of assisting biological analysis with network biology approaches as the basis for understanding a cell's functional organization up to model building for predictive systems.
Conclusion:
Underlining and extending the in silico approach with respect to the 3Rs for replacement, reduction and refinement will lead cancer research towards efficient and effective precision medicine. Therefore, we suggest refined translational models and testing methods based on integrative analyses and the incorporation of computational biology within cancer research.
... It is beyond the scope of this study to compare our method with other assays for adhesion and migration of cells, proteomics and genomics for biomarkers, isolation and detection of circulating tumor cells (see [20], and references therein). Most of these methods, used for drug sensitivity testing and for predicting metastasis, remain in the early phases of development and lack standardization [20,21]. ...
... It is beyond the scope of this study to compare our method with other assays for adhesion and migration of cells, proteomics and genomics for biomarkers, isolation and detection of circulating tumor cells (see [20], and references therein). Most of these methods, used for drug sensitivity testing and for predicting metastasis, remain in the early phases of development and lack standardization [20,21]. However, in comparison to common histopathological diagnosis, which is inherently subjective, and expression of differentiation markers, ours is a more objective, quantitative, rapid, and label-free functional technique that involves simple optical instrumentation. ...
We report an optical trapping method that may enable assessment of the differentiation status of cancerous cells by determining the minimum time required for cell-cell adhesion to occur. A single, live cell is trapped and brought into close proximity of another; the minimum contact time required for cell-cell adhesion to occur is measured using transformed cells from neural tumor cell lines: the human neuroblastoma SK-N-SH and rat C6 glioma cells. Earlier work on live adult rat hippocampal neural progenitors/stem cells had shown that a contact minimum of ~5 s was required for cells to adhere to each other. We now find the average minimum time for adhesion of cells from both tumor cell lines to substantially increase to ~20-25 s, in some cases up to 45 s. Upon in vitro differentiation of these cells with all-trans retinoic acid the average minimum time reverts to ~5-7 s. This proof-of-concept study indicates that optical trapping may be a quick, sensitive, and specific method for determining differentiation status and, thereby, the prognosis of cancer cells.
... [9][10][11][12][13][14][15] A fundamental limitation in the development of new therapies to prevent metastatic cancer is a lack of in vitro systems that can accurately recapitulate the steps of cancer cell metastasis. 16 During adhesion of CTCs under flow conditions, the biophysical forces of the circulation can dramatically alter the biochemical interactions of adhesion receptors with their ligands. 10,17,18 Currently, assays for examining the steps of metastasis are most commonly carried out in the absence of the flow of the circulatory system or using low throughput flow chambers. ...
... 10,17,18 Currently, assays for examining the steps of metastasis are most commonly carried out in the absence of the flow of the circulatory system or using low throughput flow chambers. 16 Many studies have suggested these assays to be poorly predictive of the in vivo metastatic response making them unsuitable for drug discovery or large-scale mechanistic studies. [19][20][21] Here, we present a device that enables the performance of high throughput in vitro screens for compounds that can inhibit cancer cell adhesion under physiological flow. ...
The metastatic spread of cancer is a major barrier to effective and curative therapies for cancer. During metastasis, tumor cells intravasate into the vascular system, survive in the shear forces and immunological environment of the circulation, and then extravasate into secondary tumor sites. Biophysical forces are potent regulators of cancer biology and are key in many of the steps of metastasis. In particular, the adhesion of circulating cells is highly dependent upon competing forces between cell adhesion receptors and the shear stresses due to fluid flow. Conventional in vitro assays for drug development and the mechanistic study of metastasis are often carried out in the absence of fluidic forces and, consequently, are poorly representative of the true biology of metastasis. Here, we present a novel high-throughput approach to studying cell adhesion under flow that uses a multi-well, mechanofluidic flow system to interrogate adhesion of cancer cell to endothelial cells, extracellular matrix and platelets under physiological shear stresses. We use this system to identify pathways and compounds that can potentially be used to inhibit cancer adhesion under flow by screening anti-inflammatory compounds, integrin inhibitors and a kinase inhibitor library. In particular, we identify several small molecule inhibitors of FLT-3 and AKT that are potent inhibitors of cancer cell adhesion to endothelial cells and platelets under flow. In addition, we found that many kinase inhibitors lead to increased adhesion of cancer cells in flow-based but not static assays. This finding suggests that even compounds that reduce cell proliferation might also enhance cancer cell adhesion during metastasis. Overall, our results validate a novel platform for investigating the mechanisms of cell adhesion under biophysical flow conditions and identify several potential inhibitors of cancer cell adhesion during metastasis.
... Traditional breast cancer bone metastasis models (in vitro and in vivo) contain inherent limitations with regards to controllability, reproducibility and flexibility of design. To date, many twodimensional (2-D) in vitro models and in vivo animal models have been used to investigate tumor metastasis [10][11][12]; however, a necessary balance between experimental efficiency and the biological complexity of human bone is difficult to recapitulate and control in these bone models effectively [8,13]. In fact, while they present easy and powerful methods for investigating cancer cell behavior in vitro, a 2-D cancer metastasis model oversimplifies the native 3-D microenvironment due to the lack of spatial cues which makes it difficult to recapitulate in vivo cancer progression [14]. ...
... These alterations influence the ability of cancer cells to spread, invade, survive and subsequently grow within metastatic sites. Therefore, initial cell adhesion testing is often the first assay performed when evaluating metastatic cancer cell behavior [11]. Fig. 4 shows BrCa cell adhesion on our designed HA/chitosan bone scaffolds. ...
Traditional breast cancer (BrCa) bone metastasis models contain many limitations with regards to controllability, reproducibility, and flexibility of design. In this study, a novel biomimetic bone microenvironment was created by integrating hydroxyapatite (HA) and native bioactive factors deposited by osteogenic induction of human bone marrow mesenchymal stem cells (MSCs) within a cytocompatible chitosan hydrogel. It was found that a 10% nanocrystalline HA (nHA) chitosan scaffold exhibited the highest BrCa adhesion and proliferation when compared to chitosan scaffolds with 20% nHA, 10% and 20% microcrystalline HA as well as amorphous HA. This 3D tunable bone scaffold can provide a biologically relevant environment, increase cell-cell and cell-matrix interactions as found in native bone, and retain the behavior of BrCa cells with different metastasis potential (i.e., highly metastatic MDA-MB-231, less metastatic MCF-7 and transfected MDA-MB-231). The co-culture of MSCs and MDA-MB-231 in this bone model illustrated that MSCs have the capacity to upregulate the expression of the well-known metastasis-associated gene metadherin within BrCa cells. In summary, this study illustrates the capacity of our 3D bone model in creating a biomimetic environment conducive to recapitulating the behavior of metastatic BrCa cells, making it a promising tool for in vitro BrCa cell bone metastasis study and potential therapeutics discovery.
Copyright © 2014. Published by Elsevier Ltd.
... Unfortunately, the 2D system leads to the loss of in vivo cell morphology, disrupting crucial cell-cell and cell-matrix interactions, thereby failing to accurately replicate the in vivo cancer microenvironment. [16,17] While in vivo mouse models offer a natural three-dimensional (3D) setting for studying metastasis. [18,19] However, they are costly, lack an immune system, and exhibit low efficiency in generating metastases. ...
... Similarly silencing ATF3 led to reduced levels of ATF3, CXCR3 and MMP2 proteins by 2.8, 3.0, and 2.4folds respectively, in MDA-MB231-shATF3 transduced cells compared to control MDA-MB231-shScr cells (Fig. 4F, upper & lower panels; P < 0.0001). Moreover, both the colony-forming ability and the ability of MDA-MB231 cells to invade, the properties most closely associated with metastasis [30] were reduced by 80 % (Fig. 4G, P = 0.001) and 77 % (Fig. 4H, P = 0.001), respectively for MDA-MB231-shCXCR3 transduced cells and by 2.8-fold (Fig. 4I, P = 0.006) and 2.5-fold (Fig. 4J, P = 0.002), respectively for MDA-MB231-shATF3 transduced cells over control MDA-MB231-shScr cells. These results suggest that a reduction in RAN probably reduces MMP2, at least in part, by reducing firstly ATF3 and then CXCR3, and that the latter two proteins also lie on the pathway that signals increases in cellular properties associated with metastasis. ...
RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis.
Simple summary
Ran is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.
... A majority of cancer-induced mortality is due to tumor metastasis primarily influenced by the stromal tumor microenvironment. Hence, researchers are emphasizing the understanding of tumor microenvironment, the three-dimensional (3D) architecture of ECM that regulate functional properties of cancerous cells and influence drug sensitivity (Pouliot et al., 2013). To metastasize, tumor cells must invade the surrounding tissue and basement membrane to enter the bloodstream or lymphatics, which involves a crosstalk between tumor and stroma. ...
With a slow and fast momentum, a whopping 95,55,027 cancer deaths were recorded as of 2020 globally. Subsequent detection of cancer in the patient requires a computed tomography (CT) scan to determine the spread of cancers. However, a positron emission tomography (PET) scan has been currently employed to determine the patient response to treatment and reoccurrence. PET is a medical imaging technique that allows quantitative in vivo measurements of three-dimensional distributions of positron-emitting tracers with commonly F-fluorodeoxyglucose (FDG) or other compounds as a PET tracer in oncologic and other human disease applications. PET is gaining more acceptance in recent years for its precise distinguishing capability between dead tissue and cancer cells. Over the years, PET-CT could attain an accuracy of 93% over other scans. Since a high treatment strategy alteration in cancers is observed using PET-CT, its preference in screening high-risk patients is increasing thereby making it a priority scan. The present chapter therefore enumerates the technical characteristics and usage of PET-CT in cancer and some other human diseases with the help of clinical significance and current literature.
... Overall, 2D models have incrementally increased our knowledge about ovarian cancer. The readily available assays such as proliferation, migration and invasion can be easily performed in 2D monolayers, and some of these can be quite cost-effective to run [75]. ...
Ovarian cancer (OC) is one of the most diagnosed gynecological cancers in women. Due to the lack of effective early stage screening, women are more often diagnosed at an advanced stage; therefore, it is associated with poor patient outcomes. There are a lack of tools to identify patients at the highest risk of developing this cancer. Moreover, early detection strategies, therapeutic approaches, and real-time monitoring of responses to treatment to improve survival and quality of life are also inadequate. Tumor development and progression are dependent upon cell-to-cell communication, allowing cancer cells to re-program cells not only within the surrounding tumor microenvironment, but also at distant sites. Recent studies established that extracellular vesicles (EVs) mediate bi-directional communication between normal and cancerous cells. EVs are highly stable membrane vesicles that are released from a wide range of cells, including healthy and cancer cells. They contain tissue-specific signaling molecules (e.g., proteins and miRNA) and, once released, regulate target cell phenotypes, inducing a pro-tumorigenic and immunosuppressive phenotype to contribute to tumor growth and metastasis as well as proximal and distal cell function. Thus, EVs are a “fingerprint” of their cell of origin and reflect the metabolic status. Additionally, via the capacity to evade the immune system and remain stable over long periods in circulation, EVs can be potent therapeutic agents. This review examines the potential role of EVs in the different aspects of the tumor microenvironment in OC, as well as their application in diagnosis, delivery of therapeutic agents, and disease monitoring.
... Scratch assay, also known as a wound-healing assay, is a laboratory technique that mimics cell migration and cell-cell interaction during in vivo wound healing [13][14][15][16]. HS2 cells incubated in 24-well plates for 24 hours were used for the scratch assay. ...
... Scratch assay, also known as wound healing assay, is a technique that mimics cell migration and cell-cell interaction during in vivo wound healing. [26][27][28][29] HS2 cells in 24-well plates were incubated in a humidified atmosphere with 5% CO 2 at 37°C for 24 hours and used for the scratch assay. After incubation, the medium in the wells was removed and a flat scratch in the middle of the single layer of the cells attached to the bottom of the plate was created using a sterile small pipette tip to simulate a wound. ...
Background: Photobiomodulation (PBM) depends on the use of non-ionizing light
energy to trigger photochemical changes, particularly in light-sensitive mitochondrial
structures. It triggers proliferation and the metabolic activity of the cells, primarily by
utilizing the energy from the near-infrared to the red wavelength of the light.
Purpose: This in vitro study has analyzed comparatively the most appropriate energy
doses and wavelengths to induce PBM on keratinocytes and fibroblasts for the ac-
celerated wound healing process.
Methods: 1, 3, and 5 J/cm2
energy densities of 655 and 808-nm diode lasers were
used to promote cell proliferation and wound healing process. Scratch assay and
MTT analysis were performed on keratinocytes and fibroblasts for wound closure
and cell proliferation after the triple light applications, respectively.
Results: 655-nm of wavelength was more successful on keratinocytes to induce
wound healing and cell proliferation, whereas 808-nm of wavelength was so effec-
tive on fibroblasts to heal the wounds totally and it induced cell proliferation almost
3 times compared to the untreated control group.
Conclusion: This study revealed that PBM with 655 and 808 nm of wavelengths was
effective to speed up the wound healing process at specific energy densities. In gen-
eral 808-nm of wavelength was more successful. However, the proper wavelength
and the energy density may differ according to the cell type. Thus, every light param-
eter should be chosen properly to obtain better outcomes during PBM applications.
KEYWORDS
near-infrared light, photobiomodulation, red light, wound healing
... To support this hypothesis, we could imagine an experiment in which we would compare the metastatic by injecting RKO and HCT116 cells in mice, with and without aphidicolin treatment, and see if there are differences RKO HCT116 RPE-1 ns * * *** **** ns 183 between the two cell lines. This type of experiment could also be carry out by in vitro approach with 3D multicellular tumour spheroids (Pouliot et al., 2013). c. ...
DNA replication is very well orchestrated in mammalian cells thanks to a tight regulation of the temporal order of replication origin activation, commonly called replication timing (RT). The replication timing of a given replication domain (RD) is very robust and depends on the cell type. Upon low replication stress, replication forks progress slower and it has been shown that some fragile regions are replicated later or even under-replicated. These replication delay leads to DNA damage and genetic instability, a common marker of cancers. Except for these fragile regions, the direct impact of low replication stress on the RT of the whole genome has not been explored yet. The aim of my thesis was to analyse and compare the replication timing of 6 human cell lines from different tissues (healthy or from tumours) in response to mild replication stress. Assessing this question, I have first observed heterogeneous response in between cell lines, some cancer cells were much more impacted by low replication stress. Strikingly, in some cancer cells, specific RD are undergoing a switch from late to early replication in response to replication stress. Very interestingly, this RT alteration was still detected in daughter cells. These findings disclosed a new mechanism mainly used by cancer cells in response to replication stress that brings another proof of their genome plasticity, allowing a quick response and adaptation to stress that, eventually, gives better resistance to genotoxic agents.
... While the ECM in an early tumor state can act as a tumor growth inhibitor, it supports the invasion and metastasis of cancer cells in later stages [16]. In addition, tumor cell migration can be directional when the corresponding external cues are provided in the form of a gradient of soluble signals or ECM rigidity (durotaxis/haptotaxis) [17][18][19]. There is also no doubt that the mechanical properties of the surrounding tissue material are vital in bone remodeling and repair as these phenomena are intrinsically connected to the complex force balance between bone matrix and bone cells [20]. ...
The main task of tissue engineering (TE) is to reproduce, replicate, and mimic all kinds of tissues in the human body. Nowadays, it has been proven useful in TE to mimic the natural extracellular matrix (ECM) by an artificial ECM (scaffold) based on synthetic or natural biomaterials to regenerate the physiological tissue/organ architecture and function. Hydrogels have gained interest in the TE community because of their ability to absorb water similar to physiological tissues, thus mechanically simulating the ECM. In this work, we present a novel hydrogel platform based on poly(2-ethyl-2-oxazoline)s, which can be processed to 3D microstructures via two-photon polymerization (2PP) with tunable mechanical properties using monomers and crosslinker with different degrees of polymerization (DP) for future applications in TE. The ideal parameters (laser power and writing speed) for optimal polymerization via 2PP were obtained using a specially developed evaluation method in which the obtained structures were binarized and compared to the computer-aided design (CAD) model. This evaluation was performed for each composition. We found that it was possible to tune the mechanical properties not only by application of different laser parameters but also by mixing poly(2-ethyl-2-oxazoline)s with different chain lengths and variation of the crosslink density. In addition, the swelling behavior of different fabricated hydrogels were investigated. To gain more insight into the viscoelastic behavior of different fabricated materials, stress relaxation tests via nanoindentation experiments were performed. These new hydrogels can be processed to 3D microstructures with high structural integrity using optimal laser parameter settings, opening a wide range of application properties in TE for this material platform.
... It is based on the observation that after the creation of an artificial gap (called scratch) on a confluent cell monolayer the cells on the edge of the gap will proliferate and move towards the opening progressively closing the scratch until the monolayer is reformed. This ''in vitro'' technique mimic to some extent migration and proliferation of cells ''in vivo'' and has become an easy and well-developed tool to study the behaviour of different cellular lines in many physiological or pathological contexts as for example cancer metastasis, embryonic morphogenesis [1], tissue development, immune response or wound healing [2]. It can be ultimately considered as a sensor for a great variety of conditions or chemical/physical stimuli that influence cell migration and proliferation. ...
Scratch assay is an easy and widely used “in vitro” technique to study cell migration and proliferation. In this work we focus on its modelling and on the capability to distinguish between these two phenomena that the simpler and common models are not able to disentangle.
We adapted a model based on reaction–diffusion equation for being used with common microscopy instruments/data and therefore taking place in the gap between simpler modelling approaches and complex ones. An optimized image analysis pipeline and numerical least-squares fit provide estimates of the scratch proliferation and diffusion coefficients l and D.
This work is intended as a first of a series in which the model is tested and its robustness and reproducibility are evaluated. Test samples were NIH3T3 cells scratch assays with proliferation and migration stimulated by varying the foetal bovine serum amount in the culture medium (10%, 7.5%, 5% and 2.5%). Results demonstrate, notwithstanding an expected l−D anticorrelation, the model capability to disentangle them. The 7.5% serum treatment can be identified as the model sensitivity limit. Treat–control l and D variations showed an intra-experiment reproducibility (∼±0.05∕h and ∼±200μm2∕h respectively) consistent with single fit typical uncertainties (∼±0.02∕h and ∼±300μm2∕h respectively).
... Our lab previously established a 3D vascularized microfluidic breast cancer platform incorporated with MDA-MB-231 cells and a vascularized endothelial vessel that addressed the limitation described earlier with existing in vitro tumor models. Using this vascularized platform, we determined the relationship between wall shear stress (WSS) and signaling between cancer and endothelial cells on the vasculature (Buchanan, Verbridge, Vlachos, & Rylander, 2014;Gadde, Marrinan, Michna, & Rylander, 2018;Michna, Gadde, Ozkan, DeWitt, & Rylander, 2018) but similar to other non-IBC in vitro tumor models the platform does not account for the complex tumor dynamics inherent to IBC (Bersini et al., 2014;Buchanan et al., 2012;Buchanan, Verbridge, et al., 2014;Buchanan, Voigt, et al., 2014;Duinen et al., 2017;Gadde et al., 2018;Ghousifam, Eftekharjoo, Derakhshan, & Gappa-Fahlenkamp, 2019;Huang & Chang, 2019;Jeon et al., 2015;Kim et al., 2015;Kim, Chung, Ahn, Lee, & Li Jeon, 2016;Ko et al., 2019;Koh, Stratman, Sacharidou, & Davis, 2008;Ma, Middleton, You, & Sun, 2018;Malandrino, Kamm, & Moeendarbary, 2018;Meer, Orlova, Dijke, Berg, & Mummery, 2013;Michna et al., 2018;Nguyen et al., 2013;Osaki, Serrano, & Kamm, 2018;Ozcelikkale, Moon, Linnes, & Han, 2017;Pagano et al., 2014;Pouliot, Pearson, & Burrows, 2013;Pradhan et al., 2018;Rhodes & Simons, 2007;Shang, Soon, Lim, Khoo, & Han, 2019;Sleeboom, Eslami Amirabadi, Nair, Sahlgren, & den Toonder, 2018;Sontheimer-Phelps, Hassell, & Ingber, 2019;Szot, Buchanan, Freeman, & Rylander, 2011;Szot, Buchanan, Freeman, & Rylander, 2013;Tsai, Trubelja, Shen, & Bao, 2017;Vickerman, Blundo, Chung, & Kamm, 2008). ...
Inflammatory breast cancer (IBC), a rare form of breast cancer associated with increased angiogenesis and metastasis, is largely driven by tumor‐stromal interactions with the vasculature and the extracellular matrix (ECM). However, there is currently a lack of understanding of the role these interactions play in initiation and progression of the disease. In this study, we developed the first three‐dimensional, in vitro, vascularized, microfluidic IBC platform to quantify the spatial and temporal dynamics of tumor‐vasculature and tumor‐ECM interactions specific to IBC. Platforms consisting of collagen type 1 ECM with an endothelialized blood vessel were cultured with IBC cells, MDA‐IBC3 (HER2+) or SUM149 (triple negative), and for comparison to non‐IBC cells, MDA‐MB‐231 (triple negative). Acellular collagen platforms with endothelialized blood vessels served as controls. SUM149 and MDA‐MB‐231 platforms exhibited a significantly (p < .05) higher vessel permeability and decreased endothelial coverage of the vessel lumen compared to the control. Both IBC platforms, MDA‐IBC3 and SUM149, expressed higher levels of vascular endothelial growth factor (p < .05) and increased collagen ECM porosity compared to non‐IBCMDA‐MB‐231 (p < .05) and control (p < .01) platforms. Additionally, unique to the MDA‐IBC3 platform, we observed progressive sprouting of the endothelium over time resulting in viable vessels with lumen. The newly sprouted vessels encircled clusters of MDA‐IBC3 cells replicating a key feature of in vivo IBC. The IBC in vitro vascularized platforms introduced in this study model well‐described in vivo and clinical IBC phenotypes and provide an adaptable, high throughput tool for systematically and quantitatively investigating tumor‐stromal mechanisms and dynamics of tumor progression.
... For cervical cancer, the five-year survival rate for metastatic disease is 16.5% compared to 91.5% for localized cancer, and there is no standard treatment due to its heterogeneous manifestations [39,40]. Because cell migration is an indispensable process required at every step of the metastatic cascade [41], we next explored the effect of crinamine on cervical cancer cell migration using a real-time cell migration assay based on the xCELLigence system. As shown in Figure 4a, SiHa cells treated with DMSO migrated from the upper chamber in SFM to the lower chamber containing 4% FBS, while no migration was observed in the SFM control. ...
Crinum asiaticum is a perennial herb widely distributed in many warmer regions, including Thailand, and is well-known for its medicinal and ornamental values. Crinum alkaloids contain numerous compounds, such as crinamine. Even though its mechanism of action is still unknown, crinamine was previously shown to possess anticancer activity. In this study, we demonstrate that crinamine was more cytotoxic to cervical cancer cells than normal cells. It also inhibited anchorage-independent tumor spheroid growth more effectively than existing chemotherapeutic drugs carboplatin and 5-fluorouracil or the CDK9 inhibitor FIT-039. Additionally, unlike cisplatin, crinamine induced apoptosis without promoting DNA double-strand breaks. It suppressed cervical cancer cell migration by inhibiting the expression of positive regulators of epithelial-mesenchymal transition SNAI1 and VIM. Importantly, crinamine also exerted anti-angiogenic activities by inhibiting secretion of VEGF-A protein in cervical cancer cells and blood vessel development in zebrafish embryos. Gene expression analysis revealed that its mechanism of action might be attributed, in part, to downregulation of cancer-related genes, such as AKT1, BCL2L1, CCND1, CDK4, PLK1, and RHOA. Our findings provide a first insight into crinamine's anticancer activity, highlighting its potential use as an alternative bioactive compound for cervical cancer chemoprevention and therapy.
... Migration and invasion ability of metastatic cancer cells in vitro generally reflect the metastasis ability in vivo (Justus et al., 2014;Malandrino et al., 2018;Pouliot et al., 2013). The two agonists performed well in the aspect of inhibiting metastasis. ...
... However, these models suffer from severe limitations, such as controllability, reproducibility, and flexibility of design (Pouliot, Pearson, & Burrows, 2000). In a 2D system, the cells lose their in vivo morphology, and this affects the cell-cell and cell-matrix interactions (Cukierman, Pankov, Stevens, & Yamada, 2001). ...
Breast cancer (BrCa) preferentially spreads to bone and colonises within the bone marrow to cause bone metastases. To improve the outcome of patients with BrCa bone metastasis, we need to understand better the mechanisms underlying bone metastasis. Researchers have relied heavily upon in vivo xenografts due to limited availability of human bone metastasis samples. A significant limitation of these is that they do not have a human bone microenvironment. To address this issue, we have developed a nanoclay‐based 3D in vitro model of BrCa bone metastasis using human mesenchymal stem cells (MSCs) and human BrCa cells mimicking late stage of BrCa pathogenesis at the metastatic site. This 3D model can provide a microenvironment suitable for cell–cell and cell–matrix interactions whilst retaining the behaviour of BrCa cells with different metastasis potential (i.e., highly metastatic MDA‐MB‐231 and low metastatic MCF‐7) as shown by the production of alkaline phosphatase and matrix metalloproteinase‐9. The sequential culture of MSCs with MCF‐7 exhibited 3D tumouroids formation and also occurrence of mesenchymal to epithelial transition of cancer metastasis as evidenced by gene expression and immunocytochemistry. The unique and distinct behaviour of highly metastatic MDA‐MB‐231 and the low metastatic MCF‐7 was observed at the bone metastasis site. The changes to migratory capabilities and invasiveness in MDA‐MB‐231 in comparison with tumour growth with MCF‐7 was observed. Together, a novel bone‐mimetic 3D in vitro BrCa model has been developed that could be used to study mechanisms governing the later stage of cancer pathogenesis in bone.
... Recently, it was shown that the decision to migrate as a single cell or as a collective is dependent on transforming growth factor beta (TGFβ) signaling, which is also known as a key regulator of EMT. Neoplastic cells expressing transforming growth factor beta receptor 2 (TGFβ RII) migrate predominantly as single cells or strands at the tumor-stroma border and express markers of EMT, while cells lacking the receptor show largely collective migration as large cell clusters with no evidence of EMT [25,26]. ...
A search for the “magic bullet”, a molecule, the targeting abilities of which could stop the migration of tumor cells, is currently underway, but remains in the early stages. There are still many unknowns regarding the cell migration. The main approach is the employment of mouse models, that are sources of valuable information, but still cannot answer all of the questions. One of the molecules of interest is Transglutaminase 2 (TG2). It is a well-described molecule involved in numerous pathways and elevated in metastatic tumors. The question remains whether mice and humans can give the same answer considering TG2.
... In order to study dynamic changes in cell motility and locomotion, we interrogated the permeability of endothelial cells, which infers changes to the intracellular cytoskeleton and can be performed using ECIS (17). We plated endothelial cells (HUVEC) on an ECIS microchip following standard protocol to interrogate metastasis (22). The cells were allowed to spread and form junctions and cell-cell contact on the chip. ...
Breast cancer is a leading cause of cancer-related mortality in women. Triple-negative breast cancer (TNBC; HER2-, ER-/PR-) is an aggressive subtype prone to drug resistance and metastasis, which is characterized by high intratumor microvascular density (iMVD) resulting from angiogenesis. However, the mechanisms contributing to the aggressive phenotypes of TNBC remain elusive. We recently reported that down-regulation of exchange factor directly activated by Cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in metastatic properties including proliferation and cell migration in TNBC cell lines. Here, we report that EPAC1 supports TNBC-induced angiogenesis, tumor cell migration and invasiveness as well as pro-metastatic phenotypes in endothelial cells induced through the tumor secretome. Using an approach that integrates proteomics with bioinformatics and gene ontologies, we elucidate that EPAC1 supports a tumor-secreted network of angiogenic, cell adhesion and cell migratory pathways. Using confocal microscopy we show that signaling molecules involved in focal adhesion, including Paxillin and MENA, are down-regulated in the absence of EPAC1, and electric cell substrate impedance sensing technique confirmed a role for EPAC1 on TNBC-induced endothelial cell permeability. Finally, to provide a translational bridge, we studied iMVD and therapy response using a primary human tumor explant assay, CANscriptTM, which suggests a link between therapy-modulated neovascularization and drug sensitivity. These data provide mechanistic insight into the role of EPAC1 in regulating the tumor microenvironment, iMVD and cancer cell-induced angiogenesis, a dynamic mechanism under drug pressure that may associate to treatment failure.
... It is possible to produce defined 3D cell culture matrices with a specific physical or biochemical property (tensile strength, pore pattern and size, naturally occurring components such as basement membranes) as required to study tumour metastasis. A comprehensive review on using 3D cell cultures for studying metastasis was published in 2000 (Pouliot et al., 2000). ...
Cancer cell lines have contributed immensely in understanding the complex physiology of cancers. They are excellent material for studies as they offer homogenous samples without individual variations and can be utilized with ease and flexibility. Also, the number of assays and end-points one can study is almost limitless; with the advantage of improvising, modifying or altering several variables and methods. Literally, a new dimension to cancer research has been achieved by the advent of 3Dimensional cell culture techniques. This approach increased many folds the ways in which cancer cell lines can be utilized for understanding complex cancer biology. 3D cell culture techniques are now the preferred way of using cancer cell lines to bridge the gap between the "absolute in vitro" and "true in vivo". The aspects of cancer biology that 3D cell culture systems have contributed include morphology, microenvironment, gene and protein expression, invasion/migration/metastasis, angiogenesis, tumour metabolism and drug discovery, testing chemotherapeutic agents, adaptive responses and cancer stem cells. We present here, a comprehensive review on the applications of 3D cell culture systems for these aspects of cancers. This article is protected by copyright. All rights reserved.
... Our previous study with other bee products proved that beebread and various honeys also inhibited the secretion of MMP9 and MMP2 in glioblastoma cells [24,33]. An integral part of metastasis that is required at virtually every step of the metastatic cascade is cell migration [34]. In our study, inhibition of U87MG cells migration was observed after treatment with combination of EEP and HPE. ...
Background
Propolis and Hypericum perforatum L. are natural products which contain many active compounds and have numerous beneficial effects, including an antitumor effect. Gliobmastoma multiforme (GBM) is a common primary brain tumor with poor prognosis and limited treatment options. In this study, the effect of propolis (EEP) combined with H. perforatum L. (HPE) on glioblastoma cell line U87MG was investigated for the first time. Methods
Anti-proliferative activity of EEP, HPE and their combination (EEP + HPE) was determined by a cytotoxicity test, DNA binding by [3H]-thymidine incorporation and cell migration assay. Anti-metastatic properties in U87MG treated with EEP, HPE and EEP + HPE were estimated on cells migration test (scratch assay) and metalloproteinases (MMP2 and MMP9) secretion (gelatin zymography). ResultsCombination of HPE and EEP extracts was found to have a time- and dose-dependent inhibitory effect on the viability of U87MG cells. This effect was significantly higher (p < 0.05) when compared to these two extracts applied separately, which was confirmed by the significant reduction of DNA synthesis and significantly higher mitochondrial membrane permeabilization. A significant decreasing in migration cells and in pro-MMP9 and pro-MMP2 secretion in U87MG cells were demonstrated after exposure to combination of EEP (30 μg/ml) with HPE (6.25 μg/ml). Conclusions
In this study, the combination of ethanolic extract from propolis and ethanolic extract of fresh-cut H. perforatum L. was proved the ability to reduce invasiveness of glioma cells through the inhibition of MMP2 and MMP9 secretion and suppression of cell migration. It has a more potent anti-proliferative effect on U87MG glioma cell line compared to using propolis and H. perforatum L. separately. Further studies are required to verify whether the examined extracts can activate apoptotic pathways.
... The bottom surface of the membrane is imaged and typically quantified by cells per imaging field. While this is a simplistic assay, the degree of migration through pores toward serum provides a high throughput in vitro model of tumor intravasation through leaky vasculature, with the pores in the membrane representing the gaps in the endothelium and serum representing the bloodstream (Pouliot et al., 2000). ...
In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment.
... Metastasis is one of the most complex processes in cancer and likely one of the most difficult to study and mimic using in vitro models [48,49]. Therefore, it is crucial to understand the molecular and cellular phenomena involved in the metastatic cascade [50,51]. Invasion of cancer cells through the basal membrane into a blood or lymphatic vessel (intravasion) followed by entrance in other tissue and/or organs (extravasion) are critical steps [52,53]. ...
Cancer is a major cause of morbidity and mortality worldwide, with a disease burden estimated to increase over the coming decades. Disease heterogeneity and limited information on cancer biology and disease mechanisms are aspects that 2D cell cultures fail to address. Here, we review the current 'state-of-the-art' in 3D tissue-engineering (TE) models developed for, and used in, cancer research. We assess the potential for scaffold-based TE models and microfluidics to fill the gap between 2D models and clinical application. We also discuss recent advances in combining the principles of 3D TE models and microfluidics, with a special focus on biomaterials and the most promising chip-based 3D models.
... Given the increased incidence of metastasis associated with Col3 haploinsufficiency in the 4T1 model of murine breast cancer, we examined the ability of Col3 to modulate invasion and migration of breast cancer cells through basement membrane gels supplemented with Col3, a 50:50 mixture of Col1 and Col3, or Col1 alone using standard in vitro Transwell assays. 60 Invasion of 4T1 or MDA-MB-231 cells was significantly inhibited in Col3-containing gels compared to gels with Col1 alone (Figure 6 ½F6 ½F6 ...
Breast cancer metastasis is the leading cause of cancer-related deaths in women worldwide. Collagen in the tumor microenvironment plays a crucial role in regulating tumor progression. We have shown that type III collagen (Col3), a component of tumor stroma, regulates myofibroblast differentiation and scar formation after cutaneous injury. During the course of these wound-healing studies, we noted that tumors developed at a higher frequency in Col3(+/-) mice compared to wild-type littermate controls. We, therefore, examined the effect of Col3 deficiency on tumor behavior, using the murine mammary carcinoma cell line 4T1. Notably, tumor volume and pulmonary metastatic burden after orthotopic injection of 4T1 cells were increased in Col3(+/-) mice compared to Col3(+/+) littermates. By using murine (4T1) and human (MDA-MB-231) breast cancer cells grown in Col3-poor and Col3-enriched microenvironments in vitro, we found that several major events of the metastatic process were suppressed by Col3, including adhesion, invasion, and migration. In addition, Col3 deficiency increased proliferation and decreased apoptosis of 4T1 cells both in vitro and in primary tumors in vivo. Mechanistically, Col3 suppresses the procarcinogenic microenvironment by regulating stromal organization, including density and alignment of fibrillar collagen and myofibroblasts. We propose that Col3 plays an important role in the tumor microenvironment by suppressing metastasis-promoting characteristics of the tumor-associated stroma.
Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
... Interaction with the surrounding stroma is an important factor in metastatic dissemination (Bhowmick et al., 2004;Pouliot et al., 2013;Zetter, 1993). Hence, the ability to adhere to various ECM proteins that are present in the microenvironment was measured ( Prince et al., 2002). ...
The translation of basic research into improved therapies for breast cancer patients requires relevant pre-clinical models that incorporate spontaneous metastasis. We have completed a functional and molecular characterisation of a new isogenic C57BL/6 mouse model of breast cancer metastasis, comparing and contrasting it with the established BALB/c 4T1 model. Metastatic EO771.LMB tumours were derived from parental poorly metastatic EO771 mammary tumours. Functional differences were evaluated using both in vitro assays and spontaneous metastasis assays in mice. Results were compared to non-metastatic 67NR and metastatic 4T1.2 tumours of the 4T1 model. Protein and transcript levels of markers of human breast cancer molecular subtypes were measured in the four tumour lines, as well as p53 tumour suppressor gene status and responses to Tamoxifen in vivo and in vitro. Array-based expression profiling of whole tumours identified genes and pathways that were deregulated in metastatic tumours. EO771.LMB cells metastasised spontaneously to lung in C57BL/6 mice and displayed increased invasive capacity compared to parental EO771. By immunohistochemical assessment, EO771 and EO771.LMB were basal-like, as was the 4T1.2 tumour, while 67NR had a luminal phenotype. Primary tumours from all lines were negative for progesterone receptor, Erb-b2/Neu, and cytokeratin 5/6, but positive for epidermal growth factor receptor. Only 67NR displayed nuclear estrogen receptor alpha positivity. EO771 and EO771.LMB expressed mutant p53 while 67NR and 4T1.2 were p53 null. Integrated molecular analysis of both the EO771/EO771.LMB and 67NR/4T1.2 pairs indicated that upregulation of MMP-3, Pthlh and S100a8 and downregulation of Cd36 may be causally involved in metastatic dissemination of breast cancer.
... Results are obtained by counting the number of migrating cells in the ECM gel [163]. Several authors have used the transwell chamber assay in 3D matrix for testing invasion ability of leukemia cells [164,165]. This method was later refined, by observing leukemia cell motility in 3D Transwell with time-lapse microscopy. ...
A large body of evidence indicates that three dimensional (3D) cancer models are superior to two-dimensional (2D) ones in better representing the in vivo phenomena. Indeed, 3D models allow recapitulating in vitro the in vivo features observed in solid tumors (e.g. cell polarity, cell-cell/cell-matrix interactions, biochemical/metabolic gradients, anchorage-independent growth and hypoxia). Moreover, it is well established that the microenvironment plays a fundamental role in regulating tumor development and behavior, including drug resistance. Thus, innovative models able to mimic this complexity represent attractive tools in cancer research. In this review article, we provide a comprehensive review of the application of 3D culture systems in pediatrics’ cancer research. In particular, 3D in vitro/ex vivo models of the most common pediatric tumors, such as leukemias, lymphomas and malignancies of the nervous system, will be considered.
This chapter introduces photoacoustic (PA) imaging, a noninvasive molecular imaging technology that promises immense potential in biomedical and clinical applications. It starts with the basic principle of PA imaging, a comprehensive understanding of angiogenesis, tumor invasion, and metastasis, and its applications in cancer therapy with a special focus on breast cancer. Angiogenesis is crucial for the growth of the primary tumor, and hence imaging modalities of tumor vasculature are significant for diagnostics and appropriate therapeutic intervention. The chapter also focuses on the need for clinically relevant and applicable PA imaging developments to enable clinicians and health care researchers with precise and predictable diagnostic and research outcomes like tumor progression stage that is predominantly influenced by tumor invasion and metastasis.
Cell–cell fusion is a physiological process that is hijacked during oncogenesis and promotes tumour evolution. The main known impact of cell fusion is to promote the formation of metastatic hybrid cells following fusion between mobile leucocytes and proliferating tumour cells. We show here that cell fusion between immortalized myoblasts and transformed fibroblasts, through genomic instability and expression of a specific transcriptomic profile, leads to emergence of hybrid cells acquiring dissemination properties. This is associated with acquisition of clonogenic ability by fused cells. In addition, by inheriting parental properties, hybrid tumours were found to mimic the histological characteristics of a specific histotype of sarcomas: undifferentiated pleomorphic sarcomas with incomplete muscular differentiation. This finding suggests that cell fusion, as macroevolution event, favours specific sarcoma development according to the differentiation lineage of parent cells.
Single, isolated epithelial cells move randomly; however, during wound healing, organism development, cancer metastasis, and many other multicellular phenomena, motile cells group into a collective and migrate persistently in a directed manner. Recent work has examined the physics and biochemistry that coordinates the motions of these groups of cells. Of late, two mechanisms have been touted as being crucial to the physics of these systems: leader cells and jamming. However, the actual importance of these to collective migration remains circumstantial. Fundamentally, collective behavior must arise from the actions of individual cells. Here, we show how biophysical activity of an isolated cell impacts collective dynamics in epithelial layers. Although many reports suggest that wound closure rates depend on isolated cell speed and/or leader cells, we find that these correlations are not universally true, nor do collective dynamics follow the trends suggested by models for jamming. Instead, our experimental data, when coupled with a mathematical model for collective migration, shows that intracellular contractile stress, isolated cell speed, and adhesion all play a substantial role in influencing epithelial dynamics, and that alterations in contraction and/or substrate adhesion can cause confluent epithelial monolayers to exhibit an increase in motility, a feature reminiscent of cancer metastasis. These results directly question the validity of wound-healing assays as a general means for measuring cell migration, and provide further insight into the salient physics of collective migration.
Metastasis to the central nervous system (CNS) remains a major cause of morbidity and mortality in patients with systemic cancer. Thanks to multimodality therapies, the length of survival in patients with systemic cancer continues to improve. Therefore, a focus on metastases to the CNS is paramount in order to maximize the span and quality of life for these patients. Unique interactions between the brain’s microenvironment, the blood–brain-barrier, and tumor cells themselves promote distinct molecular features in CNS metastases that may require therapeutic approaches different from those used in systemic metastases. This chapter will focus on the pathobiology, epigenetics, and immunobiology of brain metastases in order to better understand their molecular biology.
Cancer cells must escape the primary tumor site, intravasate into blood vessels, survive the hematogenous dissemination to the CNS, arrest in brain capillaries, extravasate, proliferate, and develop angiogenic abilities in order to establish brain metastases. Molecular biology, genetics, and epigenetics are rapidly expanding, enabling us to advance our know-ledge of the underlying mechanisms involved. Research approaches using cell lines that preferentially metastasize in vivo to the brain and in vitro tissue-based studies may lead to new targets for therapeutics. It is important to understand the pathobiology of the metastatic cascade in order to target research investigation and the development of more effective therapies.
This volume is essential for geneticists, molecular biologists, biochemists, and medical doctors interested in the use of mouse models in cancer research. Recent genome studies, together with refined genetic engineering techniques, have greatly increased the value of using mice for research on cancer and other human disorders. The chapters of this book will support scientists in choosing the most suitable mouse models for their research questions. The book provides detailed methodological information for genetic or chemical induction of different types of cancer, histomorphometric cancer analysis, and in vivo imaging, as well as protocols to investigate oncogene addiction, immune surveillance, and hallmarks of cancer such as angiogenesis or metastasis. Four review-like articles provide background information on mouse technologies and histopathologic differences between mouse and human cancers. The mouse models described in individual chapters will fuel the understanding of cancer initiation, immune system roles, tumor angiogenesis, invasion, metastasis, and the relevance of molecular diversity observed among human cancers. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and resourceful, Mouse Models of Cancers: Methods and Protocols, is a valuable laboratory resource for all researchers, from the graduate level upwards, who study cancer and new possibilities for its treatment. © Springer Science+Business Media New York 2015. All rights are reserved.
There is accumulating evidence indicating that aldehyde dehydrogenase (ALDH) activity selects for cancer cells with increased aggressiveness, capacity for sustained proliferation, and plasticity in primary tumors. However, emerging data also suggests an important mechanistic role for the ALDH family of isoenzymes in the metastatic activity of tumor cells. Recent studies indicate that ALDH correlates with either increased or decreased metastatic capacity in a cellular context-dependent manner. Importantly, it appears that different ALDH isoforms support increased metastatic capacity in different tumor types. This review assesses the potential of ALDH as biological marker and mechanistic mediator of metastasis in solid tumors. In many malignancies, most notably in breast cancer, ALDH activity and expression appears to be a promising marker and potential therapeutic target for treating metastasis in the clinical setting.
Lung adenocarcinoma (AC) induced by carcinogen inhalation never came close to the human counterpart. This has changed with the creation of genetically engineered models.
Different types of mouse AC were studied, some of them with a KRAS codon12 mutation background, others with HRAS mutants inserted; in most additional hits were introduced (ATG5, PTEN, PI3K, p53). Development of AC was followed for 28 weeks. Human lung AC subtypes and precursor lesions served for comparison.
AC in mice starts with papillary growth at the bronchoalveolar junction (baJ). Tumor spreads into the alveolar periphery. Due to different cell to alveolar size alveoli are filled almost completely, simulating solid growth. At a certain size hypoxic necrosis is seen in centers, followed by neoangiogenesis and desmoplastic stroma formation. Finally invasion of tumor cells into stroma occur. Metastasis is rare, due to high tumor load, causing early death. Vascular invasion is seen, if second carcinogenic hits are applied.
Dissimilarities with human precursor and AC types are: AC in mice are all non-mucinous, they are predominant papillary or solid, often with a high degree of signet ring cell formation; in addition there seems to be a requirement of large tumor size before invasion and metastasis occur. To understand AC development knowledge of the anatomy and histology of mouse and human lung is necessary, but these models open a new way of investigating lung AC.
Citation Format: Helmut H. Popper, Beatrice Grabner, Emilio Casanova, Robert Eferl, Rao Shuan, Josef Penninger. Comparison of lung adenocarcinoma development in genetically engineered mouse and in humans - similarities and differences. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1209. doi:10.1158/1538-7445.AM2014-1209
The skin is the largest organ of the mammalian body, made up of multiple layers, which include the epidermis, dermis, and subcutis (Alam and Ratner, N Engl J Med 344(13):975–983, 2001). The human interfollicular epidermis can be subdivided into five different layers: (1) stratum basale, (2) stratum spinosum, (3) stratum granulosum, (4) stratum lucidum, and (5) stratum corneum, all originating from basal keratinocytes by differentiation (Hameetman et al., BMC cancer 13:58, 2013; Ramirez et al., Differentiation 58(1):53–64, 1994). The epidermis is also able to generate different appendages: hair follicles (HF) and their associated sebaceous glands (Sibilia et al., Cell 102(2):211–220, 2000) as well as sweat glands (Luetteke et al., Genes Dev 8(4):399–413, 1994). The skin has important functions in several biological processes like environmental barrier, tissue regeneration, hair cycling, and wound repair. During these processes, stem cells from the interfollicular epidermis and from the hair follicle bulge are activated to renew the epidermis or hair. The epidermis and hair undergo continuous homeostatic regeneration and mutations, upon mutations which disturb the balance of homeostatic regeneration of epidermis and hair and lead to enhanced proliferation of keratinocytes, development of skin cancer is developed.
Tumors that arise in the skin are mainly of three types: malignant melanoma, arising from melanocytes, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC), the latter two both arising from keratinocytes or hair follicle cells. In this chapter, we will describe some genetically engineered mouse models (GEMM) that aim at modeling human BCC and SCC and their respective precancerous lesions. We will describe the experimental approaches used in our laboratory to analyze tumor-bearing mice focusing on methods necessary for the induction of tumor growth as well as for the molecular and histological analysis of tumor tissue.
The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we will describe the workflow of the wound healing assay as monitored by optical microscopy. Although the assay is straightforward, a lack of standardization in its application makes it difficult to compare results and reproduce experiments among researchers. We will recommend general guidelines for consistency, including: (1) sample preparation including the creation of the gap, (2) microscope equipment requirements, (3) image acquisition, and (4) the use of image analysis to measure the gap size and its rate of closure over time. We will also describe parameters that are specific to the particular research question, such as seeding density and matrix coatings. All of these parameters must be carefully controlled within a given set of experiments in order to achieve accurate and reproducible results.
Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated.
Materials and Methods The invasionand migrationoccurringin primaryneoplastictissue explantswerestudiedbyusinga three-dimensional collagenmatrixmodel, subsequent time-lapse videomicroscopy, and computer-assisted cell track ing. We show that not only single cells but groups of clustered cells comprising S to more than 100 cells detach from the primary tumor lesion andmigratewithintheadjacentextracellular matrix.Theseclusterswere highly polarized, resulting in a high directional persistence of migration. Locomoting cell clusters were observed in primary cultures from invasive oral squamous cell carcinomas (6 of 9), ductal breast carcinomas (2 of 3), andrhabdomyosarcoma (1of 1),whereas normaloralmucosa(0of4) was cell cluster negative. Thus, locomoting cell clusters could be a novel and potentially important mechanism of cancer cell invasion and metastasis.
Introduction
Transforming growth factor beta (TGF-β) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-β signaling regulates fibroblast recruitment and activation. Concurrently, TGF-β signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-β signaling to tumorigenesis, yet the role of TGF-β in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion.
Methods
Fluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TβRII KO) or TβRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-β signaling loss.
Results
Intravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-β signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TβRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TβRII KO carcinoma cells was identified by E-cadherin+/p120+/β-catenin+ clusters. TβRII KO tumors also exhibited a twofold greater metastasis than TβRIIfl/fl tumors, attributed to enhanced extravasation ability. In TβRII KO tumor epithelium compared with TβRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-β-insensitive mammary cancer.
Conclusion
Fibroblast-stimulated carcinoma cells utilize TGF-β signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-β signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-β signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.
Few technologies are more widespread in modern biological laboratories than imaging. Recent advances in optical technologies and instrumentation are providing hitherto unimagined capabilities. Almost all these advances have required the development of software to enable the acquisition, management, analysis and visualization of the imaging data. We review each computational step that biologists encounter when dealing with digital images, the inherent challenges and the overall status of available software for bioimage informatics, focusing on open-source options.
Current research in biology uses evermore complex computational and imaging tools. Here we describe Icy, a collaborative bioimage informatics platform that combines a community website for contributing and sharing tools and material, and software with a high-end visual programming framework for seamless development of sophisticated imaging workflows. Icy extends the reproducible research principles, by encouraging and facilitating the reusability, modularity, standardization and management of algorithms and protocols. Icy is free, open-source and available at http://icy.bioimageanalysis.org/.
BioImageXD puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXD has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes. We demonstrate its performance in a study of integrin clustering in response to selected inhibitors.
Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
We discuss the advantages and challenges of the open-source strategy in biological image analysis and argue that its full impact will not be realized without better support and recognition of software engineers' contributions to the biological sciences and more support of this development model from funders and institutions.
We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized. Reducing RhoA, Rho-associated protein kinase (ROCK), or myosin II activity switched the cells to lamellipodia-based 3D migration. These modes of 3D migration were regulated by matrix physical properties. Specifically, experimentally modifying the elasticity of the CDM or collagen gels established that nonlinear elasticity supported lamellipodia-based migration, whereas linear elasticity switched cells to lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by RhoA, ROCK, and myosin II and extrinsically by the elastic behavior of the 3D extracellular matrix.
Alveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein's transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR-mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS.
To identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells' motility. We used microarray analysis to identify genes whose expression level decreased when PAX3-FKHR was downregulated. We used mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays to determine whether PAX3-FKHR binds to the promoter region of the target gene. We used siRNA and pharmacologic inhibitor to downregulate the target gene of PAX3-FKHR and investigated the effect of such downregulation on cell motility.
We found that when PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A) decreased. We showed that PAX3-FKHR binds to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1A decreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR-mediated cell migration and metastasis.
Taken together, we have identified CPT1A as a novel transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting cell motility. CPT1A may represent a novel therapeutic target for the treatment of ARMS.
Cell morphology determines cell behavior, signal transduction, protein-protein interaction, and responsiveness to external stimuli. In cancer, these functions profoundly contribute to resistance mechanisms to radio- and chemotherapy. With regard to this aspect, this study compared the genome wide gene expression in exponentially growing cell lines from different tumor entities, lung carcinoma and squamous cell carcinoma, under more physiological three-dimensional (3D) versus monolayer cell culture conditions. Whole genome cDNA microarray analysis was accomplished using the Affymetrix HG U133 Plus 2.0 gene chip. Significance analysis of microarray (SAM) and t-test analysis revealed significant changes in gene expression profiles of 3D relative to 2D cell culture conditions. These changes affected the extracellular matrix and were mainly associated with biological processes like tissue development, cell adhesion, immune system and defense response in contrast to terms related to DNA repair, which lacked significant alterations. Selected genes were verified by semi-quantitative RT-PCR and Western blotting. Additionally, we show that 3D growth mediates a significant increase in tumor cell radio- and chemoresistance relative to 2D. Our findings show significant gene expression differences between 3D and 2D cell culture systems and indicate that cellular responsiveness to external stress such as ionizing radiation and chemotherapeutics is essentially influenced by differential expression of genes involved in the regulation of integrin signaling, cell shape and cell-cell contact.
Cell adhesion molecules play a significant role in cancer progression and metastasis. Cell-cell interactions of cancer cells with endothelium determine the metastatic spread. In addition, direct tumor cell-interactions with platelets, leukocytes and soluble components significantly contribute to cancer cell adhesion, extravasation, and the establishment of metastatic lesions.
Clinical evidence indicates that heparin, commonly used for treatment of thromboembolic events in cancer patients, is beneficial for their survival. Preclinical studies confirm that heparin possesses anti-metastatic activities that lead to attenuation of metastasis in various animal models. Heparin contains several biological activities that may affect several steps in metastatic cascade. Here we focus on the role of cellular adhesion receptors in the metastatic cascade and discuss evidence for heparin as an inhibitor of cell adhesion. While P- and L-selectin facilitation of cellular contacts during hematogenous metastasis is being accepted as a potential target of heparin, here we propose that heparin may also interfere with integrin activity and thereby affect cancer progression.
This review summarizes recent findings about potential mechanisms of tumor cell interactions in the vasculature and anti-metastatic activities of heparin.
There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed.
To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease.
We have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.
The vascular endothelium is a dynamic cellular "organ" that controls passage of nutrients into tissues, maintains the flow of blood, and regulates the trafficking of leukocytes. In tumors, factors such as hypoxia and chronic growth factor stimulation result in endothelial dysfunction. For example, tumor blood vessels have irregular diameters; they are fragile, leaky, and blood flow is abnormal. There is now good evidence that these abnormalities in the tumor endothelium contribute to tumor growth and metastasis. Thus, determining the biological basis underlying these abnormalities is critical for understanding the pathophysiology of tumor progression and facilitating the design and delivery of effective antiangiogenic therapies.
Macrophages constitute a major component of the leucocytic infiltrate of tumours. Human studies show an association between tumour-associated macrophages and tumours with poor prognostic features. In breast cancer, the presence of macrophages has been correlated with increased angiogenesis and poor prognosis but little information is available about the independent prognostic role of macrophages infiltrating breast carcinomas.
This study used immunohistochemistry and tissue microarrays to assess the density and localisation of CD68 macrophages infiltrating 1322 breast tumours and to identify any relationship with clinicopathological factors and patient outcome.
Tumour-infiltrating macrophages were present in the majority of tumours with a predominantly diffuse pattern. The density of distant stromal macrophages (infiltrating stroma away from the carcinoma, median count 14 cells) was higher than intratumoural (median zero cells) and adjacent stromal macrophages (median three cells). Higher total macrophage number was associated with higher tumour grade (r(s)=0.39, p<0.001), ER and PgR negativity, HER-2 positivity and basal phenotype (p<0.001). In univariate survival analysis, higher numbers of CD68 macrophages were significantly associated with worse breast cancer-specific survival (p<0.001) and shorter disease-free interval (p=0.004). However in multivariate model analysis, the CD68 macrophage count was not an independent prognostic marker.
Macrophages are heterogeneous with different subsets having different functions. The present study suggests that overall macrophage numbers are not related to prognosis in breast cancer. However, further studies are needed to investigate the potential role of different subsets of macrophages.
Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PIP3 and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.
Chemotaxis of tumour cells and stromal cells in the surrounding microenvironment is an essential component of tumour dissemination during progression and metastasis. This Review summarizes how chemotaxis directs the different behaviours of tumour cells and stromal cells in vivo, how molecular pathways regulate chemotaxis in tumour cells and how chemotaxis choreographs cell behaviour to shape the tumour microenvironment and to determine metastatic spread. The central importance of chemotaxis in cancer progression is highlighted by discussion of the use of chemotaxis as a prognostic marker, a treatment end point and a target of therapeutic intervention.
One of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells (ECs) into a blood-brain barrier (BBB) phenotype is shear stress (SS). With the use of a well established humanized dynamic in vitro BBB model and cDNA microarrays, we have profiled the effect of SS in the induction/suppression of ECs genes and related functions.
Specifically, we found a significant upregulation of tight and adherens junctions proteins and genes. Trans-endothelial electrical resistance (TEER) and permeability measurements to know substances have shown that SS promoted the formation of a tight and highly selective BBB. SS also increased the RNA level of multidrug resistance transporters, ion channels, and several p450 enzymes. The RNA level of a number of specialized carrier-mediated transport systems (e.g., glucose, monocarboxylic acid, etc.) was also upregulated.RNA levels of modulatory enzymes of the glycolytic pathway (e.g., lactate dehydrogenase) were downregulated by SS while those involved in the Krebs cycle (e.g., lactate and other dehydrogenases) were upregulated. Measurements of glucose consumption versus lactate production showed that SS negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration. BBB ECs are responsive to inflammatory stimuli. Our data showed that SS increased the RNA levels of integrins and vascular adhesion molecules. SS also inhibited endothelial cell cycle via regulation of BTG family proteins encoding genes. This was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. Furthermore, analysis of 2D gel electrophoresis (which allows identifying a large number of proteins per sample) of EC proteins extracted from membrane sub-cellular endothelial fractions showed that SS increased the expression levels of tight junction proteins. In addition, regulatory enzymes of the Krebb's cycle (aerobic glucose metabolism) were also upregulated. Furthermore, the expression pattern of key protein regulators of the cell cycle and parallel gene array data supported a cell proliferation inhibitory role for SS.
Genomic and proteomic analyses are currently used to examine BBB function in healthy and diseased brain and characterize this dynamic interface. In this study we showed that SS plays a key role in promoting the differentiation of vascular endothelial cells into a truly BBB phenotype. SS affected multiple aspect of the endothelial physiology spanning from tight junctions formation to cell division as well as the expression of multidrug resistance transporters. BBB dysfunction has been observed in many neurological diseases, but the causes are generally unknown. Our study provides essential insights to understand the role played by SS in the BBB formation and maintenance.
Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated.
The book describes most of the methods that are currently used in metastasis research. Both in vivo and in vitro protocols are illustrated, so that the metastatic process can be either analysed as a whole, or single events addressed separately. Each method is described in the frame of the metastatic process, therefore its significance and its limitations in the context of metastasis are always taken into account. Whenever possible, several alternative procedures are reported per each experimental issue, so that the researcher can choose the one that better suits her/his needs and possibilities.
During the past 30 years a big effort has been made to elucidate the molecular mechanisms of cancer metastatis, the leading cause of death for cancer patients. A considerable number of assays have been set up, that can be used to address specific questions concerning the single metastatic steps, or can be applied to develop and test drugs specifically interfering with selected events during the metastatic spread. This book contains an exhaustive description of most of the methods and their rationale, that are currently used in metastatic research, both to analyse metastasis in its entirety (in vivo models), or to dissect the single steps of the metastatic process (in vitro assays).
Normal human breast epithelial cells show a high degree of phenotypic plasticity in monolayer culture and express many traits that otherwise characterize tumor cells in vivo. Paradoxically, primary human breast carcinoma cells are difficult to establish in culture: most outgrowths arise from the normal tissue surrounding the tumor. These characteristics have posed major obstacles to the establishment of simple reliable criteria for mammary epithelial transformation in culture. In the present study, we show that a reconstituted basement membrane (BM) can be used to culture all normal human breast epithelial cells and a subset of human breast carcinoma cells. The two cell types can be readily distinguished by virtue of the ability of normal cells to reexpress a structurally and functionally differentiated phenotype within BM. Twelve specimens of normal breast tissue and 2 normal breast epithelial cell lines (total 14 samples) embedded in BM as single cells were able to form multicellular spherical colonies with a final size close to that of true acini in situ. Sections of mature spheres revealed a central lumen surrounded by polarized luminal epithelial cells expressing keratins 18 and 19 and sialomucin at the apical membrane. Significantly, two-thirds of normal spheres deposited a visible endogenous type IV collagen-containing BM even though they were in contact with exogenously provided BM. Growth was arrested completely within the same time period. In contrast, none of 6 carcinoma cell lines or 2 cultures of carcinoma from fresh samples (total 8 samples) responded to BM by growth regulation, lumen formation, correct polarity, or deposition of endogenous BM. These findings may provide the basis of a rapid assay for discriminating normal human breast epithelial cells from their malignant counterparts. Furthermore, we propose that the ability to sense BM appropriately and to form three-dimensional organotypic structures may be the function of a class of "suppressor" genes that are lost as cells become malignant.
The role of the integrin alpha 6 beta 1 in breast carcinoma progression was studied by targeted elimination of this integrin in MDA-MB-435 cells, a human breast carcinoma cell line that is highly metastatic in athymic mice. The strategy used is based on the finding that expression of a cytoplasmic domain deletion mutant of the beta 4-integrin subunit (beta 4-delta CYT) in MDA-MB-435 cells eliminates formation of the alpha 6 beta 1 heterodimer. MDA-MB-435 cells that lacked alpha 6 beta 1 expression (beta 4-delta CYT transfectants) formed tumors in athymic mice that were suppressed in their growth and that exhibited a significant increase in apoptosis in comparison to the control tumors. Unlike the control MDA-MB-435 cells, the beta 4-delta CYT transfectants were unable to establish metastatic foci in the lungs. Also, the control transfectants grew substantially better than the beta 4-delta CYT transfectants in the liver after intrahepatic injection because of extensive apoptosis in the beta 4-delta CYT transfectants. These data suggest that a major function of the alpha 6 beta 1 integrin in breast carcinoma is to facilitate tumorigenesis and promote tumor cell survival in distant organs.
In cancer metastasis, only a small percentage of cells released from a primary tumor successfully form distant lesions, but it is uncertain at which steps in the process cells are lost. Our goal was to determine what proportions of B16F1 melanoma cells injected intraportally to target mouse liver 1) survive and extravasate, 2) form micrometastases (4 to 16 cells) by day 3, 3) develop into macroscopic tumors by day 13, and 4) remain as solitary dormant cells. Using in vivo videomicroscopy, a novel cell accounting assay, and immunohistochemical markers for proliferation (Ki-67) and apoptosis (TUNEL), we found that 1) 80% of injected cells survived in the liver microcirculation and extravasated by day 3, 2) only a small subset of extravasated cells began to grow, with 1 in 40 forming micrometastases by day 3, 3) only a small subset of micrometastases continued to grow, with 1 in 100 progressing to form macroscopic tumors by day 13 (in fact, most micrometastases disappeared), and 4) 36% of injected cells remained by day 13 as solitary cancer cells, most of which were dormant (proliferation, 2%; apoptosis, 3%; in contrast to cells within macroscopic tumors: proliferation, 91%; apoptosis/necrosis, 6%). Thus, in this model, metastatic inefficiency is principally determined by two distinct aspects of cell growth after extravasation: failure of solitary cells to initiate growth and failure of early micrometastases to continue growth into macroscopic tumors.
To determine whether subendothelial laminins (LNs) could be impli- cated in the extravasation of neoplastic lymphocytes, we have examined the distribution of a number of LN isoforms in human vascular structures of adult individuals and have assayed the ability of the isolated LN molecules to promote adhesion of lymphoma and leukemic cells in vitro using a novel cell adhesion assay, CAFCA, Centrifugal Assay for Fluo- rescence-based Cell Adhesion (E. Giacomello et al., Biotechniques, 26: 758 -762, 1999; P. Spessotto et al., Methods Mol. Biol., 139: 321-343, 2000). The use of previously characterized LN chain-specific antibodies showed that the vast majority of the smaller vascular compartments, known to correspond to sites of lymphocyte transmigration, expressed the subunits involved in the structuring of 9 of the 12 LN isoforms known to date. Eight LN isoforms (i.e., LN-1, -2, -4, -5, -8, -9, -10, and -11) and four naturally occurring LN complexes were isolated from various tissues and cultured cells by combined gel filtration, ion exchange, and immunoaffin- ity chromatographies, and the identity/composition of the isolated LNs/LN complexes was asserted by immunochemical means and amino-acid se- quencing. Notwithstanding the widespread colocalization of LN isoforms, a panel of neoplastic B- and T-cell lines and lymphocytes isolated from patients affected by chronic lymphocytic B-cell leukemia attached prefer- entially and with high avidity to purified LN-8, purified LN-10, and LN-10-containing protein complexes, whereas lymphocytes derived from patients diagnosed with acute lymphocytic leukemia failed to bind to these LNs. All of the tested neoplastic lymphocytes failed to adhere to the isolated LN-1, LN-4, LN-9, and LN-11 and attached moderately well to purified LN-2 and LN-5. The interaction of transformed lymphocytes with LNs was cation-dependent and interchangeably mediated by the a3b1 and a6b1 integrins. The degree of engagement of the two LN receptors was dependent upon their relative levels of cell surface expression, whereas, irrespective of the phenotype, lymphocytes deprived of either of these receptors were incapable of LN binding. The findings suggest that LN-8 and LN-10 may act in an independent or complementary fashion as primary components of the endothelial basement membrane favoring the interaction of extravasating neoplastic lymphocytes. Thus, our results would demonstrate that different LN isoforms may evoke diverse cellular responses in different cell types and that this divergence may be the basis for the redundancy of LN distribution in a number of vascular structures.
Often described as incomplete or absent, the basement membrane of blood vessels in tumors has attracted renewed attention as a source of angiogenic and anti-angiogenic molecules, site of growth factor binding, participant in angiogenesis, and potential target in cancer therapy. This study evaluated the composition, extent, and structural integrity of the basement membrane on blood vessels in three mouse tumor models: spontaneous RIP-Tag2 pancreatic islet tumors, MCa-IV mammary carcinomas, and Lewis lung carcinomas. Tumor vessels were identified by immunohistochemical staining for the endothelial cell markers CD31, endoglin (CD105), vascular endothelial growth factor receptor-2, and integrin alpha5 (CD49e). Confocal microscopic studies revealed that basement membrane identified by type IV collagen immunoreactivity covered >99.9% of the surface of blood vessels in the three tumors, just as in normal pancreatic islets. Laminin, entactin/nidogen, and fibronectin immunoreactivities were similarly ubiquitous on tumor vessels. Holes in the basement membrane, found by analyzing 1- micro m confocal optical sections, were <2.5 micro m in diameter and involved only 0.03% of the vessel surface. Despite the extensive vessel coverage, the basement membrane had conspicuous structural abnormalities, including a loose association with endothelial cells and pericytes, broad extensions away from the vessel wall, and multiple layers visible by electron microscopy. Type IV collagen-immunoreactive sleeves were also present on endothelial sprouts, supporting the idea that basement membrane is present where sprouts grow and regress. These findings indicate that basement membrane covers most tumor vessels but has profound structural abnormalities, consistent with the dynamic nature of endothelial cells and pericytes in tumors.
The α3β1 integrin is elevated in several types of metastatic tumor and has been associated with increased migration and invasion. Our analysis of a series of mammary carcinomas of different histotypes and their corresponding metastases demonstrated significantly increased expression of α3β1 in the tumor metastases. We therefore studied α3β1 expression of several human breast carcinoma cell lines and its association with the invasive phenotype. The MDA-MB-231 cell line expressed high levels of the β1, α2, α3, α5, and α6 integrin subunits along with moderate levels of the αvβ3 integrin. This line was highly migratory and the most invasive using a chemo-invasion assay. In contrast, the other lines tested, MDA-MB-145, MCF-7, and SK-BR-3, showed lower migratory and invasive activity and reduced α3 integrin subunit expression. Metalloproteases capable of degrading collagen IV are necessary for the invasive process. RT-PCR showed that MDA-MB-231 cells expressed MMP-9, but not MMP-2, gelatinase/collagenase IV. Gelatin zymography demonstrated that invading MDA-MB-231 cells released high levels of MMP-9 gelatinase activity. A direct role for this gelatinase in MDA-MB-231 cell invasion was confirmed by inhibition of invasion using the metalloprotease inhibitorBatimastat. Treatment of MDA-MB-231 cells with a function-blocking anti-α3 antibody strongly inhibited migration and invasion. This correlated with a marked reduction in MMP-9 activity produced by MDA-MB-231 cells, suggesting a role for α3β1 ligand binding in cell signaling and regulation of extracellular matrix degradation. Int. J. Cancer 87:336–342, 2000. © 2000 Wiley-Liss, Inc.
The basement membrane protein, laminin (LM)-511, is a potent adhesive and migratory substrate for metastatic breast tumor cells in vitro. Its expression correlates with tumor grade and metastatic potential in vivo. These observations suggest that responsiveness to autocrine or paracrine-derived LM-511 may be an important property regulating breast cancer metastasis in vivo. To address this, we compared the metastatic potential of 4T1 mammary carcinoma cells to that of 4T1 variants isolated by repeated chemotactic migration toward LM-511 in vitro (4T1LMF4) followed by serial injection into the mammary gland and recovery of spontaneous metastases from bone (4T1BM2). Variant subpopulations exhibited a distinct morphology on LM-511 and increased expression of β1 and β4 integrins compared to parental 4T1 cells. Importantly, mice inoculated with 4T1LMF4 and 4T1BM2 variants showed a 2.5- to 4-fold increase in the incidence of spontaneous metastasis to bone compared to 4T1 tumor-bearing mice. Functionally, 4T1BM2 variants were more adherent and more invasive toward LM-511 than parental 4T1 cells. Treatment of 4T1BM2 cells with lebein-1, a disintegrin with selectivity toward LM-type integrin receptors, potently inhibited their migration and invasion toward LM-511. Similarly, α3β1 integrin-dependent migration and invasion of human MDA-MB-231 breast carcinoma cells toward LM-511 were significantly inhibited by lebein-1. Taken together, these results provide strong evidence that LM-511 contributes to the metastasis of breast tumors and suggest that targeting integrin-LM-511 interactions with lebein-1 or other inhibitors of LM-511 receptors may have therapeutic potential for patients with advanced breast cancer.
Gelled substrates can be used for a variety of in vitro and in vivo experiments. A type I collagen gelled substrate will promote cell growth and differentiation. Gelled Matrigel substrate promotes the survival of explanted cells and tissues and the differentiation of a variety of epithelial and endothelial cell types in vitro and to assess angiogenesis and increase tumor growth in vivo. Preparation of the matrices and their use are described in this unit.
Hydrogels are polymeric materials distinguished by high water content and diverse physical properties. They can be engineered
to resemble the extracellular environment of the body’s tissues in ways that enable their use in medical implants, biosensors,
and drug-delivery devices. Cell-compatible hydrogels are designed by using a strategy of coordinated control over physical
properties and bioactivity to influence specific interactions with cellular systems, including spatial and temporal patterns
of biochemical and biomechanical cues known to modulate cell behavior. Important new discoveries in stem cell research, cancer
biology, and cellular morphogenesis have been realized with model hydrogel systems premised on these designs. Basic and clinical
applications for hydrogels in cell therapy, tissue engineering, and biomedical research continue to drive design improvements
using performance-based materials engineering paradigms.
Microscale engineering technologies provide unprecedented opportunities to create cell culture microenvironments that go beyond current three-dimensional in vitro models by recapitulating the critical tissue-tissue interfaces, spatiotemporal chemical gradients, and dynamic mechanical microenvironments of living organs. Here we review recent advances in this field made over the past two years that are focused on the development of 'Organs-on-Chips' in which living cells are cultured within microfluidic devices that have been microengineered to reconstitute tissue arrangements observed in living organs in order to study physiology in an organ-specific context and to develop specialized in vitro disease models. We discuss the potential of organs-on-chips as alternatives to conventional cell culture models and animal testing for pharmaceutical and toxicology applications. We also explore challenges that lie ahead if this field is to fulfil its promise to transform the future of drug development and chemical safety testing.
We have recently demonstrated the formation of interconnecting canalicular cell processes in bone cells upon contact with basement membrane components. Here we have determined whether growth factors in the reconstituted basement membrane (Matrigel) were active in influencing the cellular network formation. Various growth factors including transforming growth factor β (TGF-β), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were identified in Matrigel. Exogenous TGF-β blocked the cellular network formation. Conversely, addition of TGF-β1 neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF, EGF, and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-E1 cells grown on Matrigel overcame the inhibitory effect of TGF-β. Some TGF-β remained bound to type IV collagen purified from the Engelbreth-Holm-Swarm tumor matrix. These data demonstrate that reconstituted basement membrane contains growth factors which influence cellular behavior, suggesting caution in the interpretation of experiments on cellular activity related to Matrigel, collagen type IV, and possibly other extracellular matrix components.
Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) α1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln α1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16–19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln α1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln α1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln α1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.
Mutationally corrupted cancer (stem) cells are the driving force of tumor development and progression. Yet, these transformed cells cannot do it alone. Assemblages of ostensibly normal tissue and bone marrow-derived (stromal) cells are recruited to constitute tumorigenic microenvironments. Most of the hallmarks of cancer are enabled and sustained to varying degrees through contributions from repertoires of stromal cell types and distinctive subcell types. Their contributory functions to hallmark capabilities are increasingly well understood, as are the reciprocal communications with neoplastic cancer cells that mediate their recruitment, activation, programming, and persistence. This enhanced understanding presents interesting new targets for anticancer therapy.
The blood-brain barrier (BBB), a unique selective barrier for the central nervous system (CNS), hinders the passage of most compounds to the CNS, complicating drug development. Innovative in vitro models of the BBB can provide useful insights into its role in CNS disease progression and drug delivery. Static transwell models lack fluidic shear stress, while the conventional dynamic in vitro BBB lacks a thin dual cell layer interface. To address both limitations, we developed a microfluidic blood-brain barrier (μBBB) which closely mimics the in vivo BBB with a dynamic environment and a comparatively thin culture membrane (10 μm). To test validity of the fabricated BBB model, μBBBs were cultured with b.End3 endothelial cells, both with and without co-cultured C8-D1A astrocytes, and their key properties were tested with optical imaging, trans-endothelial electrical resistance (TEER), and permeability assays. The resultant imaging of ZO-1 revealed clearly expressed tight junctions in b.End3 cells, Live/Dead assays indicated high cell viability, and astrocytic morphology of C8-D1A cells were confirmed by ESEM and GFAP immunostains. By day 3 of endothelial culture, TEER levels typically exceeded 250 Ω cm(2) in μBBB co-cultures, and 25 Ω cm(2) for transwell co-cultures. Instantaneous transient drop in TEER in response to histamine exposure was observed in real-time, followed by recovery, implying stability of the fabricated μBBB model. Resultant permeability coefficients were comparable to previous BBB models, and were significantly increased at higher pH (>10). These results demonstrate that the developed μBBB system is a valid model for some studies of BBB function and drug delivery.
The basement membrane protein laminin-511 has been implicated in breast cancer progression and metastasis. To identify peptides from LM-511 that modulate the metastatic properties of breast tumours, we screened laminin alpha 5 chain-derived peptides for their ability to promote adhesion of metastatic mammary carcinoma cells. Two selected adhesive peptides, α5A13b (FHVAYVLIKF) from the LN domain and A5G27 (RLVSYNGIIFFLK) from the LG-globular domain, were further characterised for their inhibitory properties against LM-511 activities in vitro and metastasis in vivo. In vitro, these peptides strongly inhibited LM-511-dependent adhesion and migration of highly metastatic 4T1.2 mammary carcinoma cells. In addition, A5G27 but not α5A13b significantly reduced breast tumour cell proliferation and inhibited laminin-511-induced matrix metalloproteinase-9 expression. Surprisingly, despite its potent inhibitory activity in vitro, A5G27 promoted rather than inhibited 4T1.2 experimental pulmonary metastasis in vivo, regardless of its route of administration. Adhesion of 4T1.2 cells to A5G27 was not inhibited by antibodies directed against α6, β1 or β3 integrins or CD44 but was significantly reduced in the presence of heparin suggesting a role for cell surface glycans. Treatment of the cells with α-L-fucosidase but not neuraminidase or heparinase II also partially inhibited cell adhesion to A5G27 and to LM-511 indicating that these interactions are mediated in part via terminal fucosyl residues. Overall, these results show that LMα5 peptides exhibit distinct functional properties in vitro and in vivo and suggest that interactions between the RLVSYNGIIFFLK sequence present in LM-511 and cell surface glycans may regulate LM-511 metastatic properties in vivo.
The following study was designed to investigate early biosynthetic and ultrastructural changes that alter functional properties of the basement membrane (BM) and affect vascular permeability in diabetic retinopathy.
To determine whether altered matrix synthesis affects cell monolayer permeability, rat retinal endothelial cells (RRECs) were grown for 4 days to confluency in normal (N, 5 mM) or high glucose (HG, 30 mM) medium on transwell inserts and subjected to an in vitro cell monolayer permeability assay. Inserts were cut out and viewed under a transmission electron microscope to assess extracellular matrix (ECM) accumulation and cell morphology. In parallel cell cultures, fibronectin and collagen IV protein expression were determined using Western Blot analysis.
Electron microscopic analysis of cells exposed to short-term HG showed no difference in inter-endothelial cell tight junctions (TJs) or in the number of vesicles or coated pits compared to those of normal cells. However, ECM accumulation underlying HG cells was significantly increased compared to that of cells grown in N medium (139 ± 7% of control, p = 0.04), with areas of focal thickening. Western blot analysis showed increased fibronectin and collagen IV expression (152 ± 24% of control, p = 0.01; 146 ± 16% of control, p = 0.02, respectively) in cells grown in HG compared to those grown in N medium. Cell monolayers grown in HG exhibited increased permeability to FITC-dextran compared to cells grown in N medium (134 ± 15% of control, p = 0.02).
High glucose-induced excess ECM accumulation and altered composition underlies structural and functional changes that allow increased permeability. This finding provides evidence for the first time that the thickened vascular basement membrane contributes to the development of excess permeability seen in diabetic retinopathy.
Many steps of the metastatic cascade can be reproduced in simple in vitro assays such as tumour cell interactions with matrix proteins, proteolysis, chemotaxis, haptotaxis, and invasion into matrices or explanted tissues. Nevertheless, there are no fully adequate substitutes for the complexity of the in vivo process. Here, we describe two "experimental" metastasis assays to yield lung or liver colonies (mimicking established micrometastatic disease), and two spontaneous metastasis assays for breast and prostate carcinomas. Examples include either murine tumour cell lines in syngeneic immunocompetent mice or human tumour xenografts in immunodeprived mice.
The scratch-wound assay is a simple, reproducible assay commonly used to measure basic cell migration parameters such as speed, persistence, and polarity. Cells are grown to confluence and a thin "wound" introduced by scratching with a pipette tip. Cells at the wound edge polarise and migrate into the wound space. Advantages of this assay are that it does not require the use of specific chemoattractants or gradient chambers and it generates a strong directional migratory response, even in cell types that do not show robust responses in "single cell" migration assays. It is most reliably analysed when performed using time-lapse imaging, which can also yield valuable cell morphology/protein localisation information.
Cells in a three-dimensional (3D) extracellular matrix environment often display different properties and behavior compared to cells cultured on a two-dimensional (2D) substrate. Recent studies characterizing the cell-matrix adhesions formed by cells within a 3D matrix have arrived at contradictory conclusions regarding the presence and composition of adhesions. Here we review this literature, and provide a comparative compilation of information found in published studies from the 3D cell-matrix adhesion field in order to identify shared and divergent conclusions and conceptually important areas that require further research. Although there is a general consensus that discrete cell-matrix adhesions exist in various 3D matrix environments, there are specific exceptions, particularly in cells undergoing amoeboid migration. There are also technical issues to consider when imaging adhesions in 3D matrix; for example, over-expression of a cytoskeletal cell adhesion component can potentially cloud the visualization of adhesions and even alter the mode of cell migration. Properties such as stiffness and local matrix topography may also affect the composition of cell-matrix adhesions. For example, even though cells contain integrin-based 3D adhesions, there can be substantial variability within these adhesions in the presence of force-dependent cytoskeletal components such as vinculin. These new findings and ideas provide promising new leads for understanding the regulation and function of cell-matrix adhesions in 3D matrix.
Beta(1) integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of beta(1) integrin signaling. We previously showed that beta(1) integrin inhibitory antibodies (e.g., AIIB2) enhance apoptosis and decrease growth in human breast cancer cells in three-dimensional laminin-rich extracellular matrix (lrECM) cultures and in vivo. Here, we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used three-dimensional lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in three-dimensional lrECM. Colonies were assayed for apoptosis and beta(1) integrin/Akt signaling pathways were evaluated using Western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in three-dimensional lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down-regulating Akt in breast cancer colonies in three-dimensional lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared with either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We previously showed that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo.
The distribution of laminin alpha1 chain in adult mouse tissue was determined by immunofluorescence using monoclonal antibody 200, reacting with the globular carboxyterminus E3 fragment of alpha1 chain. Strong reactivity was noted only in a few tissues. Reactivity was restricted to epithelial basement membranes. Expression was noted in several epithelial basement membranes of the urinary tract, and male and female reproductive organs. In addition, expression was seen in some parts of the nervous system. Expression was seen in pia mater which surrounds the brain, and in the extracellular matrices covering the vitreous chamber and the lens of the eye. Staining was seen in the adrenal gland cortex, with strongest staining in the zona glomerulosa. Staining was negative in all other studied epithelial basement membranes, such as the lung (trachea or lung epithelium), epidermis, and all parts of the gastrointestinal tract (liver, gut) except for weak staining in the ventricle and Brunner's glands. No expression was seen in basement membranes of fat, Schwann, or endothelial cells in any studied parts of the body. Both small- and large-size vessel walls were negative both in endothelial basement membranes and blood vessel walls, with the exception of some larger brain blood vessels in locations where epithelial cells have invaginated. Neither smooth muscle, myocardium or striated muscle expressed alpha1 chain. We conclude that alpha1-containing heterotrimers including laminin-1 (alpha1beta1gamma1) have a very restricted tissue distribution.
Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival in part because of the ability of individual cells to migrate significant distances into brain tissue. Invasion is a difficult process to model, because many such human tumors do not invade immunologically competent animal tissue, tumors grown in animals do not invade human tissue, and relevant human tissue substrates are not easily reproduced. We discuss models for examining invasion in vitro, and in particular review work using the tumor spheroid--fetal rat brain aggregate co-culture model, assessed with confocal microscopy and four-dimensional imaging. Quantitation of invasion in this model is discussed, as well as the invasion-inhibitory properties of tyrosine kinase (TK) inhibitors. The effects of receptor-specific tyrphostins strongly support a dominant role for Epidermal Growth Factor Receptor activation in this process and show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than is necessary for growth suppression. Inhibition of activation of the purported growth factor receptor second messenger phospholipase C- gamma 1, by pharmacological means and gene transfection, also profoundly inhibits the invasive properties of human glioblastoma and rat C6 glioma cells. We have assessed invasiveness in several human tumor specimens, which may provide information relative to prognosis and recurrence risk. Our data supports the concept of differential control of invasion and proliferation, and points to possible strategies for anti-invasive therapy for glioblastoma multiforme.
Cell migration requires sustained forward movement of the plasma membrane at the cell's front or "leading edge." To date, researchers have uncovered four distinct ways of extending the membrane at the leading edge. In lamellipodia and filopodia, actin polymerization directly pushes the plasma membrane forward, whereas in invadopodia, actin polymerization couples with the extracellular delivery of matrix-degrading metalloproteases to clear a path for cells through the extracellular matrix. Membrane blebs drive the plasma membrane forward using a combination of actomyosin-based contractility and reversible detachment of the membrane from the cortical actin cytoskeleton. Each protrusion type requires the coordination of a wide spectrum of signaling molecules and regulators of cytoskeletal dynamics. In addition, these different protrusion methods likely act in concert to move cells through complex environments in vivo.
Mortality and morbidity in patients with solid tumors invariably result from the disruption of normal biological function caused by disseminating tumor cells. Tumor cell migration is under intense investigation as the underlying cause of cancer metastasis. The need for tumor cell motility in the progression of metastasis has been established experimentally and is supported empirically by basic and clinical research implicating a large collection of migration-related genes. However, there are few clinical interventions designed to specifically target the motility of tumor cells and adjuvant therapy to specifically prevent cancer cell dissemination is severely limited. In an attempt to define motility targets suitable for treating metastasis, we have parsed the molecular determinants of tumor cell motility into five underlying principles including cell autonomous ability, soluble communication, cell-cell adhesion, cell-matrix adhesion, and integrating these determinants of migration on molecular scaffolds. The current challenge is to implement meaningful and sustainable inhibition of metastasis by developing clinically viable disruption of molecular targets that control these fundamental capabilities.
Cellular infiltration and colonization of three-dimensional (3D) porous scaffolds is influenced by many factors. One of the major factors is the internal architecture presented to the cells. In this work, we have developed and validated a microfluidic device that presents a multitude of geometric challenges to cells, mimicking the architectural aspects and characteristics of 3D porous scaffolds in a two-dimensional arrangement. This device has been utilized to investigate the influence of varying channel widths, degrees of channel tortuosity, the presence of contractions or expansions, and channel junctions on the migration of NIH 3T3 mouse fibroblasts and human bone marrow-derived mesenchymal stromal cell (hMSCs). These two cell types were observed to have vastly different migration characteristics; 3T3 fibroblasts migrate as a collective cell front, whereas hMSCs migrate as single cells. This resulted in 3T3 fibroblasts displaying significant differences in migration depending on the type of geometrical constraint, whereas hMSCs were only influenced by channel width when it approached that of the length scale of a single cell. The differences in migration characteristics were shown to be related to the expression of the intercellular junction protein N-cadherin. We observed that 3T3 fibroblasts express higher levels of N-cadherin than hMSCs and that N-cadherin inhibition modified the migration characteristics of the 3T3 fibroblasts, so that they were then similar to that of hMSCs. The results of this study both confirm the utility of the device and highlight that differences in migration characteristics of different cell types can be deterministic of how they may respond to geometric constraints within porous tissue engineering constructs.
In vitro models of the blood-brain barrier (B-BB) generally utilise murine or porcine brain endothelium and rat astrocytes which are commonly grown in foetal calf serum supplemented conditions which modulate cell growth rates. Consequently, results gained from these experimental models can be difficult to extrapolate to the human in vivo situation since they are not of human origin. The proposed in vitro Transwell model of the B-BB is a multi-culture human cell system. It requires reconstruction of the human derived B-BB components in vitro (cerebral microvascular endothelial cells, astrocytes, and brain vascular pericytes) in a three-dimensional (3D) configuration based on Transwell filters. Different cell permutations (mono-, co-, and tri-cultivation) were investigated to find the most effective model in terms of tight junction resistance of the human cerebral microvascular endothelial cells. The B-BB model permutations comprised of human astrocytes (CC-2565 and SC-1810), human brain vascular pericytes (HBVP), and human cerebral microvascular endothelial cells (hCMEC/D3), under human serum supplementation. The models were assessed by trans-endothelial electrical resistance (TEER) measurements using an epithelial voltohmmeter, to validate the tight junction formation between hCMEC/D3 cells. Mono-, co-, and tri-cultivation Transwell models constructed with human brain-derived cells under human serum supplementation demonstrated that co-cultivation of astrocytes with endothelial cells produced the most successful model, as determined by TEER. Pericytes on the other hand improved tight junction formation when co-cultured with endothelial cells but did not improve the model to such an extent when grown in tri-cultivation with astrocytes.
The increasing interest in studying the interactions between cells and the extracellular matrix (ECM) has created a need for high throughput low-cost three-dimensional (3D) culture systems. The recent development of tubeless microfluidics via passive pumping provides a high throughput microchannel culture platform compatible with existing high throughput infrastructures (e.g., automated liquid handlers). Here, we build on a previously reported high throughput two-dimensional system to create a robust automated system for 3D culture. Operational controls including temperature and sample handling have been characterized and automated. Human mammary fibroblasts (HMFs) suspended in type I collagen are loaded and cultured in microchannel arrays and used to optimize the system operational parameters. A Peltier cooler maintains the collagen as a liquid at 4 °C during cell seeding, followed by polymerization at 37 °C. Optimization of this platform is discussed (e.g., controlling collagen contraction, increasing cell viability, preventing the removal of microchannel contents), and 3D distribution of HMFs is examined by fluorescent microscopy.
Finally, we validate the platform by automating a previously developed 3D breast carcinoma coculture assay. The platform allows more efficient 3D culture experiments and lays the foundation for high throughput studies of cell—ECM interactions.
Metastasis is the leading cause of cancer death. The metastatic cascade is a complex yet inefficient process that we have only begun to understand in recent years. Several of the early steps of this cascade are not readily targetable in the clinic. Past therapeutic developmental strategies have not distinguished between micrometastases and overt metastases. This lack of understanding is apparent in therapies that have been developed for patients with metastatic disease that are not efficacious in patients with micrometastatic disease; that is, in the adjuvant setting. Moreover, drugs that target distant metastases often do not work in the adjuvant setting. This Review will discuss our current understanding of the metastatic cascade as it relates to therapy, emerging therapeutic targets in the metastatic process, and how novel antimetastatic therapies might be developed for clinical use.