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Review Article
Architecture of Cancer-Associated Fibroblasts
in Tumor Microenvironment:
Mapping Their Origins, Heterogeneity,
and Role in Cancer Therapy Resistance
Kevin Dzobo
1,2
and Collet Dandara
3
Abstract
The tumor stroma, a key component of the tumor microenvironment (TME), is a key determinant of response
and resistance to cancer treatment. The stromal cells, extracellular matrix (ECM), and blood vessels influence
cancer cell response to therapy and play key roles in tumor relapse and therapeutic outcomes. Of the stromal
cells present in the TME, much attention has been given to cancer-associated fibroblasts (CAFs) as they are the
most abundant and important in cancer initiation, progression, and therapy resistance. Besides releasing several
factors, CAFs also synthesize the ECM, a key component of the tumor stroma. In this expert review, we
examine the role of CAFs in the regulation of tumor cell behavior and reveal how CAF-derived factors and
signaling influence tumor cell heterogeneity and development of novel strategies to combat cancer. Importantly,
CAFs display both phenotypic and functional heterogeneity, with significant ramifications on CAF-directed
therapies. Principal anti-cancer therapies targeting CAFs take the form of: (1) CAFs’ ablation through use of
immunotherapies, (2) re-education of CAFs to normalize the cells, (3) cellular therapies involving CAFs
delivering drugs such as oncolytic adenoviruses, and (4) stromal depletion via targeting the ECM and its related
signaling. The CAFs’ heterogeneity could be a result of different cellular origins and the cancer-specific tumor
microenvironmental effects, underscoring the need for further multiomics and biochemical studies on CAFs and
the subsets. Lastly, we present recent advances in therapeutic targeting of CAFs and the success of such
endeavors or their lack thereof. We recommend that to advance global public health and personalized medicine,
treatments in the oncology clinic should be combinatorial in nature, strategically targeting both cancer cells and
stromal cells, and their interactions.
Keywords: cancer biology, tumor microenvironment, tumor stroma, cancer-associated fibroblasts, biomarkers,
gene expression, personalized medicine
Introduction
Recent cancer incidence and mortality statistics
indicate an always increasing burden of cancer in the
coming years, with most new cases occurring in countries of
low and middle income (Bray et al., 2018; Ferlay et al., 2019;
Fidler and Bray, 2018). Based on GLOBOCAN estimates and
others, there is a need for a new drive to find and develop new
strategies to reduce this burden worldwide ( Bray, 2016; Bray
and Soerjomataram, 2015; Bray et al., 2012, 2018; Ferlay et al.,
2019; Fidler et al., 2016). The cancer incidence and mortality
statistics provided are likely lower than the actual figures given
the limited and strained surveillance systems found in many
low- and medium-income countries (Bray et al., 2018; Ferlay
et al., 2019; Znaor et al., 2018). It is important to note that
progress has been made in raising awareness to cancer and
several cancer prevention strategies are being adopted; how-
ever, the picture still looks gloomy going forward.
Although great success has been achieved in treating
cancer via different therapeutic strategies directed at cancer
1
International Centre for Genetic Engineering and Biotechnology (ICGEB), Cape Town, South Africa.
2
Division of Medical Biochemistry, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, Institute of Infectious
Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
3
Division of Human Genetics, Department of Pathology, Faculty of Health Sciences, Institute of Infectious Disease and Molecular
Medicine, University of Cape Town, Cape Town, South Africa.
OMICS A Journal of Integrative Biology
Volume 24, Number 6, 2020
ªMary Ann Liebert, Inc.
DOI: 10.1089/omi.2020.0023
314
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cells, reports show that cancer deaths are likely to increase
globally (Bray and Soerjomataram, 2015; Bray et al., 2018;
Ferlay et al., 2019; Fidler et al., 2016; Moten et al., 2014).
Significant progress made in understanding the underlying
causes and molecular mechanisms involved in tumor initia-
tion and development has led to better patients’ outcomes
(Lesina et al., 2011; Mongiat et al., 2016; Senthebane et al.,
2017, 2018). Two major contributors to cancer deaths still
requiring better understanding are cancer relapse and me-
tastasis (Crunkhorn, 2018; Li et al., 2015; Yates et al., 2017).
Research into tumor relapse and metastasis is ongoing, with
several studies reporting novel drugs and effective strategies
aimed at limiting these processes (Mitra et al., 2015; Zhou
et al., 2009).
Further, studies have revealed the tumor microenviron-
ment (TME) as key to tumor initiation, response to therapy,
relapse, and metastasis, ultimately influencing patients’
management in the clinic (Dzobo et al., 2016a; Kalluri, 2016;
Senthebane et al., 2017, 2018). Specifically, the TME has
been shown to provide some form of protection to cancer
cells, reduce cancer cell response to therapy, and, ultimately,
promote therapy resistance (Quail and Joyce, 2013; Senthe-
bane et al., 2017, 2018). The TME can also modify cancer
cells, resulting in cancer cell heterogeneity (Dzobo et al.,
2018b; Quail and Joyce, 2013).
Studies have shown that subpopulations of cells within the
TME, including cancer-associated fibroblasts (CAFs), can
affect cancer cells differently (Ishii et al., 2016; O
¨hlund et al.,
2014, 2017). In addition, several studies have shown that the
extracellular matrix (ECM) can influence cancer cell prolif-
eration, migration, and response to therapy (Noguera et al.,
2012; Senthebane et al., 2018). This has resulted in increased
attention being given to the role of tumor-associated cells and
ECM as well as the development of therapies directed at the
tumor stroma. Recently, studies also demonstrated the gut
microbiota’s influence on cancer response to therapy through
modulation of TME (Iida et al., 2013; Ve
´tizou et al., 2015).
While underscoring the importance of microbiota in dis-
ease treatment and outcome, these studies show that modu-
lation of TME play a huge part in cancer cell response to
treatment. In addition, the activation of the immune system
has been shown to influence cancer cell sensitivity to radio-
therapy (Strom et al., 2017). Thus, the targeting or modula-
tion of the TME has real therapeutic value with the potential
to improve patients’ outcomes. A deeper understanding of the
TME and its contribution to cancer cell behavior is pertinent.
This comprehensive review presents advances in our
knowledge on CAFs and the role played by these cells in
disease initiation and progression. This review brings to the
fore the strategies being adopted to include TME-directed
therapies in the clinic, with the aim of reducing fatal disease.
The TME is used to describe the cells, ECM, and blood
vessels found within the vicinity of cancer cells in a tumor.
Included in this definition are cells such as fibroblasts, mac-
rophages, endothelial cells, lymphocytes, myeloid-derived
cells, and the ECM. Several studies have shown that de-
pending on the stage of tumor development, the TME can
provide both an inhibitory and promoting environment to
cancer cell growth (Senthebane et al., 2017, 2018). Cells
within the TME can be from the local vicinity or migrated
from distant environments (Friedl and Alexander, 2011;
Wels et al., 2008).
The CAFs are the most abundant cells within the TME and
play key roles in tumor initiation and progression (Kalluri,
2016; Su et al., 2018). Immune cells within the TME are
mostly macrophages (Hao et al., 2012; Kim et al., 2012).
Endothelial cells mostly function to form new blood vessels
that are necessary for the supply of nutrients to the growing
tumor and removal of toxic waste (De Palma et al., 2017). In
addition, endothelial cells also secrete platelet-derived
growth factor (PDGF), which attracts pericytes to tumor
blood vessels, and these pericytes are involved in the stabi-
lization of newly formed blood vessels (Armulik et al., 2011).
Stromal cells such as fibroblasts and macrophages are also
involved in the synthesis and maintenance of the ECM
(Bonnans et al., 2014; Riches, 1988). By releasing enzymes
such as matrix metalloproteinases, and growth factors, stro-
mal cells contribute to tumor ECM remodeling (Bonnans
et al., 2014). ECM remodeling enables cancer cells to migrate
and invade surrounding tissues (Gaggioli et al., 2007). As the
tumor develops, both tumor cells and the TME components
co-evolve and are transformed through the release of various
growth factors and other biomolecules, with the TME ini-
tially being tumor-restrictive but tumor-promoting at later
stages (Senthebane et al., 2017, 2018).
Several studies have demonstrated stromal cell heteroge-
neity, especially of CAFs and macrophages (Bauer et al.,
2010; O
¨hlund et al., 2017; Su et al., 2018). This heterogeneity
manifests as either tumor-restrictive or tumor-promoting
activities of the cells (O
¨zdemir et al., 2014; Rhim et al.,
2014). Elaborate studies by Su et al. as well as by O
¨hlund
et al. demonstrated the presence of distinct populations of
fibroblasts within tumors that have specific functions and that
they influence cancer cell response to therapy (Hwang et al.,
2008; O
¨hlund et al., 2017; Su et al., 2018).
Lately, the use of cell surface markers to isolate and
characterize these distinct population of fibroblasts has al-
lowed a deeper analysis of their behavior and functions (O
¨h-
lund et al., 2017; Su et al., 2018). The identification and use of
specific cell surface markers can allow targeted manipulation
of specific fibroblasts populations to achieve a specific goal
during cancer treatment. Under normal physiological condi-
tions, fibroblasts are dormant/quiescent or inactive but can be
activated by various growth factors, cytokines, and chemo-
kines (Gaggioli et al., 2007; Mueller and Fusenig, 2004).
Currently, very few clinical trials have been conducted that
target CAFs to treat cancer. This is partly because the translation
of laboratory scientific evidence into clinical use requires more
resources. It is, therefore, imperative that a deeper understand-
ing of stromal cell behavior and biology is obtained to improve
strategies targeting such cells. This review describes in detail
the involvement of CAFs in cancer pathogenesis.
Mapping CAFs’ Origins and Heterogeneity
In a landmark publication in 1858, Virchow described fi-
broblasts as cells that were spindle shaped and were re-
sponsible for the synthesis of collagen (Virchow, 1871).
Further studies showed that fibroblasts are mostly dormant
cells within the ECM and are transformed under conditions
such as inflammation, fibrosis, and wound healing (Darby
and Hewitson, 2007; Kalluri and Zeisberg, 2006). With both
inflammation and fibrosis being associated with cancer ini-
tiation and development, fibroblasts are, therefore, activated
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during these processes (Dvorak, 1986; Hanahan and Wein-
berg, 2011; Wynn and Ramalingam, 2012). These activated
fibroblasts associated with cancer are referred to as CAFs or
tumor-associated fibroblasts (Kalluri and Zeisberg, 2006;
Mueller and Fusenig, 2004).
Although initially having an anti-tumorigenic pheno-
type as normal fibroblasts, CAFs eventually become pro-
tumorigenic through mechanisms that are still under intense
investigations (O
¨hlund et al., 2014; Senthebane et al., 2017).
The CAFs will eventually become the dominant stromal cell
type within the TME and promote tumor progression via
release of several factors and the synthesis of the ECM
(Hanahan and Weinberg, 2011; Kalluri, 2016; Senthebane
et al., 2017, 2018).
Irrespective of their cell of origin, CAFs are large spindle-
shaped cells showing increased stress fibers and well-
developed cellular-ECM connections (De Wever et al.,
2008). Although their shape appears identical to normal fi-
broblasts, CAFs show increased numbers of ribosomes and a
rough endoplasmic reticulum (De Wever et al., 2008). Early
markers used for positive identification of CAFs include
alpha-smooth muscle actin (a-SMA), vimentin, and desmin
(Lazard et al., 1993; Znaor et al., 2018). Unlike their normal
equivalent, CAFs demonstrate increased proliferation and
migration capacity and show increased expression of ECM
proteins and ECM degrading enzymes (Ma et al., 2009; Saadi
et al., 2010). The resulting remodeling of the TME, referred
to as desmoplasia, causes fibrosis and stiffening of the tissue
(Kalli and Stylianopoulos, 2018; Poltavets et al., 2018).
The presence of CAFs and tissue stiffening have been
associated with cancer relapse, implying that CAFs and
desmoplasia contribute to cancer progression and therapy
resistance (Laklai et al., 2016; Tsujino et al., 2007). Calvo
et al. (2013) demonstrated that ECM remodeling is a re-
quirement for CAFs’ continuous presence within the TME.
Thus, there is a feedback loop whereby CAFs build and
maintain the TME and remodeling of the ECM whereas the
remodeling is needed for both the generation and mainte-
nance of CAFs (Calvo et al., 2013; Maller et al., 2013).
The balance between ECM synthesis and degradation is
needed for homeostasis maintenance. ECM proteins such as
collagens and fibronectin can block immune cells from in-
filtrating into the tumor (Cukierman and Bassi, 2010). In
addition, the ECM provides the ‘‘theater’’ in which all the
cellular interactions take place; blood vessels are formed; as
well as cancer cells are allowed to escape immune detection
(Cukierman and Bassi, 2010; Gorski et al., 1994).
The CAFs promote angiogenesis through the release of
MMPs, which degrade the ECM and allow the release of
vascular endothelial growth factor-A (VEGF-A) sequestered
by the ECM (Baeriswyl and Christofori, 2009). The release of
VEGF-A promotes the formation of the vascular system,
allowing the tumor to grow large with enhanced exchange of
nutrients and toxic substances (Baeriswyl and Christofori,
2009; Raica et al., 2009). The CAFs are known to release
several growth factors and cytokines that are known to pro-
mote inflammation and to assist in the evasion of the immune
system (Flavell et al., 2010; Yang et al., 2016). In short, CAFs
are the builders and are involved in the maintenance of the
TME via synthesis and release of ECM proteins and protein
factors (De Wever et al., 2008; Hwang et al., 2008; Kalluri,
2016; Kalluri and Zeisberg, 2006; Senthebane et al., 2017, 2018).
In turn, the TME promotes tumor initiation, progression,
and metastasis (De Wever et al., 2008; Hanahan and Wein-
berg, 2011; Kalluri, 2016; Kalluri and Zeisberg, 2006; O
¨h-
lund et al., 2014). Increased knowledge on the origins and
role of CAFs within the TME may allow the development of
new anti-cancer strategies (Brechbuhl et al., 2017; Bussard
et al., 2016; Casey et al., 2008; Glentis et al., 2017).
Several studies show that CAFs originate from different
cell types and this has been suggested to cause the hetero-
geneity observed in these cells (De Wever et al., 2008; Kalluri
and Zeisberg, 2006; O
¨hlund et al., 2014; Senthebane et al.,
2017). Besides the activation of fibroblasts within the vicinity
of the cancer cells, CAFs can also originate from bone
marrow-derived fibrocytes and mesenchymal stem/stromal
cells (MSCs) that are recruited to the tumor; the transdiffer-
entiation of pericytes, smooth muscle cells, and adipocytes;
epithelial cells that underwent epithelial to mesenchymal
transition (EMT); endothelial cells that underwent endothe-
lial to mesenchymal transition (EndMT); and, finally, the
activation of quiescent stellate cells (Fig. 1) (O
¨hlund et al.,
2017; Omary et al., 2007; Yin et al., 2013).
As demonstrated in breast cancer, bone marrow-derived
fibrocytes that are usually inactive and present in circulation
can be recruited to the tumor and become CAFs over time
(Barth et al., 2002).
Our recent work showed that MSCs can be converted to
‘‘CAFs’’ over time through interaction with cancer cells
(Senthebane et al., 2017). We demonstrated that transfor-
mation of MSCs into CAFs was dependent on the release of
transforming growth factor-beta (TGF-b) by both cancer
cells and the MSCs (Senthebane et al., 2017). Several other
studies have also shown that MSCs can transform/differen-
tiate into CAFs in several cancers ( Jung et al., 2013; Zhu
et al., 2014). For example, Weber et al. (2015) demonstrated
that osteopontin mediates TGF-b-dependent transformation
of MSCs into CAFs in breast cancer.
Tissue-resident MSCs and those recruited from distant
tissues and organs can interact with cancer cells and be
transformed to CAFs. Quiescent stellate cells can also
transform into CAFs when activated and contribute to the
CAF population in the liver and the pancreas (Omary et al.,
2007; Yin et al., 2013). Epithelial cells within the vicinity of
cancer cells can undergo EMT and become CAFs under fi-
brotic conditions (Iwano et al., 2002). This means that
epithelial-derived cancers can end up with a huge population
of CAFs driving tumor progression. Endothelial cells can
also undergo EndMT and become CAFs (Zeisberg et al.,
2007). Both transformed epithelial cells and endothelial cells
express CAF markers, including S100A4 (Iwano et al., 2002;
Zeisberg et al., 2007).
Through a transdifferentiation process, cells such as adi-
pocytes and pericytes can become CAFs (Dulauroy et al.,
2012; Jotzu et al., 2011). It is, therefore, plausible to speculate
that all cells that are within the vicinity of cancer cells and
those that end up at the tumor site via circulation can poten-
tially be transformed into cells that will eventually promote
tumor growth such as CAFs. We are just beginning to get a
clear picture of the components of the TME and functions in
tumor initiation and progression, and detailed mechanistic
studies are likely to reveal interesting information.
It is now accepted that CAFs show heterogeneity with
distinct subsets of cells displaying different phenotypes and
316 DZOBO AND DANDARA
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functions within the TME (O
¨hlund et al., 2014, 2017; Orimo
and Weinberg, 2007; Su et al., 2018). Further, CAFs’ het-
erogeneity is also dependent on the stage of tumor develop-
ment (Huelsken and Hanahan, 2018). Normal fibroblasts are
generally considered genetically stable, whereas the same
cannot be said about CAFs. The process of transformation,
the generation of reactive oxygen species (ROS), and the
hypoxic conditions within the tumor can result in DNA al-
terations in CAFs as well, with CAFs co-evolving with
cancer cells as the tumor develops (Campbell et al., 2011;
O
¨hlund et al., 2014).
Although rare, alterations to the genetic material of CAFs
or stromal cells have been found in different cancers
(Moinfar et al., 2000; Patocs et al., 2007). Several available
techniques, including the use of CRISPR-Cas9 technology
and tracing experiments, can be used to determine whether
genetic alterations occur in CAFs as they do in cancer.
It is plausible to speculate that as the tumor evolves so do
the cells associated with it, such as CAFs. Our study clearly
demonstrates that previously anti-cancer activity of MSCs is
diminished over time and ‘‘transformed’’ cells eventually
become pro-tumorigenic (Senthebane et al., 2017). In addi-
tion, the synthesis of the ECM over time by both CAFs and
cancer cells results in combinatorial ECM, which is pro-
tumorigenic than normal ECM (Senthebane et al., 2018).
Clearly, more research and focused analysis of the TME
components is needed, especially the tumor-associated cells.
Given their possible cellular origin, it is not surprising that
CAFs display great phenotypic heterogeneity. Different
CAFs’ subsets have been identified within the TME and are
spatially distributed throughout the tumor (O
¨hlund et al.,
2014; Patocs et al., 2007). Recent characterization of CAFs
has identified specific biological markers for specific subsets
of CAFs (Kalluri, 2016; Vennin et al., 2019). In addition, no
CAF marker is expressed by all CAFs. Early studies identi-
fied markers such as a-SMA, fibroblast activation protein-
alpha (FAP-aor just FAP), and PDGF receptor-b(PDGFR-b)
to be expressed by different subsets of CAFs, with none being
expressed by all CAFs (Akrish et al., 2016; Kalluri, 2016). To
be useful, a combination of these markers is usually used
during CAFs’ characterization.
Among the markers, a-SMA has proven useful in the
identification of CAFs as well as a marker for smooth muscle
cells and pericytes (O
¨hlund et al., 2014). Senthebane et al.
(2017) used both vimentin and a-SMA as markers to study
cancer cell-derived TGF-b-mediated transformation of MSCs
to CAFs in vitro. Over time, the expression of both markers
was significantly increased in MSCs co-cultured with cancer
cells. Cancers in which CAFs express elevated levels of a-
SMA include breast, pancreatic, and liver cancers (Ayala
FIG. 1. CAFs can be derived from different cellular sources. Fibroblasts and stellate cells can be activated through various
processes to become CAFs; mesenchymal stem cells and fibrocytes are recruited to the tumor via circulation; epithelial cells
and endothelial cells undergo epithelial to mesenchymal transition and endothelial to mesenchymal transition, respectively,
to become CAFs. Further, pericytes and adipocytes are known to undergo transdifferentiation to become CAFs. CAFs,
cancer-associated fibroblasts; MSC, mesenchymal stem cell. Figure is adapted from our earlier publication, Senthebane
et al. (2017).
CAFSIN TUMOR MICROENVIRONMENT 317
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et al., 2003; Yin et al., 2013). However, several other cells,
including normal fibroblasts, smooth muscle cells, cardio-
myocytes, and pericytes, also express a-SMA. Mostly in-
volved in the maintenance of cellular cell structure, a-SMA is
also involved in the migration and contraction of cells.
Another CAF marker vimentin is highly expressed by
CAFs in breast and prostate cancers (Kalluri and Zeisberg,
2006; Vuoriluoto et al., 2011). We recently showed that vi-
mentin can be induced via TGF-b-mediated transformation
of MSCs (Senthebane et al., 2017). Biological functions of
vimentin include promotion of migration and maintenance of
cellular structure and integrity (Guo et al., 2013; Toivola
et al., 2005; Wang and Stamenovic, 2002). In contrast to a-
SMA and vimentin, desmin is mostly downregulated in CAFs
and is also expressed by fibroblasts, muscle cells, and peri-
cytes (Armulik et al., 2011; Eyden, 2008).
Caveolin-1 is a scaffolding protein expressed in CAF
subpopulations as well as in cells such as endothelial cells,
fibroblasts, and adipocytes. Low expression of caveolin-1 is
used as a marker of a CAF subpopulation undergoing meta-
bolic reprogramming and promoting tumorigenesis (Guido
et al., 2012). In contrast, elevated levels of caveolin-1 are
observed in CAFs with the propensity to promote metastasis
(Cohen et al., 2004; Goetz et al., 2011). This clearly dem-
onstrates the challenges involved in sorting and isolating of
CAFs, a necessary step to their characterization, with huge
ramifications to their therapeutic targeting.
Recently, CD10 and G-protein-coupled receptor 77
(GPR77) have been shown to be highly expressed in CAFs
and are involved in promoting cancer stemness and resistance
to chemotherapy in breast cancer cells (Su et al., 2018).
Illustrating the challenges faced by any CAFs-targeted ther-
apies, CD10 and GPR77 are also expressed in bone marrow-
derived stromal cells and polymorphonuclear neutrophils,
respectively (Karnoub et al., 2007; Su et al., 2018).
S100A4, sometimes referred to as fibroblast-specific pro-
tein, is expressed by fibroblasts within tumors and displays
serine protease activity, allowing it to remodel the ECM
(Zhang et al., 2013a). Fibroblasts expressing S100A4 are
known to protect cancer cells via ECM production. Cells ex-
pressing S100A4 are known to be highly malignant and dis-
play a propensity for migration (Wang et al., 2005); for
example, S100A4 is highly expressed in CAFs in breast can-
cers (Orimo et al., 2005).
This marker is also expressed by other cells, including cells
undergoing EMT, macrophages, and normal fibroblasts
(Li et al., 2010; Orimo et al., 2005; Zhang et al., 2013a).
PDGFR-bis a CAF marker that has been targeted with kinase
inhibitors (Pietras et al., 2008). PDGF receptor signaling
inhibition with Imatinib was shown to abrogate malignant
progression of cervical lesions (Pietras et al., 2008). CAFs
found in colorectal and cervical cancers express high
levels of PDGFR-b(Pen
˜a et al., 2013; Pietras et al., 2008).
Macrophages have been shown to cause immune sup-
pression through the expression of FAP (Arnold et al.,
2014). The FAP is expressed in many human cancers and
is highly expressed in CAFs (Arnold et al., 2014; Huber
et al., 2003).
In summary, CAFs are a heterogeneous population of cells
clearly demonstrating their diverse cellular origin and have
been shown to have many functions within the solid tumor.
A major challenge facing scientists today is the identification
of reliable cell surface markers that can be used for thera-
peutic targeting. New data demonstrating that CAFs’ func-
tion is dependent on the location within the tumor amplify the
challenge faced (O
¨hlund et al., 2014, 2017). Irrespective of
cell of origin, tumor stage, and location within the tumor,
recent studies emphasize the need to identify cancer-specific
CAF markers or CAF subset markers for use in diagnosis and
anti-cancer targeting.
CAFs and Carcinogenesis
Data from several studies illustrate the supporting role of
TME components in tumor progression, metastasis, and for-
mation of new tumors (Bonnans et al., 2014; Cukierman and
Bassi, 2010; Senthebane et al., 2017). Although initially be-
ing prohibitive of tumor growth, we demonstrated that over
time cells within the TME promote tumor growth via the
release of growth factors and deposition of ECM (Nicosia
et al., 1993; Senthebane et al., 2017, 2018).
Several other studies confirmed that normal fibroblasts
show inhibitory effects on cancer cell growth in vitro (Stoker
et al., 1966). The expression of phosphatase and tensin ho-
molog in stromal cells was shown to be necessary for this
inhibition effect on epithelial tumors (Trimboli et al., 2009).
Several growth factors and cytokines, including vascular
endothelial growth factor (VEGF), stromal derived factor 1
(SDF-1), TGF-b, and interleukin-6 (IL-6), have been shown
to be necessary in the transformation of normal stromal cells
into cancer-supporting cells (Giannoni et al., 2010; Senthe-
bane et al., 2017).
In addition, conditions within the TME such as hypoxia
and ROS within the TME milieu also contribute to the con-
version of normal cells to pro-tumorigenic cells (Maxwell
et al., 1997; Ryan et al., 2000). Through the expression and
secretion of growth factors and cytokines and their receptors,
CAFs impact cancer cell-associated processes such as in-
flammation, angiogenesis, migration, invasion, chemoresis-
tance, and immune evasion (Fig. 2) (Casey et al., 2008; San
Francisco et al., 2004; Wang et al., 2017). Erez et al. (2010)
demonstrated that normal fibroblasts can be induced to ex-
press inflammatory genes by carcinoma cells.
microRNAs (miRNAs) are also believed to be involved in
the transformation of normal fibroblasts into CAFs. miRNAs
are delivered to their target cells via exosomes, allowing
miRNAs to induce and transform local and distant fibroblasts
(Kahlert and Kalluri, 2013; Webber et al., 2010). A recent
study by Fang et al. (2018) demonstrated that exosomal
delivered miRNA-1247 from cancer cells induces the trans-
formation of fibroblasts within lung premetastatic environ-
ments, thereby promoting metastasis.
Mitra et al. (2012) also demonstrated that cancer cells re-
program fibroblasts to CAFs via the release of miRNAs in
ovarian cancer. Pang et al. (2015) also showed that cancer
cell-derived miRNA-155 was involved in the transformation
of normal fibroblasts into CAFs in pancreatic cancer. It is
important to note that the involvement of miRNAs is bi-
directional, with stromal cells also affecting cancer cells via
the release of micro-RNAs (Musumeci et al., 2011; Nouraee
et al., 2013). The hypoxic conditions of the TME result in the
generation of ROS. Toullec et al. (2010) demonstrated that
ROS within the TME induce the expression of hypoxia in-
ducible factor 1-a(HIF1-a) and CXC-chemokine ligand 12
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by stromal fibroblasts, allowing the cells to have enhanced
metabolism and migratory capabilities.
Albrengues et al. (2015) showed that there is an epigenetic
switch that drives the transformation of normal fibroblasts into
CAFs. Leukemia inhibitory factor, a member of the IL-6 su-
perfamily, was shown to initiate the epigenetic switch, leading
to the activation of the Janus kinase–signal transducer and
activator of transcription (JAK-STAT) signaling (Albrengues
et al., 2015). The continuous activation of the JAK-STAT
signaling leads to CAFs promoting cancer cell invasive be-
havior. Several other studies have shown the importance of the
JAK-STAT signaling in the maintenance of the CAF pheno-
type (Gao etal., 2009; Huynh et al., 2017; Tan et al., 2019). The
continuous interaction between CAFs and cancer cells means
that during tumor progression CAFs also change, with CAFs
demonstrating heterogeneity at both phenotype and function
level (Bruzzese et al., 2014; Scherz-Shouval et al., 2014).
The growth and transformation of normal epithelial cells is
driven partly by CAFs (Olumi et al., 1999). Olumi et al.
(1999) demonstrated that CAFs and not normal fibroblasts
were responsible for the transformation as well as cancer
initiation and growth of prostatic epithelial cells. Ku-
perwasser et al. (2004) also demonstrated that TGF-bfrom
CAFs was necessary for tumor formation from epithelial
cells, a result that was in contrast to the use of normal fi-
broblasts. In addition, Shekhar et al. (2001) demonstrated that
normal fibroblasts inhibited tumor growth and the tumori-
genic transformation of epithelial cells.
Implantation of tumor cells and CAFs together allowed
enhanced tumor growth compared with co-implantation of
tumor cells and normal fibroblasts (Picard et al., 1986).
Several studies have shown the involvement of both cancer
cell-derived and CAF-derived TGF-band heat shock factor-1
in driving tumor cell growth and metastasis (Hawinkels et al.,
2014; Scherz-Shouval et al., 2014; Senthebane et al., 2017).
Using both esophageal and breast cancer cells, we demon-
strated that both CAFs and cancer cells released TGF-b,
which promoted the transformation of fibroblasts in addition
to promoting tumor growth (Senthebane et al., 2017). Several
cancer cells have been shown to express TGF-breceptors on
their cell surfaces, supporting the notion that cancer cells
respond to both autocrine and paracrine TGF-bsignaling
(Calon et al., 2014; Li et al., 2005).
Besides releasing growth factors, CAFs also influence tu-
mor growth through the release of enzymes such as MMPs
and urokinase-type plasminogen activator (Danø et al., 2005;
Deryugina and Quigley, 2015; Egeblad and Werb, 2002;
Martens et al., 2003; Taguchi et al., 2014).
Although further research into the role of CAFs into tumor-
igenesis is required, there is some evidence that CAFs induce
epithelial cell transformation (Wang et al., 2018). To sustain
tumor growth, new blood vessels are formed and these supply
all the nutrients required by cancer cells as well as remove toxic
substances from the tumor (Carmeliet and Jain, 2011).
CAFs release SDF-1, which has been shown to recruit
endothelial progenitor cells, leading to formation of new
blood vessels within the TME (Liu et al., 2019a; Orimo et al.,
2005). CAFs are known to release several other angiogenic
factors such as PDGF-C, VEGF-A, and secreted frizzled-
related protein-2 (SFRP2) (De Palma et al., 2017; Liekens
FIG. 2. Effect of CAFs on tumor cells, other stromal cells, and the ECM within the TM. The CAFs synthesize and secrete
the ECM, MMPs, and various factors, impacting cancer cell processes. Most of the secreted factors are growth factors and
cytokines, including HGF, FGF, TGF-b, IL-6, IL-1, COX-2, VEGF, HIF1-a, PDGF, and SDF-1, and they impact tumor
processes such as angiogenesis (1), chemoresistance (2), proliferation (3), and ECM remodeling (4). ECM, extracellular
matrix; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; HIF1-a, hypoxia inducible factor 1-a; IL-6,
interleukin-6; MMPs, matrix metalloproteases; PDGF, platelet-derived growth factor; SDF-1, stromal-derived factor 1;
TGF-b, transforming growth factor-beta; TIMP, tissue inhibitor of metalloproteinases; TME, tumor microenvironment;
VEGF, vascular endothelial growth factor.
CAFSIN TUMOR MICROENVIRONMENT 319
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et al., 2010). Through the synthesis of ECM proteins and
generation of ECM stiffness, CAFs influence blood vessel
formation and also the flow of blood through the tumor
(Egeblad et al., 2010; Senthebane et al., 2018; Twardowski
et al., 2007).
Most early-stage epithelial tumors have a basal lamina that
acts as a barrier between the tumor and the vascular system
(Bluff et al., 2009; Bossi et al., 1995). This results in early-
stage tumors lacking proper supply of nutrients and removal
of toxic substances. Migration and invasion of tumor cells
into the surrounding tissues induce angiogenesis as well
(Bergers and Benjamin, 2003). The induction of angiogenesis
is coupled to infiltration of leukocytes, proliferation and ac-
tivation of fibroblasts, as well as increased deposition of
ECM proteins (Dai et al., 2009; De Palma et al., 2017; Neve
et al., 2014; Wang et al., 2011; Zhou et al., 2006). Depending
on the tumor type, stromal components, and site in the human
body, vascularization can take different forms and patterns
(Bluff et al., 2009; Fukumura et al., 1997; Jubb et al., 2011;
Monsky et al., 2002).
In addition, the balance of expression of anti-angiogenic
factors and pro-angiogenic factors will ultimately play a key
role in determining both formation and pattern of blood
vessels within the tumor (Bussolino et al., 1991; Monsky et al.,
2002; Morrissey et al., 2008; Murakami and Simons, 2008).
Ultimately, pro-angiogenic factors and signaling are
eventually upregulated during tumor formation, leading to
aberrant and dysregulated formation of blood vessels (Ber-
gers and Benjamin, 2003; Castello et al., 2017). CAFs to-
gether with infiltrating leukocytes, macrophages, and other
stromal cells such as pericytes are known to be the major
sources of pro-angiogenic factors such as VEGFA, PDGFC,
fibroblast growth factor 2 (FGF2), Osteopontin, and MMPs
(Table 1, below) (Anderberg et al., 2009; Crawford et al.,
2009; Deroanne et al., 1997; Fukumura et al., 1998; Gerber
et al., 2000; Morikawa et al., 2002; Zhang et al., 2017; Zhao
et al., 2014).
For blood vessels to be formed there has to be the right
tumor or TME elasticity and stiffness (Egeblad et al., 2010;
Senthebane et al., 2017). The CAFs synthesize the TME
ECM; whereas enzymes released by CAFs such as MMPs,
hydroxylases, and lysyl oxidases influence both ECM syn-
thesis and degradation and, therefore, control biophysical
properties of the TME (De Palma et al., 2017; Dzobo et al.,
2012, 2014, 2018a; Liu et al., 2019b; Taguchi et al., 2014;
Twardowski et al., 2007). Growth factors such as PDGFC act
on CAFs in an autocrine manner to induce the secretion of
FGF2 as well as osteopontin (Anderberg et al., 2009; Liu
et al., 2019a; Pietras et al., 2008).
As the tumor grows in size, cancer cells migrate and invade
surrounding tissues. When cancer cells breach the basement
membrane, they enter into circulation, travel to distant tissues
and organs, and, eventually, extravasate into new tissues, a
process called metastasis. It is important to note that only a
few cancer cells survive the arduous journey to new tissues
and organs, but once there, these cancer cells colonize and
form new tumors, metastases (Chaffer and Weinberg, 2011;
Gupta and Massague
´, 2006). Grum-Schwensen et al. (2005)
demonstrated that tumors without stromal cell-derived mts1
protein do not metastasize, demonstrating the importance of
stromal cells in tumor growth and spread.
Several studies have shown that besides stromal-derived
growth factors, several cytokines are also released by stromal
cells and aid cancer cell metastasis (Chow and Luster, 2014;
Guo and Deng, 2018; Valkenburg et al., 2018). IL-6 has re-
ceived a lot of attention and has been shown to help cancer
Table 1. Several Angiogenic Factors Are Produced by Cancer-Associated Fibroblasts, Cancer-Associated
Macrophages, and Cancer-Associated Neutrophils and Influence Vascularization Within
the Tumor Microenvironment
Angiogenic factor Cellular source Effect on vascular system Effect on vascular system
VEGFA CAFs, CAMs, CANs Pro-angiogenic, recruits other
cells such as myeloid cells,
ECM production
Fukumura et al. (1998); Gerber
et al. (2000); Kalluri (2016)
PDGFC CAFs Pro-angiogenic Anderberg et al. (2009); Crawford
et al. (2009); Pietras et al.
(2008)
FGF CAFs, CAMs, CANs Pro-angiogenic Deroanne et al. (1997); Murakami
and Simons (2008)
CXCL12 CAFs, CAMs Pro-angiogenic, recruits
monocytes, endothelial cells
Liekens et al., 2010; Orimo et al.
(2005); Zhang et al. (2017)
CSF3 CAFs Pro-angiogenic Bussolino et al. (1991); Zhao et al.
(2014)
Collagen CAFs Pro-angiogenic Neve et al. (2014); Twardowski
et al. (2007); Zhou et al., 2006)
Fibronectin CAFs Pro-angiogenic Mongiat et al. (2016); Nicosia
et al. (1993)
Osteopontin CAFs Pro-angiogenic Castello et al. (2017); Dai et al.
(2009); Wang et al. (2011)
MMPs CAFs, CAMs, CANs Pro-angiogenic, creates space
for blood vessel formation
Deryugina and Quigley (2015);
Liu et al. (2019a); Taguchi et al.
(2014)
CAFs, cancer-associated fibroblasts; CAMs, cancer-associated macrophages; CANs, cancer-associated neutrophils; ECM, extracellular
matrix; FGF, fibroblast growth factor; MMPs, matrix metalloproteases; PDGFC, platelet-derived growth factor C; VEGFA, vascular
endothelial growth factor-A.
320 DZOBO AND DANDARA
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cells metastasize to the bone (Tawara et al., 2011). Chang
et al. (2013) showed that IL-6 and its downstream signaling
cascades such as JAK-STAT are involved in breast cancer
tumorigenesis and metastasis.
The CAF-derived SDF1 has been shown to aid breast
cancer cells home to the bone and aid in adaptation to this
new environment (Al-Ansari et al., 2013; Zhang et al.,
2013b). The release of stanniocalcin-1 (STC1) by CAFs
drives colon cancer metastasis (Pen
˜a et al., 2013). TGF-bhas
been implicated in many cellular processes, and Yu et al.
(2014) demonstrated that TGF-bcan induce EMT in cancer
cells. Both TGF-band hepatocyte growth factor (HGF) have
been shown to promote invasiveness in esophageal cancer
(Grugan et al., 2010; Wang et al., 2016).
By releasing huge amounts of MMPs as well as synthesizing
the ECM, CAFs can remodel the TME and allow cancer cells to
migrate and invade surrounding tissues. Increase in collagen
levels within the TME has been shown to promote cancer cell
invasiveness (Provenzano et al., 2008; Vellinga et al., 2016).
Degradation of the ECM by CAF-derived MMPs can create
highways through which cancer cells can migrate to other tis-
sues and organs (Glentis et al., 2017; Senthebane et al., 2018).
In addition, ECM stiffening is enhanced via activation of YAP1
in CAFs and has been shown to allow cancer cell invasion and
formation of tumor blood vessels (Calvo et al., 2013).
Goetz et al. (2011) demonstrated that CAF-driven bio-
mechanical remodeling of the TME can allow migration and
invasion of cancer cells. Once cancer cells reach their new
environment, it has to be remodeled to suit their needs or the
cancer cells adapt to the new conditions. As postulated by
Paget in the ‘‘seed and soil’’ theory, the new microenviron-
ment must allow cancer cells to flourish (Paget, 1889).
Kaplan et al. (2005) suggested that a premetastatic envi-
ronment must be rich in ECM proteins such as fibroblast-
derived fibronectin. In addition, stromal cell-derived factors
including TGF-band SDF1 within the premetastatic regions
may act as attractants to cancer cells (Calon et al., 2012;
Kaplan et al., 2005). Resident fibroblasts within the pre-
metastatic regions promote blood vessel formation, allowing
tumors to grow (Calon et al., 2012; O’Connell et al., 2011). It
is plausible to suggest that these fibroblasts and any other
stromal cells associated with metastatic cancer cells are res-
ident cells within the colonized regions.
The Role of CAFs in Therapy Resistance
There are two possible ways through which cancer cells
can be resistant to therapy. First, therapy resistance can be
intrinsic. The expression of several transporter proteins such
as the ABC proteins and other genetic alterations can influ-
ence drug uptake and export at the cellular levels. Second,
tumors are always evolving and this can result in previously
responsive tumors to become irresponsive. The TME has
emerged as a contributor to therapy resistance via the actions
of stromal cells and the ECM. Excessive synthesis of the
ECM by CAFs can limit drugs’ access to cancer cells and
other stromal cells. The ECM around cancer cells can form a
hindrance to drugs and can limit blood vessel formation
within the tumor (Olive et al., 2009; Zhao et al., 2018).
The binding of cancer cells to CAF-derived ECM proteins
such as fibronectin, a process called cell-adhesion-mediated
drug resistance, also aids cancer cells in avoiding drugs, thus
causing drug resistance (Damiano et al., 2001; Hazlehurst
et al., 2000). The versatile IL-6 cytokine has been implicated
in resistance to chemotherapy in lung cancer (Shintani et al.,
2016). Although it is understandable that cancer cells can
develop therapy resistance, CAFs have also been reported to
be resistant to several drugs, including gemcitabine in pan-
creatic cancer (Richards et al., 2017). Communication be-
tween CAFs and cancer cells also includes exosomes, which
transport several factors and miRNAs from CAFs to cancer
cells (Richards et al., 2017).
Most chemotherapeutic drugs target rapidly growing
cancer cells, but they cannot effectively clear all cancer cells
during treatment. In addition, chemotherapy induces DNA
damage in stromal cells, resulting in the release of several
factors that promote cancer cell survival (Huber et al., 2015).
Further, several studies demonstrated that chemotherapy can
induce fibroblasts to become CAF-like cells that promote
stem cell behavior in cancer cells (Peiris-Page
`s et al., 2015a,
2015b). Radiotherapy is known to induce the synthesis of
large amounts of ECM proteins, resulting in the survival of
cancer cells (Barker et al., 2015; Eke and Cordes, 2011;
Erkan et al., 2007). When irradiated, stromal cells can in-
crease the secretion of factors involved in chemoresistance
(Chargari et al., 2013).
The EMT process is used by cancer cells to transform and
reduce the expression of many cellular membrane proteins
such as drug transporters (Zheng et al., 2015). The net effect
of reduced drug transporter expression is reduced drug in-
take, ultimately leading to therapy resistance. The EMT is a
process that is activated by several signaling pathways and
occurs in normal events such as embryonic development.
Unfortunately, it also occurs during neoplastic conversion
of cells as well as fibrosis (Arumugam et al., 2009; Hotz et al.,
2007). Cells undergoing EMT gradually lose epithelial cell
junction proteins and begin to synthesize more vimentin, be-
coming more migratory, invasive, and chemoresistant (Ar-
umugam et al., 2009; Hotz et al., 2007). Several studies have
shown enhanced vimentin, Snail, and ZEB1expression in cells
that are chemoresistant (Guaita et al., 2002; Taube et al., 2010;
Wellner et al., 2009). Zheng et al. (2015) demonstrated that
although EMT is not necessary for metastasis, it induces the
development of chemoresistance in pancreatic cancer.
In pancreatic cancer, resistance to gemcitabine is asso-
ciated with EMT (Vega et al., 2004; Wellner et al., 2009;
Zhang et al., 2010a). Poor survival of pancreatic patients
has been associated with an increased EMT program that
causes an impaired response to chemotherapy (Hidalgo,
2010; Kleger et al., 2014). Even more sinister is the seem-
ingly ability of CAFs to uptake and accumulate high con-
centrations of drugs within the TME, resulting in a reduced
amount of drugs eventually reaching cancer cells (Hess-
mann et al., 2018). Chemotherapy can kill cancer cells via
production of ROS. The CAFs have been shown to abrogate
ROS production and therefore prevent death of cancer cells
when exposed to chemotherapy (Cheteh et al., 2017).
Several survival pathways such as MEK-ERK and PI3K-
Akt are known to be activated in cancer cells via the action of
CAF-derived HGF and cause cancer cell resistance to RAF
inhibitors (Straussman et al., 2012). In elaborate experiments,
Senthebane et al. (2017) demonstrated that CAF-derived
TGF-bplays a role in the resistance of breast and esophageal
cancer cells to paclitaxel and cisplatin. Thus, the CAF
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secretome plays a huge role in the development of therapy
resistance in cancer cells.
The presence of cancer stem cells (CSCs) within the TME
is still debatable, but several studies have shown that these
stem-like cells are able to resist therapies (Codd et al., 2018;
Dawood et al., 2014; Nassar and Blanpain, 2016; Steinbichler
et al., 2018; Yu et al., 2007a). Several studies have demon-
strated that CAFs subsets express drug transporter proteins
such as ABC transporters and also provide a protective en-
vironment to CSCs within the TME (Begicevic and Falasca,
2017; Domenichini et al., 2019; Su et al., 2018). CAF-derived
cytokines such as IL-6 are responsible for maintenance of the
CSC phenotype (Ebbing et al., 2019).
Microvesicles from CAF also deliver micro-RNAs to
generate CSCs in breast cancer (Sansone et al., 2017).
Overall, studies have demonstrated that CAFs or their sub-
sets promote therapy resistance in cancer cells via various
mechanisms from accumulating large amounts of drugs, re-
lease of protein factors, and mi-RNAs-laden exosomes to
providing physical barriers to therapy. The combined tar-
geting of these CAFs or their subsets together with cancer
cells may offer durable treatment solutions for many cancers.
CAFs and Other Stromal Cell Interactions
The interaction between TME components is through cell-
to-cell adhesions, release of growth factors, and cytokines
and indirectly through exosomes. In addition, cells interact
with the ECM via surface receptors such as integrins.
Crosstalk between stromal cells is inevitable given the
compact nature of most solid tumors. The most abundant
immune cells within the TME are the macrophages, referred
to as cancer-associated macrophages (CAMs) or tumor-
associated macrophages (TAMs).
As reported by Comito et al. (2014), CAFs and TAMs
work together to promote tumor progression in prostate
cancer. Communication between the two types of cells occurs
via the release of CXCL14 by CAFs, resulting in the re-
cruitment of TAMs to the tumor site and subsequent differ-
entiation (Comito et al., 2014). In turn, M2 TAMs are known
to activate CAFs and therefore further promote tumorigenic
growth. Hashimoto et al. (2016) demonstrated that CAFs
derived from bone marrow MSCs promote macrophage in-
vasiveness and transformation and, in turn, the resulting
TAMs can prompt the proliferation of CAFs. By working
together, these CAFs and TAMs can promote tumorigenic
growth of neuroblastoma (Hashimoto et al., 2016).
Myeloid cells within tumors can increase the expression of
S100A8 in response to IL-6 released by CAFs in colon cancer
(Kim et al., 2012). Myeloid cells within tumors can also
differentiate into suppressor cells that are linked to suppres-
sion of immunity (Kim et al., 2012). For example, circulating
myeloid-derived suppressor cells are recruited to tumors via
the release of FAP by CAFs and these cause the suppression
of immunity in hepatic cancer (Yang et al., 2016).
The CAFs are known to express the FAS ligand and this is
known to cause apoptotic death of CD8-positive T cells
(Lakins et al., 2018). Further, CAFs express programmed
cell death 1 ligand 2 (PD-L2), which causes the functional
inactivation of T cells (Lakins et al., 2018). Our study and
several others have shown that CAFs secrete huge amounts
of TGF-b, which has been suggested to cause immunosup-
pression and can induce transformation of immune cells into
immune-suppressing cells (Flavell et al., 2010; Gutcher
et al., 2011; Senthebane et al., 2017). Recently, Mariathasan
et al. (2018) provided further evidence of the involvement of
TGF-bin abrogating antitumor immunity in bladder cancer.
The CAFs attract and sequester CD8-positive T cells and
this prevents T cells from binding to and killing cancer cells
(Ene–Obong et al., 2013).
The net effect of all this is the suppression of anti-tumor
activity of immune cells. The removal of FAP-positive
CAFs was shown to reactivate the anti-tumor activity of
immune cells of T cells (Kraman et al., 2010). Overall, CAFs
are more likely to promote tumorigenic growth through their
effect on immune cells within the TME. Several growth
factors are known to be sequestered by the ECM and these
include VEGFA and TGF-b. Through production of matrix
metalloproteases (MMPs), CAFs can degrade the ECM and
cause enhanced levels of free growth factors within the TME
(Bonnans et al., 2014). Thus, through recruiting endothelial
cells in addition to releasing VEGFA, CAFs promote tumor
vascularization (Bonnans et al., 2014). The interaction of
CAFs and other cells, therefore, creates a tumor environ-
ment that promotes immune evasion, inflammation, and
vascularization.
As reported by Senthebane et al. (2017), CAFs and other
TME components can also display anti-tumorigenic effects,
especially during the early stages of tumor development. Our
data revealed that normal fibroblast-derived ECM limits
cancer cell proliferation and migration compared with an
ECM from fibroblasts co-cultured with cancer cells (Sen-
thebane et al., 2018). Further evidence of the involvement of
CAFs or their normal counterparts in anti-tumorigenic be-
haviour comes from studies of breast cancer. Brechbuhl et al.
(2017) reported two subsets of CAFs in breast cancer, with
one subset expressing CD146 and conferring sensitivity to
tamoxifen, whereas the CD146- subset confers tamoxifen
resistance. The removal of CAFs expressing a-SMA from
pancreatic ductal adenocarcinoma (PDAC) results in the
acceleration of cancer and reduced survival (O
¨zdemir et al.,
2014; Rhim et al., 2014).
With this in mind, novel therapeutic strategies aimed at
CAFs have to be selective and cognizant of the CAFs’ phe-
notypic and functional heterogeneity. Mere targeting of
CAFs for removal based on presumed pro-tumorigenic be-
havior will be detrimental and can lead to fatal disease. The
CAF subsets must be identified and only targeted therapy
should be used. In addition, it might be helpful to re-educate
or re-direct the pro-tumorigenic CAFs into anti-tumorigenic
CAFs. To be able to do this, it is important that specific
markers for anti-tumorigenic and pro-tumorigenic CAFs be
identified to distinguish the two.
Advances in Therapeutic Targeting of CAFs
CAF-directed therapies
It has long been recognized that manipulation of CAFs’
functions, especially the tumorigenic promotion part, have
therapeutic value in cancer treatment. In theory, targeting
CAFs appear easy since they are the major stromal compo-
nents of the tumor. In addition, CAFs have been described as
genetically stable, making them a better target than cancer cells
in cancer immunotherapy (Ostermann et al., 2008). In practice,
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however, major challenges need to be overcome before this
promising avenue is utilized in cancer treatment.
First, targeting CAFs will have a negative effect on normal
tissue and other components of the TME. For example, the
depletion of CAFs can result in less ECM production, cre-
ating highways through which cancer cells escape and me-
tastasize to other tissues and organs. Second, the removal of
CAFs itself suffers from the lack of specific markers that can
be used to achieve that. Third, and probably most challenging
is the existence of CAFs subsets displaying phenotypic and
functional heterogeneity. To overcome this requires the
identification of more than one specific markers of each
subset that can be used for targeting that subset.
Several approaches have been devised to target CAFs
within the TME. First, the activation and trans-differentiation
of stromal cells into CAFs can be targeted and inhibited.
Since CAFs influence both tumor cells and stromal cells via
synthesis and secretion of growth factors and cytokines, in-
hibition of this synthesis and secretion is under intense
scrutiny, with the aim of inhibiting CAF function. Second,
CAFs’ normalization through the use of several molecules
has been tried.
For example, all-trans-retinoic acid (ATRA) can be used to
induce quiescence in CAFs or activated fibroblasts/stromal
cells, preventing aberrant secretion of growth factors and
cytokines. Although CAFs are pro-tumorigenic, they can be
used as carriers of drugs to kill cancer cells. Together with
other cells such as CAMs, MSCs, and pericytes, CAFs can
carry anti-cancer viruses and apoptosis-inducing ligands.
Several studies, including our recent publication, have
demonstrated that MSCs can be transformed into CAFs
(Borriello et al., 2017; Chan et al., 2019; Ridge et al., 2018;
Senthebane et al., 2017; Tan et al., 2019). Senthebane et al.
(2018) demonstrated the influence of the ECM in tumor
growth and chemoresistance.
Importantly, the presence of ECM proteins, including type
I collagen and fibronectin, was shown to affect drug delivery
to cancer cells, with the knockdown of both ECM proteins
showing increased drug-induced cancer cell death (Senthe-
bane et al., 2018). Previous studies implicated signaling
pathways such as the MEK-ERK and MMPs in regulating
ECM synthesis (Dzobo et al., 2012, 2014). The ablation of
stromal ECM can, therefore, be used to increase cancer cell
sensitivity to anti-cancer drugs. Direct targeting of CAFs or
their subsets is still challenging, with difficulties in the
identification of markers to be used to identify the CAFs.
Currently, immunotherapy, use of immunotoxins, and DNA
vaccines can be used to target CAFs, with the duly still out on
the effectiveness of such therapy.
Senthebane et al. (2017) recently utilized both vimentin
and a-SMA as markers to identify MSC-derived CAFs. Other
studies have shown that a-SMA only identifies a specific
subset of CAFs (Choi et al., 2013; Ding et al., 2014; O
¨zdemir
et al., 2014). O
¨zdemir et al. (2014) described that the removal
of a-SMA-positive CAFs in pancreatic ductal adenocarci-
noma (PDAC) was linked to decreased angiogenesis. The
same authors observed that angiogenesis decreased whereas
there was increased hypoxia within solid tumors leading to
increased EMT and the presence of CSCs (O
¨zdemir et al.,
2014). Several lung and colon cancer animal models have
shown that removal of FAP from within solid tumors is as-
sociated with decreased tumor growth (Santos et al., 2009).
Santos et al. (2009) demonstrated that a preclinical FAP
inhibitor, PT630, can inhibit the growth of tumors as well as
stromagenesis in lung and colon cancers. Similarly, the re-
moval of FAP in PDAC also reduced tumor growth (Feig
et al., 2013). Ostermann et al. (2008) developed a monoclonal
antibody against FAP that they conjugated to maytansinoid
and this treatment has been used in pancreatic, head, and neck
cancers where it has shown its effectiveness.
In addition, extensive experiments by Loeffler et al. (2006)
using a mouse DNA vaccine against FAP resulted in ablation
of CAFs and enhanced drug uptake by tumors. The DNA
vaccine induced a CD8
+
T cells to kill a large part of the
CAFs expressing FAP. Further evidence for the utility of the
DNA vaccine against FAP-positive CAFs was provided by
Reisfeld (Reisfeld, 2007). The utility of chimeric antigen
receptor T (CAR T) cell treatment for solid tumors is still to
be proven.
Several reports, however, show that targeting FAP-positive
CAFs using CAR T cells results in antitumor activity (Kakarla
et al., 2013; Lo et al., 2015; Wang et al., 2014). However,
CAFs are not the only cells expressing FAP. Bone marrow-
derived MSCs are also known to express FAP (Chung et al.,
2014). Thus, it is possible that anti-tumor CAR T cells will also
target other cells. Although reports show that CAR T cells
targeting FAP-positive CAFs are able to kill cancer cells in
lung and pancreatic cancers (Lo et al., 2015), Roberts et al.
(2013) demonstrated that CAR T cells killed bone marrow-
derived stem cells and can cause cachexia and anemia.
Increased knowledge and analytical tools have enabled
scientists to identify new and better surface markers for
CAFs. For example, Su et al. (2018) were able to characterize
tumorigenic CAF subsets based on the expression of CD10
and GPR77. Used together with FAP and a-SMA, these new
surface markers are likely to define specific CAF subsets,
leading to their killing.
The same authors reported reduced cancer cell stemness
when GPR77 was blocked (Su et al., 2018). Several strategies
are being developed to target cellular sources of CAFs.
Senthebane et al. (2017) demonstrated that it is possible that
MSCs are transformed into CAFs through their interaction
with cancer cells. Thus, targeting MSCs may prevent the
accumulation of CAFs in tumors, reducing pro-tumorigenic
cells within the tumor. The removal of pro-tumorigenic
support is likely to reduce tumor growth and metastasis.
Several clinical trials have been undertaken to target CAFs
sources with the duly still out on their effectiveness.
The re-education of activated CAFs has been suggested as
a strategy to direct CAFs from being pro-tumorigenic to anti-
tumorigenic. The inactivation of CAFs can induce quies-
cence, with the resulting CAFs or fibroblasts dividing slowly
and releasing normal levels of growth factors and other fac-
tors. Akin to quiescent satellite cells in muscle, these re-
educated CAFs can attain a tumor-suppressive phenotype.
The induction of quiescence in pancreatic stellate cells in
PDAC through the use of ATRA reverts the activated cells to
normal fibroblasts and arrests tumor growth (Froeling et al.,
2011). The mechanism of action of ATRA involves inhibition
of the versatile Wnt-b-catenin signaling cascade (Froeling
et al., 2011).
Inactivated pancreatic stellate cells also release seques-
tered CD8
+
T cells, allowing infiltration into PDAC and re-
sulting in tumor growth arrest (Ene–Obong et al., 2013).
CAFSIN TUMOR MICROENVIRONMENT 323
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Carapuca et al. (2016) showed that coupling ATRA, a vita-
min A analogue, and gemcitabine can be effective at treating
PDAC in animal models through inhibition of several path-
ways, including Wnt-b-catenin and Hedgehog signaling.
Sherman et al. (2014) demonstrated that global reprogram-
ming of the stroma through the use of calcipotriol, a vitamin
D receptor ligand, and gemcitabine resulted in reduced in-
flammation and ECM synthesis in PDAC tumors. Reduced
ECM synthesis allowed better delivery of gemcitabine into
the tumor, leading to tumor regression (Sherman et al., 2014).
Both ATRA and calcipotriol are at the preclinical stage of
investigations. Thus, depending on the tumor type, different
strategies from ablation of CAFs to normalization of acti-
vated CAFs may be undertaken to achieve better cancer
treatment. This provides a new avenue of cancer treatment
that focuses not just on cancer cells but on the stromal
component as well. Combinations of strategies focused on
cancer cells and stromal cells may offer a durable cure for
cancer treatment.
Targeting signaling pathways activated in CAFs
and their downstream effectors
One of the remaining challenges to CAFs’ ablation and
normalization is the huge heterogeneity observed in many
tumors, meaning some CAFs subsets will remain unaltered
by treatment. Instead of targeting the CAFs, several ap-
proaches have been developed to target signaling pathways
activated in CAFs and factors released by tumor and stromal
cells. Tumor cells and stromal cells release huge amounts of
factors during tumor initiation and progression (Senthebane
et al., 2017, 2018; Sullivan et al., 2010). Senthebane et al.
(2017) demonstrated that TGF-breleased by tumor cells and
transforming MSCs can be involved in the transformation of
stromal cells into potential CAFs. The same study also
showed that once transformed, activated CAFs release in-
creased levels of the same growth factor, TGF-b, which is
involved in tumor growth and development of chemoresis-
tance (Senthebane et al., 2017).
One signaling cascade that has been studied in detail in
different cellular processes is the IL-6-JAK-STAT pathway
(Giannoni et al., 2010; Sansone and Bromberg, 2012; Steyn
et al., 2019). Sanz-Moreno et al. (2011) showed that On-
costatin M, a member of the IL-6 superfamily, activates
STAT3 signaling and drives ECM remodeling. ECM re-
modeling allows cancer cells to invade surrounding tissues,
escape, and metastasize to other sites (Sanz-Moreno et al.,
2011). A detailed description of inhibition of the IL-6-JAK-
STAT signaling pathway as well as the TGF-bsignaling is
given next.
Pietras et al. (2008) demonstrated that the reduction of
FGF2 and fibroblasts growth factor-7 (FGF7) in animal
models of cervical cancer via Imatinib-mediated inhibition of
PDGF receptor slowed cancer cell division and disrupted
angiogenesis. The authors showed that targeting CAFs can
act in complement to conventional therapeutic strategies and
improve the management of cancers that are difficult to cure.
Research by Anderberg et al. (2009) further showed that
PDGF-CC induces the expression of osteopontin in CAFs,
leading to tumor growth acceleration.
The chemokine SDF1, exclusively produced by FAP-
positive CAFs, binds to its receptor CXCR4, resulting in the
suppression of immunity within the TME through the pre-
vention of CD8
+
T cell infiltration (Fearon, 2015; Feig et al.,
2013; Kraman et al., 2010). Several inhibitors of SDF1-
CXCR4 interactions have been developed and these can
reactivate the anti-tumor immunity by enhancing the infil-
tration of CD8
+
T cells into the TME (Feig et al., 2013).
Inhibition of SDF1 and CXCR4 interactions include the use
of inhibitors such as AMD3100 and may enhance the anti-
tumor effects of monoclonal antibodies against PDI/PDL1 in
pancreatic duct adenocarcinoma (Feig et al., 2013).
Another strategy under consideration to control CAF-
derived factors includes the inhibition of protein synthe-
sis. The inhibition of protein synthesis through the use of
SOM230, an inhibitor of protein synthesis in a-SMA-positive
CAFs, resulted in reduced levels of several molecules (Duluc
et al., 2015). Indeed, the inhibition of protein synthesis in
CAFs led to less ability of cancer cells to resist chemotherapy
in a PDAC model (Duluc et al., 2015).
Targeting the IL-6-JAK-STAT signaling in cancer
It has been shown that the IL-6-JAK-STAT3 is abnor-
mally activated in several cancers, with enhanced activation
of the pathway linked to poor patients’ survival (Chen et al.,
2013b; Kusaba et al., 2006; Macha et al., 2011). Within the
TME, the IL-6-JAK-STAT3 signaling has been shown to be
responsible for cancer cell invasive and metastatic behavior.
In addition, the IL-6-JAK-STAT3 signaling represses im-
munological response to tumors. Anti-cancer agents or in-
hibitors against members of the IL-6-JAK-STAT3 have
been approved by the FDA and the EMA, whereas others are
still under investigation.
Several pathological conditions display high levels of
IL-6 and increased activation of the IL-6-JAK-STA3, in-
cluding rheumatoid arthritis as well as many cancers (Ku-
mari et al., 2016; Masjedi et al., 2018). Mutations in genes
of several JAK-STAT pathway members activate the path-
way in many neoplasms. Increased IL-6 in circulation and
within the TME is a result of the JAK-STAT pathway being
constitutively activated (Kusaba et al., 2006; Taniguchi and
Karin, 2014; Wang et al., 2013).
Besides CAFs, multiple other cells have been shown to
produce IL-6 within the TME, from pericytes, immune cells,
and even tumor cells (Bournazou and Bromberg, 2013; Na-
gasaki et al., 2014; Nozawa et al., 2006). The activation of
STAT3 by IL-6 in tumor cells results in the expression of
proliferation and survival genes, including cyclin D1 and
BCL2-like protein 1 (BCL-xL), respectively (Fisher et al.,
2014). Since both CAFs and tumor cells show activation of
IL-6-JAK-STAT signaling, the description given next and the
targeting of the IL-6-JAK-STAT signaling can therefore be
applied to both CAFs and tumor cells to achieve durable
cancer treatment. IL-6 activated STAT3 can induce VEGF,
MMPs, and TGF-bexpression, promoting angiogenesis, in-
vasion, and development of therapy resistance, respectively
(Fisher et al., 2014; Yu and Jove, 2004; Yu et al., 2009).
Besides direct effects on cancer cells, IL-6 and JAK-STAT
signaling influences the behavior of stromal cells, including
CAFs, CAMs, TANs, effector T cells, and killer cells (Harris
et al., 2007; Iwata-Kajihara et al., 2011; Kortylewski et al.,
2005; Kujawski et al., 2010; Lee et al., 2010; Yu et al.,
2007b). By affecting both CAFs and immune cells, the
324 DZOBO AND DANDARA
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IL-6-JAK-STAT signaling contributes to the suppression of
the immune response in the TME.
The importance of IL-6 in TME is underscored by its
various effects on tumor cell and stromal cell survival, pro-
liferation, and invasiveness (Kortylewski et al., 2005; Le-
derle et al., 2011). Two IL-6 signaling pathways are known:
the classical IL-6 signaling pathway, which involves IL-6
binding to the IL-6 receptor on the cell surface and interacts
with transmembrane protein gp130; the trans-signaling
pathway whereby IL-6 binds to a soluble form of the IL-6
receptor and then interacts with gp130. Importantly, although
similarities exist in the ways in which both signaling path-
ways regulate cell behavior, the trans-signaling pathway
controls the recruitment and activation of stromal cells
(Campbell et al., 2014; Hunter and Jones, 2015). Activation
of the IL-6 signaling results in activation of mostly JAK1 and
JAK2, which then phosphorylate members of the STATs
family ( Johnson et al., 2018; Steyn et al., 2019).
Of the STATs proteins, STAT3 is the most studied and has
been linked with tumor progression and suppression of the
immune system (Bromberg, 2002; Yu et al., 2009). Activa-
tion of STATs proteins is regulated by PIAS proteins, sup-
pressor of cytokine signaling proteins, phosphatases, and
miRNAs (Dorritie et al., 2014; Kim et al., 2010; Sekine et al.,
2006; Zhang et al., 2007).
The IL-6 levels are increased in cancers, including breast,
lung, colorectal, esophageal, ovarian, and prostate cancers
(Chen et al., 2013a; Chung and Chang, 2003; Culig and Puhr,
2012; Dethlefsen et al., 2013; Maccio
`and Madeddu, 2013).
Several preclinical studies utilizing models and patient
samples have demonstrated the critical roles of circulating
IL-6 in tumor development (Becker et al., 2004; Lesina et al.,
2011; Sansone et al., 2007). For example, IL-6 enhances CSC
proliferation in breast cancer (Sansone et al., 2007). In ad-
dition, IL-6 is known to promote EMT and metastasis in
breast cancer (Chang et al., 2013; Sullivan et al., 2009).
Consequently, IL-6 levels in circulation have been used as
prognostic indicators in patients’ survival and can predict
response to therapy (Gao et al., 2016; Knupfer and Preiss,
2010; Maccio and Madeddu, 2013; Sanguinete et al., 2017).
Several studies utilizing models and patients’ samples
have demonstrated the critical role played by CAF- and
tumor-derived IL-6 cancers, including breast, colorectal,
esophageal, lung, and skin cancers (Gao et al., 2007; Gri-
vennikov et al., 2009; Lederle et al., 2011; Sansone et al.,
2007). Aberrant levels of STAT3 activation have been shown
in many cancers (Frank, 2007; Roeser et al., 2015). Besides
being involved in tumor progression, several studies have
shown that STAT3 can promote therapy resistance (Sen et al.,
2012; Spitzner et al., 2014).
Several strategies have been developed to target the IL-6-
JAK-STAT signaling pathway (Fig. 3). Siltuximab, for
example, targets IL-6 directly and Tocilizumab targets the
IL-6 receptor. Siltuximab has shown efficacy against sev-
eral solid tumors, including lung and prostate cancers (Ca-
varretta et al., 2008; Song et al., 2014). Siltuximab have
been shown to reduce the levels of STAT3 and other sig-
naling cascades such as MEK-ERK in tumors (Fizazi et al.,
2012; Karkera et al., 2011). Tocilizumab is used in patients
with rheumatoid arthritis and has efficacy against colorectal
and pancreatic cancers (Dijkgraaf et al., 2015; Grivennikov
et al., 2009).
FIG. 3. Some of the strategies targeting both CAF- and tumor cell-derived IL-6-JAK-STAT signaling pathways are
indicated. JAK, Janus kinase; STAT, signal transducer and activator of transcription; X, blocking of signaling step.
CAFSIN TUMOR MICROENVIRONMENT 325
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Inhibitors of JAKs include Pacritinib, Ruxolitinib, and
Tofacitinib and these have shown varied efficacy against
cancers such as colitis, myelofibrosis, liver, pancreatic, and
ovarian cancers (Hedvat et al., 2009; Komrokji et al., 2015;
Neubauer et al., 1998; Sandborn et al., 2017; Tavallai et al.,
2016; Vannucchi et al., 2015). The STAT3 inhibitors dem-
onstrated antitumor activities and promoted apoptosis against
several cancer cells (Chen et al., 2007; Fuh et al., 2009; Pan
et al., 2013; Schust et al., 2006; Siddiquee et al., 2007; Zhang
et al., 2010b).
Targeting the CAF-derived TGF-bsignaling
pathway in cancer
Several reports document the lack of cancer patients’ re-
sponse to therapy being associated with TGF-bsignaling in
CAFs and CAMs (Brunen et al., 2013; Colak and Ten Dijke,
2017; Ganesh and Massague, 2018; Hao et al., 2019; Ja-
kowlew, 2006; Mariathasan et al., 2018; Reiss, 1999). In
normal cells and during early stages of tumor development,
the TGF-bsignaling displays anti-tumor functions, with re-
ports of cancer cell cycle arrest and induction of apoptosis
(Colak and Ten Dijke, 2017; Gu and Feng, 2018; Senthebane
et al., 2017).
Our study demonstrated that as tumors develop, TGF-b
from both stromal cells and tumor cells participates in the
transformation of normal fibroblasts and MSCs into CAFs
and thus contributes to further tumor development, metasta-
sis, and therapy resistance (Fig. 4) (Bussard et al., 2016;
Castells et al., 2012; Senthebane et al., 2017). Thus, de-
pending on the stage of tumor development, TGF-bsignaling
can have both tumor-promoting and tumor-suppressive
functions. Targeting this pleiotropic signaling cascade is
therefore difficult, with further research required to determine
the specific members of the pathway to target as well as the
right doses. Importantly, the effects of inhibition of this sig-
naling cascade on both cancer cells and stromal components
must be further investigated. The elucidation of possible
biomarkers that can point to the usefulness of potential in-
hibitors during and after treatment must be determined.
In their elaborate study, Mariathasan et al. (2018) dem-
onstrated that a combination of TGF-bsignaling blockade
and anti-PD-L1 repressed TGF-bsignaling in stromal cells,
leading to enhanced tumor suppression. Importantly, the
authors demonstrated the need to combine treatment strate-
gies to attain durable cure in cancer. Several drugs targeting
TGF-bare under trial and these include fresolimumab
(NCT02581787).
Targeting CAF-derived ECM proteins
and associated signaling
The CAFs are the principal cells responsible for synthe-
sizing the ECM (Barbazan and Matic Vignjevic, 2019; Er-
dogan and Webb, 2017; Houthuijzen and Jonkers, 2018;
Khan et al., 2018; Liu et al., 2019b). In normal tissue, fibrillar
collagens, fibronectin, hyaluronan, and tenascin C are de-
posited in large amounts to make the ECM. Increased syn-
thesis of ECM proteins results in desmoplastic reactions and
these are known to drive tumor growth (Ronnov-Jessen et al.,
1996). Besides inducing desmoplastic reactions, increased
ECM synthesis creates a barrier for the delivery of drugs to
cancer cells (Laklai et al., 2016; Senthebane et al., 2017). In
detailed in vitro experiments, we demonstrated that the
presence of an ECM increased the resistance of breast and
esophageal cancer cells to several chemotherapeutic agents
(Senthebane et al., 2018).
Although it is plausible that a reduction in ECM synthesis
in CAFs and other stromal cells such as macrophages is an
appealing strategy, improper ECM synthesis can create
FIG. 4. Both tumor and stromal cells secrete TGF-binto the TM milieu. TGF-band other growth factors can be
sequestered by the ECM. Increased TGF-bwithin the TM leads to transformation of MSCs and fibroblasts into CAFs.
Increased CAF- and tumor-derived TGF-blevels cause increased tumor growth, chemoresistance, and metastasis. Figure
adapted from Senthebane et al. (2017).
326 DZOBO AND DANDARA
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‘‘highways’’ through which cancer cells may metastasize
( Jabłon
´ska-Trypuc
´et al., 2016; Senthebane et al., 2017,
2018). Increased amounts of both collagen and hyaluronan in
tumors can result in compressed vascular networks, inad-
vertently reducing the flow of drugs to cancer cells (Chauhan
et al., 2013; Dzobo et al., 2014). Targeting both collagen and
hyaluronan production by both CAFs and macrophages can
allow drugs to reach cancer cells (Chauhan et al., 2013). The
inhibition of collagen through the use of halofuginone has
been shown to reduce desmoplasia, impacting tumor pro-
gression ( Juarez et al., 2012).
Enzymatic degradation of ECM proteins, such as collagen
via MMPs occurs during ECM remodeling. The use of MMPs
as an anti-cancer therapy has largely failed to materialize
(Vandenbroucke and Libert, 2014). For a start, there is so
much overlap in MMP activities and indiscriminate degra-
dation of ECM proteins can have a negative effect during
treatment (Vandenbroucke and Libert, 2014).
Currently, several MMPs inhibitors are under investigation
in many cancers (Chiappori et al., 2007). Deciphering the
exact MMPs activities and their specific targets can aid in the
development of novel use of MMPs in cancer treatment
(Coussens et al., 2002; Dzobo et al., 2012; Wojtowicz-Praga
et al., 1997). Hyaluronan can be depleted through the use of
recombinant hyaluronidase enzyme to allow drugs to reach
cancer cells ( Jacobetz et al., 2013; Provenzano et al., 2012).
Targeting hyaluronic acid via the use of PEGPH20 is still
under investigation (NCT01453153). New clinical trial data
(HALO-109-301) show that when used together with gem-
citabine and nab-paclitaxel in patients with advanced pan-
creatic cancer, PEGPH20 is promising (Doherty et al., 2018).
Caution, however, must be heeded as the resulting expan-
sion of the vascular system can result in invasion and metas-
tasis of cancer cells. A combination of the removal of ECM
proteins during treatment can enhance patients’ survival,
demonstrated in PDAC by Hingorani et al. (2016). In vitro
work by Senthebane et al. (2018) demonstrated that the re-
moval of both collagen and fibronectin from ECM resulted in
increased apoptosis in esophageal cancer cells. Antibodies
have been used to target fibronectin to reduce vascularization
and tenascin C to prevent metastasis (Ebbinghaus et al., 2004;
Reardon et al., 2006). Targeting Tenascin C together with
chemotherapy improves the survival of glioma patients
(Reardon et al., 2006). The inhibition of signaling pathways
perturbed in TME such as the IL-6-JAK-STAT and sonic
hedgehog cascade can aid in reducing CAFs and their secreted
factors, resulting in better therapy response (Olive et al., 2009).
For example, IPI-926, an inhibitor of the Hedgehog pathway,
together with Gemcitabine has been shown to influence resis-
tance to chemotherapy in PDAC (Olive et al., 2009). Currently,
most stromal-directed therapy is still in its infancy and removal
of any stromal elements must be studied carefully. Stromal
cells and their products such as the ECM are needed for normal
tissue architecture and homeostasis (Dzobo et al., 2012;
Senthebane et al., 2017, 2018; Sherman et al., 2014). Any
stromal-directed therapy will have to work in combination with
tumor-directed therapy to achieve durable cancer treatment.
Targeting CAF- or stromal-induced angiogenesis
Given the diverse cells and mechanisms through which
tumor vasculature is induced and develops, the inhibition of
tumor angiogenesis is challenging. Although angiogenesis
inhibition has been successful in some cases, partial to total
failure has also been reported in some studies (Bergers and
Hanahan, 2008; Ferrara and Adamis, 2016; Ferrara, 2016;
Jayson et al., 2016; Okaji et al., 2008; Patrikidou et al., 2014;
Saharinen et al., 2017; Schmittnaegel et al., 2017). Studies
demonstrated that tumor angiogenesis is regulated by several
factors and mechanisms and does not solely dependent on
VEGF-A signaling (Bergers and Hanahan, 2008; Ferrara,
2016; Potente et al., 2011). Several studies have also shown
the existence of tumor growth requiring no angiogenesis
(Pa
`ez-Ribes et al., 2009; Rubenstein et al., 2000).
Angiogenesis-independent tumor growth is also achieved
through the action of immune cells such as macrophages and
neutrophils (De Palma and Lewis, 2013; Gabrusiewicz et al.,
2014; Liang and Ferrara, 2016). Coupling anti-angiogenesis
specific therapy with strategies that disrupt any compensatory
factors and signaling from cells such as macrophages and
neutrophils offers better potential at inhibiting angiogenesis
in tumors (Baer et al., 2016; Biswas and Mantovani, 2010;
Lewis et al., 2016). Blockage or elimination of both macro-
phages and neutrophils that confer VEGF-A-independent
angiogenesis to tumors has been shown to enhance tumor
response to VEGF-A specific therapy (Rivera et al., 2015).
Kaneda et al. (2016) demonstrated that the inhibition of
PI3K signaling, highly expressed in myeloid cells, success-
fully inhibited angiogenesis in cancer treatment. Caution
must be taken, however, as several studies have shown that
the inhibition of angiogenesis may enhance the ability of
tumor-associated cells to promote tumor growth (Bergers and
Hanahan, 2008; De Palma and Lewis, 2013; Ferrara and
Adamis, 2016). Crawford et al. (2009) demonstrated that
some tumors can overcome angiogenesis inhibition through
upregulation of PDGF-C
Currently, Bevacizumab (anti-VEGF) is under clinical
trials for the treatment of different cancers from colorectal
cancer (NCT02885753), lung cancer (NCT03100955) to
cholangiocarcinoma cancer (NCT03251443) in combination
with 5-fluorouracil and panitumumab. Ramucirumab, also
known as LY3009806, is under clinical trial in combination
with paclitaxel for the treatment of gastric adenocarcinoma
(NCT02898077). RAD001 is also under clinical trial for the
treatment of renal cell carcinoma (NCT01206764).
CAFs as Delivery Vehicles of Therapeutic Agents
As described by Miao et al. (2017), CAFs can be used to
deliver anti-tumor drugs or nanoparticles and cause cancer
cell death via apoptosis. It has also been shown that fibro-
blasts within a tumor can scavenge and accumulate thera-
peutic drugs, resulting in increased drug concentrations
within tumors (Hessmann et al., 2018). Senthebane et al.
(2017) demonstrated that MSCs can be transformed into
CAFs and thus are a major source of CAFs found in tumors.
The authors and others demonstrated that although MSCs
initially demonstrate anti-tumor behavior they are easily
transformed into tumor-supporting cells (CAFs) when in
contact with cancer cells (Dzobo et al., 2016b; Shi et al., 2017).
Due to their likelihood of being found within tumors,
MSCs have been suggested as drug-delivery vehicles or can
be manipulated to secrete anti-tumor factors (Shi et al., 2017).
Amniotic MSCs have been suggested to deliver therapeutic
CAFSIN TUMOR MICROENVIRONMENT 327
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agents to tumors (Bonomi et al., 2015). Several clinical trials
on the use of MSCs in gene therapies for different cancers are
underway (Abrate et al., 2014; Jobst et al., 2017; Niess et al.,
2015). Overall, the use of MSC-based gene therapies requires
further investigations as Senthebane et al. (2017) demon-
strated. The authors’ results show that MSCs can be pro-
tumorigenic or anti-tumorigenic depending on tumor stage
(Dzobo et al., 2016b; Senthebane et al., 2017). Other studies
support this dual effect of MSCs (Clarke et al., 2015; Hong
et al., 2014; Park et al., 2008).
Of late, CAFs are now being used as a stromal indicator of
the disease stage during diagnosis and as an indicator of tu-
mor response to treatment strategies. Tsujino et al. (2007)
demonstrated that the number of myofibroblasts within TME
can be used as a clinical biomarker of disease relapse in co-
lorectal cancer. Surowiak et al. (2007) showed that the pres-
ence of CAFs within breast cancer tissue is a poor prognostic
factor. Contrasting data on the prognostic value of CAFs in
tumors may be due to CAFs’ heterogeneity as well as the co-
evolution of CAFs with tumor cells during tumor progres-
sion (Brechbuhl et al., 2017; Paulsson and Micke, 2014).
Outlook and Conclusion
To summarize, we provide the definitions of the key con-
cepts in the present analysis in Box 1. Several lines of re-
search are being initiated to evaluate the effectiveness of
many therapeutic drugs on CAFs. Epigenetic drugs, includ-
ing histone deacetylase inhibitors, are under investigation
for their effect on several signaling pathways such as the
JAK-STAT pathway in both cancer cells and CAFs (Dzobo,
2019). These epigenetic drugs are being evaluated on their
potential to prevent CAFs’ generation and increase in abun-
dance within tumors (Younes et al., 2016). Another appealing
strategy includes the use of normal fibroblasts in cancer
treatment or the reversion of activated CAFs to normal fi-
broblasts (Kirsner et al., 2012).
Many questions remain to be answered regarding the
origin of CAFs or their subsets, as this can influence CAF-
directed therapeutic strategies adopted during cancer treat-
ment. It is hoped that as new information becomes available,
new markers and signaling pathways specific only to CAFs
or their subsets can be identified.
The identification that MSCs, besides fibroblasts, can be a
source of CAFs might explain CAFs’ heterogeneity and
could be the reason why several CAFs subsets are observed in
tumors (Senthebane et al., 2017). If different cells contribute
to the CAFs’ subsets observed in tumors, which cell(s) of
origin gives rise to pro-tumorigenic CAFs and which cell(s)
gives rise to anti-tumorigenic CAFs? Based on data revealed
by Senthebane et al. (2017), it appears that despite different
cell(s) of origin, stromal cells may start as anti-tumorigenic
but are transformed through several secreted factors to be-
come pro-tumorigenic. Whether different cell types con-
tribute to different subsets requires further investigation.
In addition, the identification of specific markers for
different subsets can allow retrospective determination of
cells of origin as well as the development of specific anti-
tumorigenic therapies against these CAFs’ subsets. Stromal
cells can also co-evolve with tumor cells. Is tumor cell het-
erogeneity also partly driven by different CAF subsets?
Epigenetic regulation of CAFs must also be investigated to
allow the development of drugs targeting epigenetics of
CAFs (Dzobo, 2019). Many clinical trials conducted for
different candidate drugs have focused on targeting cancer
cells and up to now no trial has been done on candidate drugs
targeting CAFs.
One major challenge faced by scientists during the de-
velopment of novel cancer therapies has been the absence of
adequate cancer models that recapitulate the TMEs as seen
in vivo to use in preclinical studies. This has prevented the
proper understanding of tumor cell–stromal cell interactions
as well as cell–matrix interactions. For example, very few
available cancer models include cells such as CAFs, CAMs,
and cancer-associated neutrophils (Dzobo et al., 2018a). As
shown by recent reports, the addition of ECM proteins to
cancer models as well as the development of tumor spheroids
and organoids promises to reveal more about tumor initiation
and development than the use of cancer cells in in vitro ex-
periments (Dzobo et al., 2019).
New advances, including microfluidic technology, will
allow the development of what is known as ‘‘human organs
on chips.’’ With the ability to change parameters as required
during tumor development as well as potential additions of
Box 1. Definitions of the Key Concepts
in the Present Expert Review
Endothelial to mesenchymal transition: The process
through which endothelial cells are transformed into
mesenchymal cells. This is achieved through the loss
of endothelial properties and the gaining of
mesenchymal properties.
Epithelial to mesenchymal transition: The process
through which epithelial cells are transformed into
mesenchymal cells. This is achieved through the loss
of epithelial properties and the gaining of
mesenchymal properties.
Extracellular matrix (ECM): Fibrous proteins secreted by
stromal cells including fibroblasts, mesenchymal stem
cells, and pericytes and its main function is to provide
structural support and biochemical cues to surrounding
cells. The ECM accumulates growth factors,
cytokines, and chemokines and these, in turn, influence
tumor and stromal cell behavior.
Desmoplasia: The resulting state of tissue after a
response to an injury and includes fibrosis as stromal
cells synthesize huge amounts of ECM proteins.
Matrix metalloproteases: A large family of zinc-
containing protease enzymes referred to as
endopeptidases and are responsible for degrading
ECM proteins such as fibronectin, collagens, and
laminins.
Tumor microenvironment: Includes all cellular and
noncellular components of the environment
surrounding/within a tumor and includes all stromal
cells, blood vessels, and the ECM. Stromal cells
include cancer-associated fibroblasts, cancer-
associated macrophages, cancer-associated
neutrophils, pericytes, and other immune cells. The
tumor microenvironment (TME) provides the
‘‘theater’’ within which tumor cells can survive and
tumors develop and eventually spread.
Stromal cells: Includes all cells found within the TME
and act to support tumor cells by secreting tumor-
promoting growth factors, cytokines, chemokines, and
ECM proteins.
328 DZOBO AND DANDARA
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several cells, including CAFs, these new cancer models will
potentially reveal accurate information about tumor initiation
and development. In addition, microfluidic technology as
applied to tumor development will also allow collection of
samples at specific times with ease.
Author Disclosure Statement
The authors declare they have no conflicting financial
interests.
Funding Information
The funding for this research was provided by the National
Research Foundation (NRF) of South Africa (Grant no.
91457: RCA13101656402), the International Centre for
Genetic Engineering and Biotechnology (ICGEB) (Grant no.
2015/0001), and the University of Cape Town.
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Address correspondence to:
Kevin Dzobo, PhD
International Centre for Genetic Engineering
and Biotechnology (ICGEB)
Cape Town Component
Wernher and Beit Building (South)
Anzio Road
Observatory
Cape Town 7925
South Africa
E-mail: kdzobosnr@yahoo.com
Collet Dandara, PhD
Division of Human Genetics
Pharmacogenetics Research Group
Division of Human Genetics
Department of Pathology
Faculty of Health Sciences
Institute of Infectious Diseases and Molecular Medicine
University of Cape Town
Anzio Road
Observatory
Cape Town 7925
South Africa
E-mail: collet.dandara@uct.ac.za
Abbreviations Used
a-SMA ¼alpha-smooth muscle actin
ATRA ¼all-trans-retinoic acid
CAFs ¼cancer-associated fibroblasts
CAMs ¼cancer-associated macrophages
CANs ¼cancer-associated neutrophils
CAR T ¼chimeric antigen receptor T
CSCs ¼cancer stem cells
ECM ¼extracellular matrix
EMT ¼epithelial to mesenchymal transition
EndMT ¼endothelial to mesenchymal transition
FAP ¼fibroblast activation protein
FGF ¼fibroblast growth factor
GPR77 ¼G-protein-coupled receptor 77
HGF ¼hepatocyte growth factor
HIF1-a¼hypoxia inducible factor 1-a
IL-6 ¼interleukin-6
JAK ¼Janus kinase
miRNAs ¼microRNAs
MMPs ¼matrix metalloproteases
MSCs ¼mesenchymal stem cells
PDAC ¼pancreatic ductal adenocarcinoma
PDGF ¼platelet-derived growth factor
PDGFR-b¼PDGF receptor-b
ROS ¼reactive oxygen species
SDF-1 ¼stromal-derived factor 1
SFRP2 ¼secreted frizzled-related protein-2
STAT ¼signal transducer and activator of transcription
STC1 ¼stanniocalcin-1
TAMs ¼tumor-associated macrophages
TGF-b¼transforming growth factor-beta
TIMP ¼tissue inhibitor of metallo proteinases
TME ¼tumor microenvironment
VEGF ¼vascular endothelial growth factor
VEGFA ¼vascular endothelial growth factor-A
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