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Abstract and Figures

Chemoresistance is a leading cause of morbidity and mortality in cancer and it continues to be a challenge in cancer treatment. Chemoresistance is influenced by genetic and epigenetic alterations which affect drug uptake, metabolism and export of drugs at the cellular levels. While most research has focused on tumor cell autonomous mechanisms of chemoresistance, the tumor microenvironment has emerged as a key player in the development of chemoresistance and in malignant progression, thereby influencing the development of novel therapies in clinical oncology. It is not surprising that the study of the tumor microenvironment is now considered to be as important as the study of tumor cells. Recent advances in technological and analytical methods, especially 'omics' technologies, has made it possible to identify specific targets in tumor cells and within the tumor microenvironment to eradicate cancer. Tumors need constant support from previously 'unsupportive' microenvironments. Novel therapeutic strategies that inhibit such microenvironmental support to tumor cells would reduce chemoresistance and tumor relapse. Such strategies can target stromal cells, proteins released by stromal cells and non-cellular components such as the extracellular matrix (ECM) within the tumor microenvironment. Novel in vitro tumor biology models that recapitulate the in vivo tumor microenvironment such as multicellular tumor spheroids, biomimetic scaffolds and tumor organoids are being developed and are increasing our understanding of cancer cell-microenvironment interactions. This review offers an analysis of recent developments on the role of the tumor microenvironment in the development of chemoresistance and the strategies to overcome microenvironment-mediated chemoresistance. We propose a systematic analysis of the relationship between tumor cells and their respective tumor microenvironments and our data show that, to survive, cancer cells interact closely with tumor microenvironment components such as mesenchymal stem cells and the extracellular matrix.
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Int. J. Mol. Sci. 2017, 18, 1586; doi:10.3390/ijms18071586 www.mdpi.com/journal/ijms
Review
The Role of Tumor Microenvironment in
Chemoresistance: To Survive, Keep Your
Enemies Closer
Dimakatso Alice Senthebane
1
, Arielle Rowe
2
, Nicholas Ekow Thomford
3
, Hendrina Shipanga
1
,
Daniella Munro
3
, Mohammad A. M. Al Mazeedi
4
, Hashim A. M. Almazyadi
4
,
Karlien Kallmeyer
5
, Collet Dandara
3
, Michael S. Pepper
5
, M. Iqbal Parker
1,2
and Kevin Dzobo
1,2,
*
1
Division of Medical Biochemistry and Institute of Infectious Disease and Molecular Medicine,
Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town,
Cape Town 7925, South Africa; SNTDIM001@myuct.ac.za (D.A.S.); hmshipanga@gmail.com (H.S.);
iqbal.parker@uct.ac.za (M.I.P.)
2
International Centre for Genetic Engineering and Biotechnology (ICGEB), Cape Town Component,
Wernher and Beit Building (South), UCT Medical Campus, Anzio Road, Observatory, Cape Town 7925,
South Africa; arielle.rowe@icgeb.org
3
Pharmacogenetics Research Group, Division of Human Genetics, Department of Pathology and
Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town,
Cape Town 7925, South Africa; thmnic023@myuct.ac.za (N.E.T.); MNRDAN002@myuct.ac.za (D.M.);
collet.dandara@uct.ac.za (C.D.)
4
Batterjee Medical College, Prince Abdullah AlFiasal St, Obhur Al-Shamaliyah, Jeddah 23819, Saudi Arabia;
almazeedi.mohammad@yahoo.com (M.A.M.A.M.); hashemmazeedi@gmail.com (H.A.M.A.)
5
Institute for Cellular and Molecular Medicine, Department of Immunology and
South African Medical Research Council (SAMRC) Extramural Unit for Stem Cell Research and Therapy,
Faculty of Health Sciences, University of Pretoria, Pretoria 0002, South Africa;
karlienkallmeyer@gmail.com (K.K.); michael.pepper@up.ac.za (M.S.P.)
* Correspondence: kd.dzobo@uct.ac.za; Tel.: +27-021-406-7689
Received: 3 July 2017; Accepted: 19 July 2017; Published: 21 July 2017
Abstract: Chemoresistance is a leading cause of morbidity and mortality in cancer and it continues
to be a challenge in cancer treatment. Chemoresistance is influenced by genetic and epigenetic
alterations which affect drug uptake, metabolism and export of drugs at the cellular levels. While
most research has focused on tumor cell autonomous mechanisms of chemoresistance, the tumor
microenvironment has emerged as a key player in the development of chemoresistance and in
malignant progression, thereby influencing the development of novel therapies in clinical oncology.
It is not surprising that the study of the tumor microenvironment is now considered to be as
important as the study of tumor cells. Recent advances in technological and analytical methods,
especially ‘omics’ technologies, has made it possible to identify specific targets in tumor cells and
within the tumor microenvironment to eradicate cancer. Tumors need constant support from
previously ‘unsupportive’ microenvironments. Novel therapeutic strategies that inhibit such
microenvironmental support to tumor cells would reduce chemoresistance and tumor relapse. Such
strategies can target stromal cells, proteins released by stromal cells and non-cellular components
such as the extracellular matrix (ECM) within the tumor microenvironment. Novel in vitro tumor
biology models that recapitulate the in vivo tumor microenvironment such as multicellular tumor
spheroids, biomimetic scaffolds and tumor organoids are being developed and are increasing our
understanding of cancer cell-microenvironment interactions. This review offers an analysis of recent
developments on the role of the tumor microenvironment in the development of chemoresistance
and the strategies to overcome microenvironment-mediated chemoresistance. We propose a
systematic analysis of the relationship between tumor cells and their respective tumor
Int. J. Mol. Sci. 2017, 18, 1586 2 of 29
microenvironments and our data show that, to survive, cancer cells interact closely with tumor
microenvironment components such as mesenchymal stem cells and the extracellular matrix.
Keywords: chemoresistance; tumor microenvironment; tumor heterogeneity; mesenchymal stem
cells; angiogenesis; extracellular matrix; clinical oncology
1. Introduction
Cancer is a multifactorial disease and is one of the leading causes of death worldwide. It results
from both genetic and epigenetic transformation of normal cells leading to abnormal proliferation.
Cancer deaths outnumber the combined deaths from diseases such as HIV/AIDS, malaria and
tuberculosis worldwide [1,2]. Despite the development of potent chemotherapeutics against many
cancer types in recent years, durable or long lasting cure is still out of reach for many patients [3,4].
This is partly due to the development of drug/therapeutic resistance which stems from the
remarkable adaptive behavior of cancer cells and is driven by both genetic and epigenetic factors.
There are many distinct cancer types and these differ significantly in their genetic makeup, behavior
and treatment responses [5]. Differences in cancer cells behavior stem from the dysregulation of a
number of growth signals that are involved in the direct entry into and progression through the cell
cycle. Due to the diverse nature of cancer, many treatment strategies have been developed and each
takes advantage of a different aspect of the disease. However, most cancer drugs still target DNA
replication and DNA repair mechanisms.
Cancer cells proliferate at a much higher rate than normal cells and invade nearby tissues or
spread to distance organs. A number of oncogenes and tumor suppressor genes such as p53, c-Myc
and transforming growth factor-β (TGF-β) are mutated in cancer cells and have been observed to
play key roles in cancer cell proliferation, invasion and metastasis. Most of these oncogenes and
tumor suppressor genes are considered as major contributors to drug resistance [6]. Resistance is
usually accompanied by recurrence of the disease. Different cancer types respond to treatment in
different ways and therefore some are better treated than others. The most common treatments for
cancer are surgery, radiotherapy and chemotherapy. Surgery can successfully remove the cancerous
tissue from the body and combined with chemotherapy and radiotherapy can be successful in
treating any cancer [7]. Radiotherapy uses radiation to kill cancer cells. Chemotherapy remains the
preferred method due in part to its effectiveness and low cost. Its lack of selectivity however hampers
its success as normal cells are also killed in the process. Patients undergoing chemotherapy suffer
many side-effects such as loss of hair, bleeding, nausea and death. Due to its genotoxic effects,
chemotherapy induces changes in both normal and cancer cells creating further cancer cell
heterogeneity and ultimately affecting the efficiency of chemotherapy [8].
A huge challenge in cancer treatment is the development of chemoresistance resulting in cancer
cells that are more aggressive and able to metastasize [9]. Mechanisms that contribute to
chemoresistance include tumor heterogeneity, drug inactivation, apoptosis evasion, enhanced
deoxyribonucleic acid (DNA) repair, increased drug efflux, epithelial-to-mesenchymal transition and
the involvement of the tumor microenvironment (TM) [8]. Though cancer cell chemoresistance is
usually attributed to heterogeneity within the cancer cell population, mutations and epigenetic
alterations influencing the metabolism and retention of drugs by cancer cells [10–17], additional
causes could play important roles in the development of this phenomenon. Most important is the
diversity within the tumor microenvironment (TM) in terms of the stromal cells present, the amount
of oxygen available and the acidity of the environment [18–24]. Another important difference is the
amount of the extracellular matrix (ECM) proteins around the cancer cells [25–27]. ECM proteins can
create a barrier through which the drugs must pass in order to reach the cancer cells while their
presence promote tumor metastasis [28–34]. As the tumor grows, it becomes difficult for
chemotherapeutic agents to reach cancer cells near the center of the tumor. All these factors can have
a huge influence on how cancer cells respond to drugs.
Int. J. Mol. Sci. 2017, 18, 1586 3 of 29
The genetic makeup of cancer cells and cellular processes occurring within cancer cells
contribute immensely to the inability of most chemotherapeutic drugs used in clinical oncology to
effectively clear these cells from the body [12,14–17,35–37]. Several mutations to key genes encoding
important proteins responsible for xenobiotic metabolism, as well as import and export of drugs from
cells such as the ABC transporters have been identified and shown to influence how tumor cells
respond to several drugs [12,14–17,35–37]. However, with remarkable advancement in technology
and analytical methods seen in the last decade, attention has shifted to the TM contribution towards
the development of chemoresistance [38–46]. Chemotherapeutic drugs need to access all cancer cells
in a solid tumor to be effective, thus components of the tumor microenvironment become important
players in the response of these cells to drugs [33,47–53]. The TM is a dynamic entity and is
characterized by cellular heterogeneous, the amounts of oxygen, nutrients and ECM proteins [41,54–
59]. The heterogeneous nature of cancer and stromal cells within the TM is reflected in the ECM
produced by these cells. The variability of the ECM within the TM also makes targeting the ECM
difficulty and might explain why therapeutic targeting of the ECM has not had much success in
several clinical trials. Both cancer cells and stromal cells do deposit the ECM in a tumor [60,61]. Novel
strategies need to be developed to specifically target the ECM from different cells within the tumor.
In addition, understanding the response of cancer cells to the ECM at different stages of tumor
development would allow for the understanding of the contribution of each ECM protein during
tumor progression. Determining the most effective time point when cancer cells respond to the ECM
is also necessary in the intervention to stop cancer growth. In addition several studies have shown
that matrix metalloproteinases play a huge role in inducing processes such as epithelial-
mesenchymal transition with the end result being malignant transformation [60–62].
This review focusses on the contribution of the TM constituents in the development of
chemotherapeutic resistance especially the role played by mesenchymal stem cells and the ECM. To
overcome chemoresistance, it is imperative that the TM contribution be studied and specified as only
then can we attain long lasting treatment in clinical oncology.
2. Cancer Cell Chemoresistance
The accumulation of genetic aberrations over time has been recognized as the main cause of
cancer [63–70]. A combination of genetic mutations and epigenetic alterations results in tumor
heterogeneity [67,71–77]. Tumor heterogeneity can contribute towards chemoresistance in many
ways. Tumor heterogeneity is one of the recent addition to the list of drivers of chemoresistance
[78,79]. Tumors are made up of cancer cells that differ in their phenotype and therefore
chemotherapeutic responses. Differences in phenotypes may also arise due to cancer cell-
microenvironment interactions besides the obvious genetic differences [78,79].
The implication of intratumor heterogeneity is that cancer cells within a tumor have different
responses to the same chemotherapeutic drug. Variants of cancer cells that do not respond to a drug
can result in relapse. Epigenetic modifications can take the form of DNA methylation and histone
modification. Hypermethylation of the multi-drug resistance protein 1 (MDR1) gene promoter has
been reported to cause downregulation of certain genes involved in apoptosis. Methylation of the
O(6)-methylguanine DNA methyltransferase (MGMT) gene is known to cause silencing of several
genes. A small fraction of undifferentiated cancer cells have anti-drug properties. These drug-
resistant cancer cells are known to be present in circulation as well as in solid tumors. In addition,
solid tumors have been shown to be a complex mixture of tumor cells, stromal cells and the ECM
[80–85].
Chemotherapy destroys cancer cells mostly through induction of apoptosis by damaging DNA
and inhibiting cell cycle progression [5,86,87]. Over time, cancer cells can acquire diverse strategies
that decrease the efficacy of many therapeutic agents leading to chemoresistance [88]. Resistance to
therapy occurs either as de novo or acquired. Acquired resistance occur when changes in the genetic
makeup of cells over time result in therapy-resistant cells. De novo drug resistance can either be
soluble-factor mediated drug resistance or cell-adhesion mediated drug resistance. Chemokines,
growth factors and cytokines are known to induce the soluble factor mediated drug resistance. The
Int. J. Mol. Sci. 2017, 18, 1586 4 of 29
interaction of cancer cells and stromal components such as fibroblasts and the ECM via surface
receptors such as integrins induce cell-adhesion mediated drug resistance.
The bi-directional communication between cancer and stromal cells is much more complex than
initially perceived. Our data and that from others have shown that the cancer cell-stromal cell
relationship is transient and ever changing [27,55,89,90]. Both tumor cells and stromal cells within
the TM are exposed to different conditions over time including different concentrations of drugs.
Eventually cancer and stromal cells develop a cooperative relationship that appear to benefit cancer
cells. Through the release of soluble factors and the ECM, stromal cells determine the conditions
within the TM. Stromal cells such as fibroblasts and mesenchymal stem cells have been the subject of
many drug resistance studies to date. A summary of the various mechanisms known to be involved
in cancer cell chemoresistance is shown in Figure 1. These mechanisms include enhanced survival
signaling, enhanced drug inactivation, reduced drug uptake, enhanced DNA repair processes and
inhibition of apoptosis [91].
Figure 1. Schematic representation of processes that have been implicated in the development of
chemoresistance. Some of these processes include enhanced survival signaling, enhanced drug
inactivation, enhanced drug export, reduced drug uptake, inhibition of apoptosis, and increased
production of extracellular matrix (ECM) proteins and inhibition of cytoskeleton organization
(adapted from [92]).
3. Tumor Microenvironment
The dynamic nature of the TM during malignant progression underscores the need to study its
role in disease progression. Importantly, the role of the cellular and non-cellular components in
tumor initiation and progression needs to be investigated. Solid tumors are more than just a lump of
cancer cells. Beside stromal cells, non-cellular components of the TM include the ECM and soluble
growth factors [93–98]. The interaction between cells and their respective microenvironment is key
for cellular growth and the maintenance of homeostasis. So it is for tumor growth. Though the
gradual accumulation of genetic lesions creates the initial ‘spark’ necessary for disease initiation it is
widely acknowledged that the TM play a critical role at every stage of malignant progression. Cancer
cell-microenvironment interactions impacts on disease initiation, development and ultimately
metastasis. Understanding the role of the TM in disease progression and chemotherapy is now
Int. J. Mol. Sci. 2017, 18, 1586 5 of 29
considered central to cancer eradication. Initially thought to be only due to genetic lesions in cancer
cells, the heterogeneous nature of tumors is now understood to be of microenvironmental origin as
well. Both cellular and non-cellular components of the TM contribute towards the tumor
heterogeneity observed in solid tumors. By contributing towards the tumor heterogeneity the TM
components ultimately play a part in the development of chemoresistance. The crosstalk between
tumor cells and their microenvironment makes this relationship very complex. However, the
plasticity of the tumor stroma affords scientists an opportunity to devise therapeutic strategies that
can allow most TM members to acquire anti-tumorigenic properties. It is also possible to convert pro-
tumorigenic TM constituent members to become anti-tumorigenic.
In normal tissues a homeostatic environment is maintained with most cells maintaining their
differentiated states and well defined boundaries between tissue compartments. Tumor initiation and
progression is associated with disruption of tissue architecture and organization [99–101]. An
environment that was tumor inhibiting becomes permissive and supportive to tumor growth and
metastasis [80,83,90,102–104]. The TM (Figure 2) is now identified as a leading factor that influences
cancer cell proliferation, metastasis and anticancer drug efficacy [105–107]. Normal cellular processes
and tissue homeostasis are reversed in tumors, as tumor cells bypass or override necessary
homeostatic control measures. Cellular mechanisms of surrounding cells and the effect of non-
cellular components is basically hijacked by cancer cells with the ultimate goal of ensuring cancer
cells survival. Several anti-tumorigenic cells such as fibroblasts and macrophages are converted into
tumor-promoting cells, releasing soluble factors such as growth factors and proteases needed by
tumor cells to burrow through the ECM and support accelerated tumor cell growth [60,108].
Fibroblasts and macrophages are converted to cancer associated fibroblasts (CAFs) and tumor
associated macrophages (TAMs) via the action of tumor-released factors such as TGF-β and platelet
derived growth factor (PDGF). Both tumor-associated fibroblasts and macrophages are known to
participate in this pro-tumorigenic process. Importantly CAFs are known to synthesize and deposit
large quantities of thick ECM fibers, thus contributing to deregulated homeostasis. CAFs also
contribute towards cancer cell invasion and metastasis through synthesis of metastasis-promoting
ECM proteins such as fibronectin and periostin and the release of matrix metalloproteases. This
allows tumor cells to lose their attachment to the ECM and acquire mesenchymal behavior. The origin
of CAFs in solid tumor is controversial. The most straight forward suggestion is that they are of
fibroblast origin. Through the action of tumor-derived factors normal fibroblasts are converted into
‘activated fibroblasts’ also termed CAFs with the function of bidding for tumor cell survival. Several
studies have suggested that they are of endothelial origin. Yet other studies appear to show that
mesenchymal stem cells can be converted to CAFs. Our studies support this suggestion. Tumor-
released TGF-β appears to contribute to mesenchymal stem cells conversion to α-smooth muscle
producing CAFs.
TAMs are known to locate to the leading edge of tumors where they release matrix
metalloproteases needed to degrade the ECM. TAMs also contribute to the increased levels of growth
factors such as EGF leading to tumor cell migration. The plasticity of macrophages allows them to act
both as pro-tumorigenic and anti-tumorigenic depending on the surrounding environments and
existing conditions. Through the release of pro-inflammatory cytokines, macrophages present
antigens and play an anti-tumorigenic role in the TM. However, activated macrophages can be pro-
tumorigenic through production of type II cytokines. Macrophages also help tumor cells intravasate
into blood vessels. TM conditions such as hypoxia and acidity play significant roles in the activation
of macrophages, with macrophages appearing to be attracted to regions of low oxygen tension.
Localized selective pressures such as hypoxia and acidity select for stromal cells that ensure the
survival of cancer cells. Several reports have also s hown that the presence of gr owth factors and micro
RNAs can drive activated macrophages back to normal leading to tumor regression. Thus resident
macrophages can be targeted in the TM to have anti-tumorigenic properties. Due to the presence of
several components within the TM, tumor cells are exposed to chemotherapeutic drugs in a gradient
fashion. The ECM by forming thick fibers within the tumor present a physical barrier to diffusion of
chemotherapeutic drugs [109–114].
Int. J. Mol. Sci. 2017, 18, 1586 6 of 29
Figure 2. Tumor Microenvironment. The tumor microenvironment consists of several cells including
cancer cells, mesenchymal stem cells (MSCs), endothelial cells, fibroblasts, cancer stem cells (CSC),
bone marrow-derived cells (BMDC) as well as Extracellular matrix (ECM). All the cells in the tumor
microenvironment (TM) contribute to tumor progression.
The invasive nature of cancer cells cannot be exhibited without the interplay between tumor
cells and their microenvironment. With an increase in our knowledge of molecular targets and
medicine, clinical therapeutic targets have increased to include components of the TM [28,54,89]. The
crosstalk between stromal and tumor cells involves growth factors, chemokines and cytokines as well
as ECM and can affect the sensitivity of anticancer drugs and pathways involved in apoptosis
[115,116]. Tumors depend on the formation of new blood vessels, through a process called
angiogenesis, in order to increase in size as tumor cells need a constant supply of oxygen and
nutrients [117–119]. Due to tissue disorganization associated with tumors, they tend to have lower
blood flow and therefore less drugs reaches the tumor cells than is administered.
For chemotherapeutic drugs to reach tumor cells in vivo, they have to travel through the blood
vessels [120–123]. Blood flow through a solid tumor is varied and disorganized [124,125]. Most blood
vessels in tumors are dilated and “leaky” compared to those in normal tissues. Thus the vasculature
also influences the response of tumor cells to drugs. The compactness of a solid tumor increases blood
flow resistance and this causes gradients of nutrients and oxygen, meaning that there are different
proliferation rates in different regions of the tumor [126–131]. Nutrient deprivation is also a result of
blood flow resistance. Most chemotherapeutic drugs are designed against highly proliferating cells
and not quiescent cells [132–135]. Thus having tumor cells proliferating at different rates has a major
impact on the effectiveness of chemotherapy. Due to impaired blood flow the clearance of breakdown
products within the tumor can lead to a toxic microenvironment [136–138].
The compactness of a solid tumor and the reduced blood flow leads to either temporary or
chronic hypoxia [139–145]. Cells in hypoxic conditions tends to divide slowly, making them
unresponsive to chemotherapeutic reagents. Hypoxia can result in the activation of genes associated
with angiogenesis and cell survival [146–148]. Many chemotherapeutic drugs cause DNA damage
through generating free radicals [149–151]. Without oxygen however, the cytotoxicity of several
chemotherapeutic drugs whose activity depends on generating free radicals is reduced. In addition,
it has been shown that cancer stem cells (CSCs) tend to be located at the center of a solid tumor [152–
157]. These CSCs are able to withstand lower oxygen levels than the general population of cancer
cells [18,80]. Thus the inability of drugs to reach the center of the solid tumor can result in recurrence
Int. J. Mol. Sci. 2017, 18, 1586 7 of 29
of the tumor even after an apparent successful treatment. It has also been observed that hypoxia can
lead to increased expression of P glycoprotein, which is involved in drug inactivation, resulting in
drug resistance [158]. Hypoxia inducible factor (HIF)-1 is stimulated under low oxygen conditions
and this transcription factor controls many genes involved in survival mechanisms such as
angiogenesis and apoptosis. Several pro-drugs have been developed to be activated under complete
or partial hypoxic conditions. For example, Tirapazamine (TPZ) is activated over a range of oxygen
levels. Due to the varying amounts of oxygen in solid tumors, TPZ activation over time. Thus at some
point tumor cells are exposed to sub-lethal levels of TPZ with the consequent development of
chemoresistance. The pH in the TM can affect the cytotoxicity of anticancer drugs. An acidic
microenvironment can inhibit the activation of many chemotherapeutic drugs [159,160]. Changes in
pH inside and outside of cancer cells can have a lasting effect on chemotherapeutic drugs. The
pressure gradient that exists within the microenvironment also influences the distribution of many
anticancer drugs. The ability of cancer cells to manipulate their microenvironment enables them to
acquire important hallmark properties that are necessary for tumor growth and development.
3.1. Cancer-Associated Fibroblasts (CAFs)
Cancer-associated fibroblasts (CAFs) are activated fibroblasts found in association with cancer
cells. CAFs are the most abundant cells within the TM and are involved in tumor initiation, by
activating signals involve in growth and differentiation, and evade cancer therapy [161,162]. CAFs
secrete growth factors, such as hepatocyte growth factor (HGF), epidermal growth factor (EGF), and
cytokines such as stromal cell-derived factor 1 (SDF-1) and IL-6. Wang et al. showed that secretion of
HGF by CAFs induced resistance to EGF-tyrosine kinase inhibitors in lung cancer cells [163].
Secretion of chemokines and cytokine by CAFs can lead to immune cells infiltration which contribute
to angiogenesis and metastasis [164]. CAFs are known to stimulate the growth of new blood vessels
through the release of growth factors such as vascular endothelial growth factor. Enhanced invasion
of pancreatic cancer cells was observed in the presence of fibroblast-derived SDF-1 and IL-8 was also
found to induce angiogenesis in vitro [165]. CAFs can regulate ECM composition via expression of
matrix metalloproteinases (MMPs), which allows cancer cell adhesion and migration as well as
inhibition of apoptosis by activating PI3K-Akt/PKB as seen in breast cancer models [158]. The
presence of CAFs or transformed fibroblasts is known to activate migratory behavior in cancer cells
through upregulation of integrin expression and cell survival signaling pathways such as the MEK-
ERK and the PI3K-Akt pathways. In prostate cancer, increased secretion of MMP-2 and MMP-9 by
CAFs was associated with the induction of epithelial-mesenchymal transition (EMT), known to be
involve in cancer cell invasion and metastasis [166]. CAFs also secrete IL-6, which promotes cancer
metastasis and chemoresistance through induction of EMT [167]. A study by Conze and colleagues
showed that IL-6 is overexpressed in multidrug resistant breast cancer [168]. In vitro and in vivo
studies have shown that CAFs derived from breast cancer induced tamoxifen resistance through
decreasing estrogen receptor-α (ER-α) levels and IL-6 secretion [169].
3.2. Mesenchymal Stromal/Stem Cells (MSCs)
MSCs have received a lot of attention in cancer biology partly because of their primitive nature
and their ability to generate several other cells types. Through the action of tumor cell-derived factors,
MSCs are recruited to the tumor site where they produce factors needed by cancer cells. MSCs are
found in many adult tissues including bone marrow and adipose tissues [170]. MSCs can self-renew
and differentiate into specialized tissue-specific cell types such as adipocytes, chondrocytes,
fibroblasts and osteoblast [167,170–172]. MSCs are also found in the TM and are known to play an
important role in tumor progression and chemoresistance [172]. MSCs promote tumor growth either
by the secretion of growth factors, or by differentiating into tumor associated fibroblasts (TAFs)
[55,170,173,174]. The origin of TAFs or CAFs in the TM is still debatable. TAFs are a heterogeneous
cell population and are commonly identified by α-smooth muscle actin (α-SMA) and vimentin
expression which is indicative of an ‘activated’ myofibroblast-like phenotype [175,176]. One source
of TAFs that has been touted is MSCs present in the tumor stroma [175,176]. We present data from
Int. J. Mol. Sci. 2017, 18, 1586 8 of 29
an extension of our previous publication [90], showing that long term co-culture of esophageal
WHCO1 and breast cancer MDA MB 231 cells with human MSCs trigger hMSCs differentiation into
‘tumor associated fibroblasts’ via the TGF-β/Smad signaling pathway.
In our study, we evaluated the effect of esophageal WHCO1 and breast MDA MB 231 cancer
cells on Wharton’s Jelly-derived mesenchymal stromal/stem cells (WJ-MSCs) gene expression over
24 days of co-culture. The expression of α-SMA, the most reliable marker of tumor associated
fibroblasts (TAFs) and vimentin showed a clear and gradual increase in WJ-MSCs up to a maximum
at day 16 in our co-culture system (Figure 3A,B). TGF-β is one of the growth factors released by cancer
cells in order to evade immune detection in vivo and can increase expression of proteins such as α-
SMA and vimentin. Treatment of WJ-MSCs with 1 µM 5-azacytidine resulted in their differentiation
into myofibroblastic lineages expressing increased levels of α-SMA and type I collagen. Addition of
exogenous TGF-β (10 µM) and treatment of naïve MSCs with 5-azacytidine (1 µM) up to 48 h resulted
in increased levels of α-SMA and type I collagen similar to MSCs co-cultured for 16 days (Figure 3C–
F). Our observations show that over time MSCs exposed to esophageal and breast cancer cells
differentiate and express markers of the myofibroblastic lineage. Many studies have shown that
ACTA2 (α-SMA) gene transcription is regulated through the interactions of several signaling
pathways. To substantiate these results, the TGF-β inhibitor SB 431542 (10 nM) was added to the co-
culture media. Addition of SB 431542 decreased the α-SMA protein levels in MSCs exposed to
WHCO1 and MDA MB 231 cells (Figure 4A,B). As an orthogonal approach, suppression of TGF-β
expression in co-cultured MSCs through the use of TGF-β siRNA resulted in decreased α-SMA
protein levels (Figure 4C,D). In addition, TGF-β knockdown in both WHCO1 and MDA MB 231 cells
during co-culture decreased α-SMA protein levels in MSCs (Figure 4E,F). We also observed that
Smad2 increased in WJ-MSCs cocultured with WHCO1 and MDA MB 231 cells (data not shown).
These results demonstrate that the TGF-β/Smad signaling pathway is involved in the differentiation
of MSCs into TAFs and that TGF-β probably is probably produced by both MSCs and cancer cells.
Thus it is possible that cancer cells can attract MSCs to the tumor site and the MSCs can become
part of the TM as TAFs. However other cells can also be a source of TAFs. TAFs are known as
accomplices in increased tumor growth, metastasis and chemoresistance [177,178]. MSCs can also
promote drug resistance both by secreting protective cytokines, and by preventing cancer cell
apoptosis [177]. Our data show that MSCs can be transformed to CAFs by cancer cells through release
of growth factors such as TGF-β (Figure 5). Importantly, both WHCO1 and MDA MB 231 cells co-
cultured with “cancer cell activated” WJ-MSCs survive treatment with paclitaxel and cisplatin better
than WHCO1 and MDA MB 231 cancer cells alone (Figure 6).
Int. J. Mol. Sci. 2017, 18, 1586 9 of 29
Figure 3. Cancer cells trigger MSCs differentiation into tumor associated fibroblasts via the
transforming growth factor-β (TGF-β) /Smad signaling pathway. For the co-culture experiments cells
were co-cultured in 6-transwell plates (size of pore: 0.4
µ
m, Polycarbonate membrane, Costar,
Corning, Cambridge, MA, USA). Mesenchymal stem cells (5 × 10
5
cells) were cultured in the upper
insert and cancer cells (WHCO1 and MDA MB 231) (5 × 10
5
cells) were cultured in the lower compartment.
Empty inserts were used for the control group (no cells) and a mixture of MSCs medium and cancer
cell medium (1:1) was used. Medium was changed every 3 days for longer incubation periods and
fresh TGF-β and reagents were added. TGF-β and all reagents were added to the media to the final
concentrations as shown. At specific time points or at the end of the experiment, cells (cancer cells
and MSCs) were harvested and used in various analyses. (A) Western blot analysis of lysates from
MSCs co-cultured with WHCO1 cells for 24 days showing α-smooth muscle actin (α-SMA) and
vimentin protein levels. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a
loading control. (B) Real time quantitative reverse transcription polymerase chain reaction (RT-qPCR)
analysis was performed to assess the expression of Actin, alpha2, smooth muscle, aorta (ACTA2) (α-
SMA gene) in MSCs co-cultured with WHCO1 and MDA MB 231 cancer cells over a 24 day period;
(C,D) western blot analysis of lysates from MSCs co-cultured with WHCO1 cells for 16 days or after
the addition of 10 nM TGF-β (C) or 1 µM 5-azacytidine (D) for 48 h showing the expression of type I
collagen and α-SMA; (E,F) western blot analysis of lysates from MSCs co-cultured with MDA MB 231
cells for 16 days or after the addition of 10 nM TGF-β (E) or the addition of 1 µM 5-azacytidine (F) for
48 h showing the expression of type I collagen and α-SMA.
Both WHCO1 and MDA MB 231 cancer cells co-cultured with the above WJ-MSCs for 16 days
survived treatment with cisplatin and paclitaxel better than WHCO1 and MBA MB 231 cell alone
(Figure 6). It is evident that the presence of WJ-MSCs, possibly through the release of protein factors,
protected the cancer cells from the effect of the drugs used.
Int. J. Mol. Sci. 2017, 18, 1586 10 of 29
Figure 4. WHCO1, MDA MB 231 cells and MSCs secrete TGF-β. Mesenchymal stem cells (5 × 10
5
cells)
were cultured in the upper insert and cancer cells (WHCO1 and MDA MB 231) (5 × 10
5
cells) were
cultured in the lower compartment as described in Figure 3. At specific time points or at the end of
the experiment, cells (cancer cells and MSCs) were harvested and used in various analyses. (A,B) TGF-
β inhibitor SB431542 was added to the co-culture media to a final concentration of 10 µM. Co-culture
was continued for 16 days after which α-SMA protein levels was determined by western blot analysis.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C,D) MSCs
were treated with TGF-β siRNA to a final concentration of 100 nM and co-culture was continued for
16 days. To maintain knockdown of TGF-β, subsequent transfections were done every other three
days till the end of the experiment. Western blot analysis was performed to evaluate the α-SMA
protein levels in MSCs lysates; (E,F) WHCO1 and MDA MB 231 cells were treated with TGF-β siRNA
to a final concentration of 100 nM and co-culture was continued for 16 days. To maintain knockdown
of TGF-β, subsequent transfections were done every other three days till the end of the experiment.
Western blot analysis was performed to evaluate the α-SMA protein levels in MSCs lysates.
Figure 5. Our co-culture experiments have shown that TGF-β plays an important role in the
interaction between cancer cells and MSCs. Our data show that in the long term, WHCO1 and MDA
MB 231 cancer cell exposed-Wharton’s Jelly derived-MSCs differentiate into tumor associated
fibroblasts (TAFs) through a TGF-β/Smad-mediated process.
Int. J. Mol. Sci. 2017, 18, 1586 11 of 29
Figure 6. Co-cultured cancer cells survive treatment with cisplatin and paclitaxel better than WHCO1
and MDA MB 231 cells alone. WHCO1 and MDA MB 231 cancer cells (5 × 10
5
) were cultured alone or
co-cultured with WJ-MSCs for 16 days as described in Figure 3. Empty inserts were used for the
control group (no MSCs) and a mixture of MSCs medium and cancer cell medium (1:1) was used.
Medium was changed every 3 days for longer incubation periods. At the end of the incubation, the
same number of WHCO1 and MDA MB 231 cancer cells were treated with increasing concentrations
of paclitaxel and cisplatin for 48 h as shown above. After 48 h, cells were counted with a Countess
Cell counter using the Trypan Blue exclusion method. Cells were expressed as a percentage of cells
treated with 0.1% DMSO (control). Experiments were repeated three times. * p < 0.05.
3.3. The Role of the Extracellular Matrix in Chemotherapeutic Resistance
The ECM is the crucial non-cellular component of the TM and consists of mainly glycoproteins,
proteins and proteoglycans [179]. The ECM plays key roles in tissue maintenance and function. The
ECM regulates cellular behavior directly and indirectly [179]. Due to the crucial roles the ECM plays
in vivo, a number of mechanisms are involved in the regulation of ECM production, degradation and
remodeling [180]. Perturbation of these mechanisms can promote pathological conditions such as
fibrosis and cancer [179,181]. The physical properties of the ECM determines its role as a scaffolding
to maintain tissue structure and function [179]. It also controls the behavior of cells through
proliferation, differentiation and signaling pathways [182,183]. The signaling abilities of the ECM’s
biochemical properties permits interactions between cells and their environment [179]. The
composition and structure of the ECM is precisely tuned according to the needs of the surrounding
cells. This is achieved through the release of soluble factors such as growth factors and chemokines.
Besides serving as a physical scaffold onto which cells are anchored, the ECM provides signals
necessary for cellular growth, migration and differentiation. Both physical and chemical properties
of the ECM can influence cellular behaviors and these properties can be altered in cancer. ECM
remodeling involves many enzymes, including matrix degrading enzymes including MMPs, lysyl
oxidase (LOX), tissue inhibitors of metalloproteinases (TIMPs) and cathepsins [60]. Thus the
composition of the ECM in cancer is a very important factor in deciding the efficacy of many drugs.
Most cancer cell behavior is affected by the surrounding ECM. Effective cancer treatment requires
knowledge of the cancer-ECM interactions in addition to the interactions with other TM components.
Due to its plasticity, the ECM has been ascribed both pro-tumorigenic and anti-tumorigenic
properties. Initially thought to be a passive bystander, the ECM is emerging as a key player in
malignant initiation, progression and chemoresistance. It is likely that the ECM inhibits early tumor
Int. J. Mol. Sci. 2017, 18, 1586 12 of 29
growth and at later stages becomes pro-tumorigenic. Several studies have shown that the ECM
present in the TM influence disease progression and is a major indicator of clinical prognosis. High
levels of protease inhibitors within the ECM is associated with a good clinical outcome whilst high
levels of surface receptors such as integrins and MMPs are associated with a poor outcome and
relapse of disease. The ECM and its associated proteins, now referred to as the ‘matrisome’, is
synthesized by different types of cells within the TM. The manipulation of the ECM and its ligands
offers an attractive therapeutic avenue to eradicate cancer. Many studies have shown that matrix
stiffness can influence cellular adhesion to surfaces, migration, differentiation and even proliferation
[27,89,184–186]. Cells migrating to other regions have been shown to be softer and more pliable than
benign cells. In general the tumor surrounding-ECM has been found to be stiffer than the ECM
surrounding healthy tissues [23,51,186–191]. The stiffening observed in cancer is thought to be linked
to fibrosis and deposition of collagen as shown in breast cancer [139,192–195]. Studies have showed
that a stiff microenvironment induces tumor progression and malignancy through integrin signaling
as a result of ECM tumor-associated remodeling [106,107,179,196]. Several studies have reported that
tumor metastasis is promoted by ECM stiffening through the action of lysyl oxidase and the increased
deposition of collagens and fibronectin. Stiffened ECM downregulates the expression of genes
associated with cell cycle inhibition. MicroRNAs are reportedly induced by matrix stiffening and
these microRNAs downregulates the expression of PTEN, a tumor suppressor protein. This has the
effect of increasing PI3K-Akt activity, a survival pathway implicated in tumor growth and metastasis.
Inhibition of lysyl oxidase (LOX) softens the ECM [197]. ECM abnormality is known to result in
cancer cell growth, survival, migration and anticancer drug resistance [179]. However, the ECM is
composed of many constituents and as such it is difficult to pinpoint the role of each component on
tumor progression. It is likewise difficult to recapitulate the in vivo situation in an in vitro setting in
order to study the effect of each individual TM component on tumor progression and
chemoresistance.
Several ECM proteins have been associated with resistance to chemotherapy. Fibronectin has
been associated with increased migration of several cancer cells [198–201]. Changes in ECM elasticity
and stiffness are some of the factors known to affect drug delivery to cancer cells. ECM stiffness has
been associated with tumor initiation in many cancer types. Dysregulation of ECM remodeling can
result in the evasion of apoptosis by mutant cells, enlargement of CSC pool and disruption of tissue
polarity [179]. Like normal cells, tumor cells need nutrients, oxygen and waste exchange [179]. These
needs are met by angiogenesis, which results in increase in tumor size, and by lymphangiogenesis,
the growth of lymphatic blood vessels [179]. Diffusion and pressure are associated with drug delivery
in the interstitial spaces. ECM remodeling promotes drug resistance in the form of physical barrier
that dissolve or delay drug delivery [202]. Interaction of the ECM with other cells has been considered
to be involved in the promotion of chemoresistance through the activation of survival proteins [203].
These survival pathways include PI3K/AKT, p53 and MAPK which have been demonstrated to be
activated upon the binding to ECM. Cancer should no longer be viewed as a disease of mis-regulated
or mutated genomes. That tumors are like organs is illustrated more by their dependent on
angiogenesis. The tumor’s need for nutrients and oxygen, supplied via the bloodstream, makes
angiogenesis a necessity for tumor growth. Several factors are released by stromal cells within the
TM to initiate and to allow tumor vascularization to occur.
3.3.1. Collagen
Collagen is the main ECM protein synthesized in several tissues. Collagen is known to promote
cancer cell clustering and invasiveness. Collagen is an ECM protein for scaffolding and provides
tissue strength and support. The structural organization and level of collagens within tissues can
indirectly influence drug efficacy. Type I and IV collagen can promote drug resistance through the
interaction with integrins on cancer cells [204,205]. An environment rich in collagen is known to
activate several signaling pathways such as MEK-ERK and the Wnt/B-catenin pathways. Increased
expression of ECM proteins such as collagen by cancer cells further limits the diffusion of
chemotherapeutic drugs into cancer tissues [206–208]. Drug delivery is significantly limited by
Int. J. Mol. Sci. 2017, 18, 1586 13 of 29
tortuous and dense tumor ECM [207,208]. The expression of collagen play a crucial role in both drug
resistance at the cellular level and tissue mediated drug resistance [206]. Interaction of ECM proteins
including collagens with cancer cells can alter the cancer cell response to the presence of
chemotherapeutic reagents [206,209]. Several studies have shown that high levels of expression of
collagen genes was associated with drug resistance in ovarian and breast cancer cell lines [206,210].
The time needed for drugs to penetrate through collagen fibers before reaching cancer cells is
lengthened, which can result in drug resistance [211].
ECM that contains large amount of collagen enhances tumor progression and invasiveness
[204,210,212]. Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive human
malignancies and a leading cause of cancer mortality. A unique molecular hallmark associated with
PDA is the presence of dense collagen-rich fibrosis [213]. Increased expression of type I collagen has
been associated with increased risk of metastasis in several cancer types [213–215]. Pancreatic cancer
cells cultured in 3D collagen showed decreased sensitivity to gemcitabine therapy and increased
proliferation despite drug treatment [213,216]. Collagen type XI α1 (COL11A1) is a member of
collagen family, which is the important component of the interstitial ECM. Overexpression of
COL11A1 is associated with progression of several cancers and poor survival [177,178,217]. COL11A1
expression has been demonstrated to be high in cisplatin-resistance ovarian cancer cells [218,219]. In
addition, COL11A1 promotes ovarian cancer cell chemoresistance through the activation of signaling
pathways such Akt and PDK1 pathways [219]. The deposition of collagens, expression of LOX and
increased ECM stiffness in breast cancer resulted in increased adhesion and PIK3 activity [197]. These
findings suggest that the TM induces chemo-protection and increases cancer cell survival through
remodeling of components in the ECM.
3.3.2. Laminin
Laminin constitutes a major family of the ECM proteins in the basal lamina and is known to
affect cellular processes such as differentiation, adhesion and migration [220,221]. This family of ECM
proteins plays a key role in the invasive behavior of several cancer cells. Laminin-332 (LN-332) is a
heterotrimer made of β3, α3 and γ2 chains that has been shown to be key in cell adhesion and cancer
metastasis [220–223]. Laminin-332 is involved in maintaining the self-renewal abilities of CSCs and
has been implicated in resistance to sorafenib and doxorubicin [223]. Laminin β3 chain expression is
associated with poor outcome in colorectal cancer and is related to chemoresistance to 5-FU-based
chemotherapy regimens [222]. Another family member, Laminin β1 (LAMB1), is increased in
paclitaxel-resistance cell lines [220].
LN-332 can bind the integrin α3β1 receptor which is reported to be enhanced in gefitinib
resistance in hepatocellular carcinomas (HCCs) [224]. LN-integrin interactions increase cell survival
and chemoresistance through the activation of mTOR [223,225]. It was demonstrated that LN-332
does not only protect hepatic cancer cells against therapeutic drugs but it promotes cell proliferation
upon sorafenib exposure [223]. LN-332 and its γ2-chain play a key role in CSC self-renewal and
differentiation and in maintaining and supporting quiescence as part of the human hepatic cancer
stem cell niche [223]. LN protects pancreatic cells from gemcitabine induced apoptosis and
cytotoxicity [226]. The protection of pancreatic cells by LN is a result of the activation of focal
adhesion kinase (FAK), itself a result gemcitabine resistance induced apoptosis [226].
3.3.3. Fibronectin
Fibronectin (FN) plays a crucial role in growth, differentiation, adhesion and migration [227].
Fibronectins are glycoproteins that attach cells to collagen fibers in the ECM, facilitating movement
of cells through the ECM [227]. Fibronectin binds to cell surface integrins and collagen resulting in
reorganization of the cell’s cytoskeleton allowing movement of cells. Fibronectin has been found to
be key in wound healing and in cancer initiation and progression [200]. Increased tumorigenicity and
resistance to apoptosis-inducing therapeutic drugs in lung cancer is achieved when lung carcinoma
cells adhere to FN [228]. Overexpression of FN at the invasion front and in tumor stroma is observed
in head and neck squamous cell carcinomas (HNSCCs) [229]. Increased expression of FN in HSCC is
Int. J. Mol. Sci. 2017, 18, 1586 14 of 29
associated with decreased survival of patients [200]. FN induced migration of carcinoma collectives
through αvβ6 and α9β1 integrins [200]. Small-cell lung cancer cells (SCLC) that adhered to laminin,
collagen and fibronectin were found to be protected from apoptosis induced by chemotherapeutic
drugs compared to those that were grown on plastic [228]. FN facilitated non-small cell lung
carcinoma cell (NSCLC) growth and reduced apoptosis through induction of cyclooxygenase-2
(COX-2) and activation of integrin α5β1 [230]. These effects were correlated with activation of many
kinase signaling pathways such as MEK-ERK and Rho kinase signaling pathways [231]. FN adhesion
led to protection of tumor cells against docetaxel-induced apoptosis [223,231,232].
3.3.4. Periostin
Periostin, a secretory protein also known as osteoblast-specific factor 2, is expressed as an
extracellular matrix protein [233,234]. It is a cell adhesion protein that plays important roles in tooth
and bone tissue homeostasis and development. It has also been found to be key in cardiac
development and healing [220,233,234]. Overexpression of periostin has been implicated in many
types of cancer such as gastric, colon, esophageal, ovarian, thyroid, lung, breast and head and neck
carcinomas [233,234]. It regulates cell-matrix interactions through binding to fibronectin, type I/V
collagen and tenascin C [234]. Periostin is a ligand for integrins such as αvβ3, αvβ5 and α6β4 [235].
It interacts with several signaling pathways such as Notch 1 and B-catenin signaling. It has been
demonstrated that periostin in normal esophagus is significantly lower than in esophageal squamous
cell carcinoma (ESCC) [234,235]. Periostin induces the PI3K-Akt signaling pathway by binding as a
ligand to αvβ3 and αvβ5 integrins in esophageal cancer [176]. Periostin also increases cancer cell
proliferation and EMT in nicotine-induced gastric cancer [176]. Periostin is overexpressed in gastric
cancer cells that are resistance to cisplatin and 5-fluorouracil (5-FU) [234]. It was shown that periostin
levels correlated with tumor angiogenesis and tumor recurrence [236]. In epithelial ovarian
carcinoma, periostin induced Akt phosphorylation to increase resistance to paclitaxel [237]. Periostin
not only serves as a prognostic factor for clinical outcome but also plays a role in resistance in several
tumor cell types [238].
4. Strategies to Overcome Chemoresistance
To date most remedies for cancer have tended to focus directly on intrinsic characteristics of
cancer cells. This is despite the fact that a tumor is a heterogeneous mixture of cancer cells, stromal
cells and the extracellular matrix. Indeed, heterogeneity occurs at every level in cancer cells. Targeting
stable stromal cells, with less or no genetic mutations, therefore is appealing. Stromal cells, due to
their stable genetic makeup, are less likely to develop resistance to therapeutic agents. Perturbing or
removing all the supporting cells and non-cellular components in the TM should ultimately lead to
tumor regression or tumor cell reversion. Most stromal components can be engineered to be anti-
tumorigenic due to their pliable behaviors. Given the heterogeneity evident in all cancers, it is
imperative to study the TM as a possible avenue for cancer treatment. Indeed, several reports on
combination therapies against both cancer and stromal cells appear to show promising outcomes in
animal studies and in early phases of clinical trials. Several important aspects of TM-directed
therapies need to be researched further. For example, the use of MSCs in cancer treatment needs to
be studied further as several studies have shown that over time MSCs can be converted to ‘cancer
associated fibroblasts’. Thus benefits derived from MSCs might be negated in the long run if MSCs
are converted to CAFs. We advocate for the inclusion of TM components in in vitro experimental
systems in order to delineate the role of TM components on cancer cell growth and metastasis. Novel
animal models that are able to initiate tumors within native tissues will advance our understanding
of the involvement of the TM in malignant initiation and development.
The efficacy of chemotherapeutic drugs may be impaired in several ways including limited
delivery of drugs, cell death inhibition, drug inactivation, drug target alteration, EMT, the
involvement of the TM or any combination of these factors. Therefore, combination therapy appear
to be a reasonable solution to prevent drug resistance in many cancer types. Several novel ways have
been suggested to overcome drugs resistance due to microenvironmental factors. Before treatment,
Int. J. Mol. Sci. 2017, 18, 1586 15 of 29
administration of antiangiogenic therapy can help to remove extra capillaries and abnormal blood
vessels leading to a reduction in the pressure of the interstitial fluid [239–242]. Other suggestions
include damaging already existing blood vessels leading to solid tumor vessel permeability and
increased drug delivery [184,243–245]. It should be recalled however that several strategies that target
tumors inadvertently affect normal tissues as well.
Another effective way to improve drug penetration and efficacy is to inhibit sequestrations of
drugs in cellular compartments such as endosomes [246–251]. Yet another strategy involve modifying
the ECM to enable enhanced penetration of drugs into solid tumors [187,252–254]. Caution must be
exercised however as ECM modification can promote cancer metastasis [255–258]. Modifying or
degrading even part of the ECM might create a highway through which cancer cells can migrate to
other tissues or organs [257,259–261].
The ABC transporters are an important mechanism for drug resistance. As mentioned above,
ABC transporters play a role in protecting tissues from toxins but they also play a role in the uptake
of drugs and delivery to their target molecules. As a result, targeting ABC transporters could be used
in the treatment of cancer in future. Great interest has been shown towards the manufacture of anti-
ABC drugs. Inhibitors against P glycoprotein may be considered to be the best treatment of cancer
and prevention of MDR. Additionally, Chen et al. showed that activation protein kinase D isoform 2
(PKD2) is an important modulator of MDR and P-glycoprotein expression in paclitaxel-treated breast
cancer cell lines [262]. The same study also demonstrated that shRNA knockdown of PDK2 in breast
cancer cell lines resulted in significant decrease in resistance to anticancer agent paclitaxel [262].
These results suggest that inhibition of MDR and P-gp through the inactivation of PKD2 might be a
potential strategy to overcome chemoresistance. However, due to the specificity of the anti-ABC
drugs, each patient’s ABC profile and expression levels will need to be determined before treatment
using these drugs. The use of antibodies has been successful in increasing the efficacy of anticancer
drugs and reducing growth factors which are overexpressed in breast cancer. The use of trastuzumab
against the human epidermal growth factor receptor 2 (HER2), a protein involved in the development
of breast cancer, was found to increase the efficacy of chemotherapy in metastatic breast cancer that
overexpresses HER2 [263]. Another example is the monoclonal antibody cetuximab, which
specifically blocks EGFR that is overexpressed in several cancers. Cetuximab is effective in patients
who were resistant to treatment with fluorouracil and irinotecan in colorectal cancer [264].
Epithelial-to-mesenchymal transition (EMT) is one of the factors that contributes to
chemoresistance and therefore future drug discovery targeting EMT should be considered [62].
Drugs targeting the TM are better potential strategies to overcome chemoresistance. More especially
by targeting the hypoxic regions of tumors to improve drug delivery. Using combinational therapies
targeting different stromal cells (such as MSCs, CAFs, ECM) found in the TM can enhance the efficacy
of many antitumor agents. This can be achieved by understanding the mechanisms of cell-ECM
interactions and using drugs that inhibit components involved in ECM remodeling. How
chemotherapy affects the TM stromal components is only now becoming clear. Several strategies
targeting stroma-initiated signaling are being explored to combat drug resistance. Very few studies,
however, have focused on how stromal components respond to chemotherapy and how this
contribute to chemoresistance. Recent studies have shown that stroma cells develop drug resistance
in the same way as cancer cells, and that stromal cell-drug resistance is vital for cancer cell drug
resistance. Targeting the TM and stroma-initiated signaling might be effective ways of killing cancer
cells if done in combination with conventional therapy. The protective effect provided by stromal
cells to cancer cells can be blocked through selective inhibition of specific receptors.
Lastly, many in vitro models utilized during drug development do not recapitulate the in vivo
tumor environment. Most drug development assays are done on 2D cancer cell monolayer cultures
where cancer cells are fully exposed to chemotherapeutic reagents do not show drug resistance. Of
late, several 3D models have been developed to study cancer cell behavior in vitro. Multicellular
tumor spheroids are being used as in vitro tumors and novel information is being obtained. While
3D culture of cancer cells recapitulate the in vivo tumor environment better than 2D culture, cancer
cell drug resistance in 3D culture is not only due to cellular changes. Drug distribution in 3D is
Int. J. Mol. Sci. 2017, 18, 1586 16 of 29
affected by many other factors such as the presence of ECM components and soluble factors within
the microenvironment milieu. The involvement of biomedical engineers in the development of 3D
culture models is important since many of these models take into account ECM biophysical
properties and controllability in designing the best model. Finally, to fully recapitulate the 3D setting
it will be important to include a vascular component such as endothelial cells.
5. Conclusions
The future success of cancer therapy is dependent in part on the ability to identify and target
mechanisms and pathways involved in chemotherapy resistance. Several targeted strategies
including the use of monoclonal antibodies still require a proper understanding of chemoresistance
before successful treatment is achieved. Strategies to inhibit processes such as EMT and the removal
of supporting stromal cells and the ECM are some of the ways being envisaged to treat cancer in the
future. Importantly, the role of the TM components in tumor development and metastasis is now
under greater scrutiny. The efficacy of chemotherapy is impaired by reduced delivery of drugs to
tumor cells leading to resistance of many anticancer agents. Drug inactivation, inhibition of
apoptosis, EMT and the TM play an essential role in events leading to drug resistance and relapse.
Tumor associated ECM also plays a role in chemoresistance by providing an environment that
stimulates survival pathways. Unlike tumor cells, the components of the TM do not harbor genetic
mutations. TM- or stromal directed therapies appear to be gaining ground as they can be used in
combination with conventional therapies to control malignant progression. Unfortunately, as many
studies have shown, several therapeutic targets identified in stromal cells are common to tumor cells
as well, presenting a huge conundrum to scientists. Clinical trials targeting a dysregulated TM show
some avenues that can be taken to engineer stromal cells to modulate conventional therapeutic
efficacy. Therefore, multiple drugs targeting the TM and inhibiting tumor-stroma interactions may
be important strategies to overcome chemoresistance and improve cancer treatment.
Acknowledgments: The funding for this research was provided by the National Research Foundation (NRF) of
South Africa (Grant Number: 91457: RCA13101656402), International Centre for Genetic Engineering and
Biotechnology (ICGEB) (Grant Number: 2015/0001), the South African Medical Research Council in terms of the
MRC’s Flagships Awards Project SAMRC-RFA-UFSP-01-2013/STEM CELLS, the SAMRC Extramural Unit for
Stem Cell Research and Therapy Unit, the National Research Foundation and the Institute for Cellular and
Molecular Medicine of the University of Pretoria and the University of Cape Town.
Author Contributions: Dimakatso Alice Senthebane and Kevin Dzobo performed all experiments and analyzed
the data. Kevin Dzobo, Collet Dandara, Michael S. Pepper, and M. Iqbal Parker developed the experimental
design. All authors proofread and corrected the manuscript. Dimakatso Alice Senthebane and Kevin Dzobo
wrote the main body of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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... It is universally accepted that cancer has major hallmarks including the presence of genomic instability and mutations, unrestricted growth, evasion of growth suppressors, resisting cell death, enhanced inflammation, enhanced metabolism, the ability to promote angiogenesis, invasion and metastasis [1,2]. It is also scientifically accepted that tumors are more than just tumor cells and include recruited stromal cells and the non-cellular component, the extracellular matrix (ECM) ( Figure 2) [3][4][5][6]. Stromal cells and the ECM are active participants during tumorigenesis, starting as anti-tumorigenic during the initial stages to being pro-tumorigenic over time and contributing to the attainment of specific cancer hallmarks [3][4][5][6]. Thus, the study and understanding of cancer and tumorigenesis now extends beyond tumor cells to include the stromal cells and the ECM, which make up the tumor microenvironment (TME) [3,[5][6][7][8][9][10][11][12][13][14][15][16][17][18]. ...
... It is also scientifically accepted that tumors are more than just tumor cells and include recruited stromal cells and the non-cellular component, the extracellular matrix (ECM) ( Figure 2) [3][4][5][6]. Stromal cells and the ECM are active participants during tumorigenesis, starting as anti-tumorigenic during the initial stages to being pro-tumorigenic over time and contributing to the attainment of specific cancer hallmarks [3][4][5][6]. Thus, the study and understanding of cancer and tumorigenesis now extends beyond tumor cells to include the stromal cells and the ECM, which make up the tumor microenvironment (TME) [3,[5][6][7][8][9][10][11][12][13][14][15][16][17][18]. ...
... Stromal cells and the ECM are active participants during tumorigenesis, starting as anti-tumorigenic during the initial stages to being pro-tumorigenic over time and contributing to the attainment of specific cancer hallmarks [3][4][5][6]. Thus, the study and understanding of cancer and tumorigenesis now extends beyond tumor cells to include the stromal cells and the ECM, which make up the tumor microenvironment (TME) [3,[5][6][7][8][9][10][11][12][13][14][15][16][17][18]. Stromal cells include normal fibroblasts, cancer associated fibroblasts (CAFs), cancer associated macrophages (CAMs), mesenchymal stem cells (MSCs), inflammatory cells and endothelial cells [3,7,11,13,17,19,20]. ...
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Tumorigenesis is a complex and dynamic process involving cell-cell and cell-extracellular matrix (ECM) interactions that allow tumor cell growth, drug resistance and metastasis. This review provides an updated summary of the role played by the tumor microenvironment (TME) components and hypoxia in tumorigenesis and highlight various ways through which tumor cells reprogram normal cells including into phenotypes that are pro-tumorigenic including cancer associated- fibroblasts, -macrophages and -endothelial cells. Tumor cells secrete numerous factors leading to transformation of a previously anti-tumorigenic environment into a pro-tumorigenic environment. Once formed, solid tumors continue to interact with various stromal cells including local and infiltrating fibroblasts, macrophages, mesenchymal stem cells, endothelial cells, pericytes, and secreted factors and the ECM within the tumor microenvironment (TME). The TME is key to tumorigenesis, drug response and treatment outcome. Importantly, stromal cells and secreted factors can initially be anti-tumorigenic but over time promote tumorigenesis and induce therapy resistance. To counter hypoxia, increased angiogenesis leads to formation of new vascular networks in order to actively promote and sustain tumor growth via supply of oxygen and nutrients whilst removing metabolic waste. Angiogenic vascular network formation aid in tumor cell metastatic dissemination. Successful tumor treatment and novel drug development require the identification and therapeutic targeting of pro-tumorigenic components of the TME including cancer-associated- fibroblasts (CAFs) and -macrophages (CAMs), hypoxia, blocking ECM-receptor interactions, in addition to targeting of tumor cells. Re-programming of stromal cells and the immune response to be anti-tumorigenic is key to therapeutic success. Lastly, this review highlights potential TME- and hypoxia-centred therapies under investigations.
... Dermal fibroblasts can also be induced into myofibroblasts; themselves can form dermal adipocytes, preventing scar formation in the process [280]. Several reports indicate that activated fibroblasts, demonstrated by α-SMA expression can originate from MSCs [281,282]. ...
... In the granulation tissue, growth factors and MMPs have been shown to play important roles in the transition of fibroblasts to myofibroblasts [287,288]. It has been suggested that only a small subpopulation of local fibroblasts can convert into myofibroblasts during the process of wound healing, but several reports including our own show that MScs can be converted to activated fibroblasts given the right microenvironmental signals [281,289,290]. Normal fibroblasts go through several transient stages via proto-fibroblasts and eventually into myofibroblasts through the action of TGF-B and interactions with ECM components such as fibronectin [291]. ...
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Based on its large surface area and covering the whole human body, the skin body’s largest organ and its main function is protection. Injuries and wound healing involving the skin offer valuable lessons shared with and of relevance to other organ systems and the diseases that impact them. Arguably the most complex human body process, wound healing is a multifaceted process that involves multiple cells and the extracellular matrix (ECM), with each component playing a specific role in the different stages of the healing process. Importantly, studies indicate that cells with stem cell-like properties are present within many of the human tissues and play key roles in case of tissue and cellular injury. Cell-to-cell and cell-to-ECM interactions are salient in wound healing subsequent to an injury. Microenvironment related factors and the variations therein including hypoxia or the abundance of oxygen, the presence/absence of growth factors and cytokines add to the complexity of the wound healing process and impact cell function and result in compromised or enhanced wound healing. This expert review critically examines the advances in biochemical and analytical tools that are enable the analysis of numerous cells and molecules within the wound microenvironment, revealing great cellular heterogeneity as well as novel molecular targets of importance to enhance wound healing. In a broader angle, we emphasize the ways in which wound healing is significant in the search for perfect skin after injury and in many common complex human diseases including cancer. In all, wound healing is a centrepiece of integrative biology research and applications in medicine as well as dermatology as discussed in this review.
... Recently, several researches have shown that immune cells in tumor microenvironment (TME) play an important role in tumor immune escape and immunotherapy resistance (18,19). As previous studies were focused on only one type of immune cell and one gene, the understanding of the CRC microenvironment may be incomplete (20,21). ...
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... The occurrence, growth, metastasis, and drug resistance of cancers are impacted by these mechanisms. Growing evidence shows targeting tumor cells and ignoring the surrounding TME is not effective enough to overcome the cancer disease (5,(8)(9)(10)(11)(12). ...
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IntroductionModern targeted cancer therapies are carefully crafted small molecules. These exquisite technologies exhibit an astonishing diversity of observed failure modes (drug resistance mechanisms) in the clinic. This diversity is surprising because back of the envelope calculations and classic modeling results in evolutionary dynamics suggest that the diversity in the modes of clinical drug resistance should be considerably smaller than what is observed. These same calculations suggest that the outgrowth of strong pre-existing genetic resistance mutations within a tumor should be ubiquitous. Yet, clinically relevant drug resistance occurs in the absence of obvious resistance conferring genetic alterations. Quantitatively, understanding the underlying biological mechanisms of failure mode diversity may improve the next generation of targeted anticancer therapies. It also provides insights into how intratumoral heterogeneity might shape interpatient diversity during clinical relapse.Materials and Methods We employed spatial agent-based models to explore regimes where spatial constraints enable wild type cells (that encounter beneficial microenvironments) to compete against genetically resistant subclones in the presence of therapy. In order to parameterize a model of microenvironmental resistance, BT20 cells were cultured in the presence and absence of fibroblasts from 16 different tissues. The degree of resistance conferred by cancer associated fibroblasts in the tumor microenvironment was quantified by treating mono- and co-cultures with letrozole and then measuring the death rates.Results and DiscussionOur simulations indicate that, even when a mutation is more drug resistant, its outgrowth can be delayed by abundant, low magnitude microenvironmental resistance across large regions of a tumor that lack genetic resistance. These observations hold for different modes of microenvironmental resistance, including juxtacrine signaling, soluble secreted factors, and remodeled ECM. This result helps to explain the remarkable diversity of resistance mechanisms observed in solid tumors, which subverts the presumption that the failure mode that causes the quantitatively fastest growth in the presence of drug should occur most often in the clinic.Conclusion Our model results demonstrate that spatial effects can interact with low magnitude of resistance microenvironmental effects to successfully compete against genetic resistance that is orders of magnitude larger. Clinical outcomes of solid tumors are intrinsically connected to their spatial structure, and the tractability of spatial agent-based models like the ones presented here enable us to understand this relationship more completely.
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