TITLE: post-translational modifications. This poses a significant challenge

TITLE:
UNDERSTANDING TUMOUR MICROENVIRONMENT AND ITS ROLE IN THERAPEUTIC RESISTANCE

Cancer has long been regarded as an
independent disease of only neoplastic cells i.e. a disease of single cell
type. Substantial information has been gathered about the genetic changes in
the neoplastic cells that promote tumourigenesis and metastasis. However,
advanced studies have shown that tumours are heterogeneous neoplasms composed
of different types which not only includes cancer cells but also mesenchymal
cells, endothelial cells, immune cells, fibroblasts/myofibroblasts, glial, epithelial
cells, fat, vascular, smooth muscle, extracellular matrix(ECM) and its secreted
extracellular molecules. Cancerous cells modify and recruit non-cancerous cells
from local or distant host tissue to the tumour microenvironment. These
non-neoplastic cells that constitute the tumour microenvironment are the
supporting cells that help the neoplastic cells in tumour development by
providing ECMs, growth factors, cytokines, vascular networks and extracellular
molecules acting in autocrine and/or paracrine manner.  Recent studies have shown that along with the
cancer cells within a tumour, the non-cancerous cells in the tumour
microenvironment are also heterogeneous in their molecular signatures which are
termed as tumour microenvironment heterogeneity. These    result in cell-to-cell variations in genetic
expression, gene signature and post-translational modifications. This poses a
significant challenge in the treatment of cancer because of drug resistance
which occurs extensively in all types of cancer.

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The main reason for this is that targeted
drugs have been developed against single or multiple aberrant molecular
signatures based on the diagnosis of a mixed population of cancer cells in most
cases from a single biopsy. Understanding the specific driving forces behind
different sub-types of intra-tumour heterogeneity

there will be greater improvement in cancer
treatment. Here the extrinsic factors that include the components of the tumour
microenvironment and that act on the cancer cells to influence their genotypes
and phenotypes.

have been discussed.

 

CELLS OF THE
STROMA

 

FIBROBLASTS

 

Fibroblasts are the primary cell types in the
normal connective tissue stroma and are the primary producers of the
non-cellular scaffold – the extracellular matrix.

Fibroblasts are responsible for  the deposition of fibrillar ECM that includes
type I, type III and type V collagen and fibronectin. They also contribute to
the formation of the basement membrane by secreting type IV collagen and
laminin.

The connective tissue and the ECM are
continually remodeled through a dynamic process of ECM protein production and
degradation by fibroblast-derived matrix metalloproteinases (MMPs). Fibroblasts
are also responsible for wound repair.

However, the cancer-associated fibroblasts are
functionally and phenotypically distinct from the normal fibroblasts which are
in the same tissue but not in tumour microenvironment.

CAFs 
within the tumour stroma are identical to the activated fibroblasts in
wounds by their spindyloid appearance and the expression of ?-SMA (smooth
muscle actin). They are distinct in the sense that CAFs are perpetually activated
neither reverting to normal phenotype nor undergoing apoptosis and elimination.
There are several theories regarding the origin of CAFs . Firstly, the
activation of the tissue-resident fibroblasts and secondly, the local cancer
cells or epithelial cells undergoing epithelial-to-mesenchymal transition (
EMT)  and finally, the migration and
activation of a marrow-derived cell .

    Experimentally,  factors such as TGF-? can induce normal
fibroblasts to express ?-SMA. However, it is not clear that these
experimentally activated cells acquire other characteristics of CAFs and also
if the phenotype will be stable. The second theory is the EMT . EMT is the
biologic process in which epithelial cells lose their cell-to-cell polarity and
cell-cell adhesion and gain migratory and invasive properties to become
mesenchymal stem cells. Apart from EMT of cancer cells to CAFs , EMT of
surrounding normal epithelial cells may also be an additional source of cells
for CAF formation. Recent studies have also shown that bone marrow-derived
precursor cells invade tumours and function as CAFs . It is not clear whether
these cells are activated as a result of influence of the tissue environment or
if they are a subset of cells within the marrow with an already activated
phenotype and are recruited to the site of tumours.

         
CAFs produce many growth factors like transforming growth factor (TGF-?)
, hepatocyte growth factor , insulin growth factor  , which lead to  proliferation and invasion of cancer cells .
They also secrete chemokines such as monocyte chemotactic protein 1,
interleukin 1 to stimulate proliferation of tumour cells.

CAFs also produce MMPs mostly MMP-9, MMP-2 and
other matrix modifying enzymes which include urokinase- type plasminogen activator
(uPA) that degrade the ECM  and support
tumour invasion and metastasis . CAFs also produce SDF-1 that stimulates tumour
growth directly. SDF-1 signalling can also stimulate angiogenesis by recruiting
EPCs into the tumour stroma. (Table 1)  

           

 

IMMUNE CELLS
OF THE STROMA

 

Immune cells like monocytes, macrophages,
neutrophils, lymphocytes are recruited by the tumour cells and thereafter, reside
in the tumour stroma. Infact, at the early stage of tumourigenesis the immune
system of the host can eliminate a significant portion of the premalignant
cells even before their initiation. However, sometimes the cancerous cells
evade the immune system and stay at a dormant stage for a long time called the
equilibrium stage. When these cells are mutated they escape the immune defence
system and start to proliferate rapidly to form a tumour. In other words, the
tumour microenvironment is in an immunosuppressive state where the suppressed
immune cells benefit the tumour by promoting angiogenesis,

TABLE 1.  SECRETIONS OF CAFs

NAME

TYPE

FUNCTION

TGF-?

Cytokine

Secreted protein
that controls cell growth, cell proliferation, differentiation, and apoptosis.
Responsible for tumour metastasis by inducing chemo attraction of cancer
cells to distant organs.

HGF

Cytokine

Promotes cancer
cell migration and angiogenesis

IGF 1/2

Mitogen

Increased cell
proliferation, suppression of apoptosis

Monocyte
chemotactic protein 1

Cytokine

Responsible for
macrophage/ monocyte infiltration in tumour tissue

Interleukin-1

Cytokine

Responsible for
stimulating immune responses such as inflammation

MMPs

Protease or matrix
modifying enzyme

Controlled
remodelling of ECM which is responsible for growth, invasion and metastasis
of malignant tumours

SDF-1

Cytokine

Stimulate
angiogenesis by recruiting circulating EPCs into the tumour stroma

SPARC

Glycoprotein

Suppresses immune
cell infiltration in tumours, promotes cell-cell deadhesion, angiogenesis, ECM
modelling

tumour survival and metastasis.   After
the monocytes are actively recruited into tumours along defined chemotactic
gradients which are  chemotactic ligands
that create chemical concentration gradients that organisms move towards or
away  , they reside and differentiate
into  tumour-associated macrophages (
TAMs) . TAMs constitute the major portion of the immune cells in the tumour
stroma. TAMs appear to be preferentially attracted to and retained in areas of
necrosis (unprogrammed cell death caused due to
external factors such as infection,trauma and unregulated digestion of cellular
components),  and hypoxia (
deficiency of oxygen reaching the tissues) where they become phenotypically
altered and upregulate hypoxia-induced transcription factors. TAMs rather than
being tumouricidal also adopt a protumoural phenotype at both primary and metastatic
sites.

Macrophages also release VEGF, HGF, MMP2 and
IL-8 that influence endothelial cell behaviour and ultimately stimulate the formation
of blood vessels. Neutrophils are also stimulators of angiogenesis. Additional immune
cells do not play much important role in carcinogenesis. They are not
consistent residents of the stroma and they are restricted to only specific
types of cancer.

 

VASCULAR ENDOTHELIAL CELLS

Angiogenesis
is the physiological process through which new blood vessels form from
pre-existing vessels. For cancer cell growth angiogenesis is important because
it supplies nutrients and oxygen which is needed for tumour growth. Many
components of stroma are responsible for initiation of angiogenesis out of which
CAFs play an important role in synchronizing events of angiogenesis by
secretion of many ECM molecules and growth factors such as TGF-?, VEGF,
fibroblast growth factor. It also secretes SDF-1 where SDF-1 signalling is
responsible for recruiting endothelial progenitor cells (EPCs) into the tumour
stroma to form new blood vessels known as vascular mimicry. CAFs also produce a
significant amount of SPARC ( secreted protein acidic and rich in cysteine)
responsible for the regulation of angiogenesis. CAFs also secrete many MMPs
which  causes the  initiation of angiogenesis by the degradation
of the basement membrane,  sprouting of
endothelial cells, regulation of pericyte attachment. As the tumour grows
rapidly there are increased chances of intratumoural hypoxia which promotes
angiogenesis by the production of many secreted factors such as
hypoxia-inducible factors , angiopoietin 1, angiopoietin 4, placental growth
factor, platelet-derived growth factor B. However, these neoangiogenic vessels
are non-uniformly distributed, irregularly shaped, inappropriately branched, and
tortuous often ending blindly. These do not have the classical hierarchy of
arterioles, capillaries, venules and often have arteriovenous shunts. These
vessels are variably fenestrated and leaky which are pathways for cancer cells
to enter circulation to initiate metastasis.

RESISTANCE TO THERAPIES MEDIATED BY
TUMOUR STROMA

In
recent years medical oncology has focused largely on specific therapeutic
approaches with the aim of identifying patient subpopulations that would
benefit from these therapeutic strategies. The exploration of therapeutic
resistance has largely focused on tumour cells . However, recent studies have
suggested that the mechanisms of therapeutic resistance not only depend on
alterations in the tumour cells but also in the tumour stroma

FIBROBLAST MEDIATED RESISTANCE

Early
co-culture experiments showed that within a solid tumour fibroblasts are not
passive elements and could potentially respond and affect therapy. It has been
seen that irradiated or damaged fibroblasts could better support tumour cell
growth than non-irradiated fibroblasts. Stromal derived hepatocyte growth
factor ( HGF) is responsible for rendering tumour cells resistant to BRAF
inhibition in cell lines  that harbour
the BRAF mutation. Moreover, abundance of HGF expression in patients correlated
with reduced responsiveness to drug treatment. These examples show the
importance of assessing potential stromal mediators of inherent resistance and
highlight the challenge of elucidating how therapeutic treatment could elicit
resistance through unforeseen stromal changes. The response of the supporting
stroma to treatment may show a more complicated picture in which
stress-response programs in these cells may be limiting treatment efficacy by
providing an effective  protective
environment for tumour cells.

VASCULAR-MEDIATED RESISTANCE

It
is speculated that tumour vasculature serves as a barrier to optimal drug
delivery. The blood vessels in the tumour are compressed as the dense nature of
tumour  restrains the tumour vasculature
and disrupts the efficient blood flow, thus elevating the interstitial pressure
. This may obstruct movement within and across tumour vessels . Recent studies
have shown that in order to reduce the interstitial pressure and improve vessel
 flow, cytoreduction of stroma  through the enzymatic destruction of
hyaluronan is a possible option. Cytoreduction is literally the reduction in
the number of cells. Moreover, normalization of leaky vascular beds through
VEGFA pathway inhibition has also been suggested to transiently increase drug
delivery in solid tumours.  Paracrine signalling from endothelial cells within
this niche has been shown to increase chemoresistance by inducing a
stem-cell-like phenotype in a subset of colorectal tumour cells. Similarly,
hypoxic regions of tumours can harbour and support the survival of colon cancer
stem cells during chemotherapy.  These
studies suggest that distinct niches within a tumour could support and instruct
tumour regrowth following treatment and highlight the unanticipated effects
that therapeutic interventions can have on non-tumour cell components, which
can then limit treatment efficacy.

IMMUNE MEDIATED RESISTANCE

The
immune system is an active component of the disease as it recognizes cancer
cells. However, tumour cells evade the immune system due to defects in antigen
presentation and loss in antigenicity. This leads to malignancies and is one of
the major reasons for patient to become refractory to treatment. Tumour cells
also escape from the immune system by modifying tumour microenvironment in an
immune-suppressive state. Infact, immunotherapy refers to the harnessing of the
patient’s immune surveillance to cure the cancer. Several of them have shown
promising results.However, there are some reports of resistance to
immunotherapy. Intrinsic resistance
is shown in patients who fail to evoke T cell responses and antitumour
activity. Generally,patients with immunodeficient virus infection, who have
received transplants, or  elderly people
may not have a strong systemic immune response because of a decrease in their
total T-cell pool . Moreover, many tumour antigens are also expressed in
healthy cells, which would lower the response of T cells to these antigens . In
the tumour microenvironment, secretion of TGF-? and IL-10 could inhibit the
function of T cells .  In the context of
anti-angiogenic therapy, tumours may be rendered refractory to anti-VEGF
therapy by a pro-inflammatory micro-environment that includes multiple cell
types such as myeloid cells and TAMs that secrete factors compensating for VEGF
loss to support angiogenesis. Depletion of MDSC expansion and recruitment that
is mediated predominantly by secretion of G-CSF in anti-VEGF insensitive
experimental models could rescue responsiveness to VEGF depletion, leading to
decreased vessel density and tumour growth. Expression of checkpoint molecules including lymphocyte
activation gene 3, T cell membrane protein 3, and B and T lymphocyte attenuator
is able to inhibit the activity of T cells in the tumour immature. The adoptive cell transfer (ACT) as
the name suggests is the transfer of cells into patients. The cells may
originate from the patient or a different individual. This is a way to therapeutically
harness the anti-tumour effects of adaptive immunity in patients. The aim of
ACT is to boost a patient’s anticancer immunity by transplanting T cells that
recognize tumour-specific antigens, leading to elimination of cancer cells.  It is a very effective method but responses
are not always sustained. Recent work  suggests that inflammation, especially the
presence of TNF (tumour necrosis factor-?) secreted by infiltrating macrophages
resulting from the initial tumour response leads to environmental changes that
induce loss of the targeted tumour antigens. In summary, the immune system can
be implicated in both inherent, as well as acquired resistance to targeted
therapies.

FUTURE PERSPECTIVES AND STRATEGIES TO
OVERCOME THERAPEUTIC RESISTANCE

The contribution of TME in the cancer therapeutic
resistance has been discussed above . This discussion elaborates  on the different types of cells that  contribute to the induction of therapeutic
resistance through their independent mechanisms .  Intercellular communication within the tumour
and its heterogeneity both result in increased resistance to various
therapeutic options . Moreover, tumour cells often utilize secreted molecules
such as exosomes to communicate. Thus, it would be promising to target intratumoural
interactions in anticancer therapies because they are largely responsible for
the therapeutic resistance of tumour cells. It was seen that ?-elemene
treatment  inhibits transfer of multidrug
resistance-associated miRNAs and thus blocks intercellular communication in the
tumour . Myeloid cells develop tumour therapeutic resistance through alteration
of the characteristics of tumour cells, ECM remodelling and angiogenesis . Co-culture
of MTLn3 cancer cells derived from primary bone marrow-derived macrophages and isolated
from cathepsin B- or S-deficient mice gave results of  impaired cancer cell invasion than co-culture
with macrophages from wild-type mice . When combined with sorafenib (an
inhibitor of tyrosine protein kinases) treatment, TANs depletion suppressed cancer
growth and angiogenesis . However, this 
is not all as other pathways of myeloid cells-induced therapeutic
resistance should be investigated.  Tumour microenvironment has been
considered to be of great importance in various therapeutic attempts including
one investigating the use of nanomedicine . Other parts of the tumour
microenvironment also have been targets of treatment . Bevacizumab (Avastin)
which is a variant of anti-VEGF antibody  was approved by the United States Food and
Drug Administration as a therapy for metastatic colorectal cancer. 

CONCLUSION

Tumour microenvironment has been implicated in tumour
growth, invasion, and metastasis. There has been a great deal of progress in
understanding how myeloid cells and CAFs in TME can affect cancer itself . In
particular, the mechanism by which tumours 
are generally resistant to conventional therapies has been a subject of
interest in the recent days. We summarized some recent reports revealing signal
cascades relevant to tumour therapeutic resistance. In addition to the
contribution of tumour microenvironments in causing therapeutic resistance
described in this review, other features such as interaction of cancer cells
with the ECM should be evaluated. Utilizing appropriate models that reflect
characteristics of the microenvironment would help us treating cancer
effectively. Moreover, it is desirable to develop therapeutic approaches
targeting multiple signal pathways rather than those involved in sustaining tumour
microenvironments. This will ultimately help improve cancer treatment and save
many lives.

 

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