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

TITLE:UNDERSTANDING TUMOUR MICROENVIRONMENT AND ITS ROLE IN THERAPEUTIC RESISTANCECancer has long been regarded as anindependent disease of only neoplastic cells i.e. a disease of single celltype. Substantial information has been gathered about the genetic changes inthe neoplastic cells that promote tumourigenesis and metastasis.

However,advanced studies have shown that tumours are heterogeneous neoplasms composedof different types which not only includes cancer cells but also mesenchymalcells, endothelial cells, immune cells, fibroblasts/myofibroblasts, glial, epithelialcells, fat, vascular, smooth muscle, extracellular matrix(ECM) and its secretedextracellular molecules. Cancerous cells modify and recruit non-cancerous cellsfrom local or distant host tissue to the tumour microenvironment. Thesenon-neoplastic cells that constitute the tumour microenvironment are thesupporting cells that help the neoplastic cells in tumour development byproviding ECMs, growth factors, cytokines, vascular networks and extracellularmolecules acting in autocrine and/or paracrine manner.  Recent studies have shown that along with thecancer cells within a tumour, the non-cancerous cells in the tumourmicroenvironment are also heterogeneous in their molecular signatures which aretermed as tumour microenvironment heterogeneity. These    result in cell-to-cell variations in geneticexpression, gene signature and post-translational modifications. This poses asignificant challenge in the treatment of cancer because of drug resistancewhich occurs extensively in all types of cancer.

The main reason for this is that targeteddrugs have been developed against single or multiple aberrant molecularsignatures based on the diagnosis of a mixed population of cancer cells in mostcases from a single biopsy. Understanding the specific driving forces behinddifferent sub-types of intra-tumour heterogeneity there will be greater improvement in cancertreatment. Here the extrinsic factors that include the components of the tumourmicroenvironment and that act on the cancer cells to influence their genotypesand phenotypes.have been discussed.

 CELLS OF THESTROMA FIBROBLASTS Fibroblasts are the primary cell types in thenormal connective tissue stroma and are the primary producers of thenon-cellular scaffold – the extracellular matrix.Fibroblasts are responsible for  the deposition of fibrillar ECM that includestype I, type III and type V collagen and fibronectin. They also contribute tothe formation of the basement membrane by secreting type IV collagen andlaminin.

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The connective tissue and the ECM arecontinually remodeled through a dynamic process of ECM protein production anddegradation by fibroblast-derived matrix metalloproteinases (MMPs). Fibroblastsare also responsible for wound repair.However, the cancer-associated fibroblasts arefunctionally and phenotypically distinct from the normal fibroblasts which arein the same tissue but not in tumour microenvironment.CAFs within the tumour stroma are identical to the activated fibroblasts inwounds by their spindyloid appearance and the expression of ?-SMA (smoothmuscle actin). They are distinct in the sense that CAFs are perpetually activatedneither reverting to normal phenotype nor undergoing apoptosis and elimination.There are several theories regarding the origin of CAFs . Firstly, theactivation of the tissue-resident fibroblasts and secondly, the local cancercells or epithelial cells undergoing epithelial-to-mesenchymal transition (EMT)  and finally, the migration andactivation of a marrow-derived cell .

    Experimentally,  factors such as TGF-? can induce normalfibroblasts to express ?-SMA. However, it is not clear that theseexperimentally activated cells acquire other characteristics of CAFs and alsoif the phenotype will be stable. The second theory is the EMT . EMT is thebiologic process in which epithelial cells lose their cell-to-cell polarity andcell-cell adhesion and gain migratory and invasive properties to becomemesenchymal stem cells. Apart from EMT of cancer cells to CAFs , EMT ofsurrounding normal epithelial cells may also be an additional source of cellsfor CAF formation. Recent studies have also shown that bone marrow-derivedprecursor cells invade tumours and function as CAFs . It is not clear whetherthese cells are activated as a result of influence of the tissue environment orif they are a subset of cells within the marrow with an already activatedphenotype 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 andother matrix modifying enzymes which include urokinase- type plasminogen activator(uPA) that degrade the ECM  and supporttumour invasion and metastasis . CAFs also produce SDF-1 that stimulates tumourgrowth directly. SDF-1 signalling can also stimulate angiogenesis by recruitingEPCs into the tumour stroma. (Table 1)              IMMUNE CELLSOF THE STROMA Immune cells like monocytes, macrophages,neutrophils, lymphocytes are recruited by the tumour cells and thereafter, residein the tumour stroma. Infact, at the early stage of tumourigenesis the immunesystem of the host can eliminate a significant portion of the premalignantcells even before their initiation. However, sometimes the cancerous cellsevade the immune system and stay at a dormant stage for a long time called theequilibrium stage.

When these cells are mutated they escape the immune defencesystem and start to proliferate rapidly to form a tumour. In other words, thetumour microenvironment is in an immunosuppressive state where the suppressedimmune 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.   Afterthe monocytes are actively recruited into tumours along defined chemotacticgradients which are  chemotactic ligandsthat create chemical concentration gradients that organisms move towards oraway  , they reside and differentiateinto  tumour-associated macrophages (TAMs) . TAMs constitute the major portion of the immune cells in the tumourstroma. TAMs appear to be preferentially attracted to and retained in areas ofnecrosis (unprogrammed cell death caused due toexternal factors such as infection,trauma and unregulated digestion of cellularcomponents),  and hypoxia (deficiency of oxygen reaching the tissues) where they become phenotypicallyaltered and upregulate hypoxia-induced transcription factors. TAMs rather thanbeing tumouricidal also adopt a protumoural phenotype at both primary and metastaticsites.Macrophages also release VEGF, HGF, MMP2 andIL-8 that influence endothelial cell behaviour and ultimately stimulate the formationof blood vessels. Neutrophils are also stimulators of angiogenesis. Additional immunecells do not play much important role in carcinogenesis.

They are notconsistent residents of the stroma and they are restricted to only specifictypes of cancer. VASCULAR ENDOTHELIAL CELLSAngiogenesisis the physiological process through which new blood vessels form frompre-existing vessels. For cancer cell growth angiogenesis is important becauseit supplies nutrients and oxygen which is needed for tumour growth. Manycomponents of stroma are responsible for initiation of angiogenesis out of whichCAFs play an important role in synchronizing events of angiogenesis bysecretion of many ECM molecules and growth factors such as TGF-?, VEGF,fibroblast growth factor.

It also secretes SDF-1 where SDF-1 signalling isresponsible for recruiting endothelial progenitor cells (EPCs) into the tumourstroma to form new blood vessels known as vascular mimicry. CAFs also produce asignificant amount of SPARC ( secreted protein acidic and rich in cysteine)responsible for the regulation of angiogenesis. CAFs also secrete many MMPswhich  causes the  initiation of angiogenesis by the degradationof the basement membrane,  sprouting ofendothelial cells, regulation of pericyte attachment. As the tumour growsrapidly there are increased chances of intratumoural hypoxia which promotesangiogenesis by the production of many secreted factors such ashypoxia-inducible factors , angiopoietin 1, angiopoietin 4, placental growthfactor, platelet-derived growth factor B. However, these neoangiogenic vesselsare non-uniformly distributed, irregularly shaped, inappropriately branched, andtortuous often ending blindly. These do not have the classical hierarchy ofarterioles, capillaries, venules and often have arteriovenous shunts. Thesevessels are variably fenestrated and leaky which are pathways for cancer cellsto enter circulation to initiate metastasis.

RESISTANCE TO THERAPIES MEDIATED BYTUMOUR STROMAInrecent years medical oncology has focused largely on specific therapeuticapproaches with the aim of identifying patient subpopulations that wouldbenefit from these therapeutic strategies. The exploration of therapeuticresistance has largely focused on tumour cells . However, recent studies havesuggested that the mechanisms of therapeutic resistance not only depend onalterations in the tumour cells but also in the tumour stroma FIBROBLAST MEDIATED RESISTANCEEarlyco-culture experiments showed that within a solid tumour fibroblasts are notpassive elements and could potentially respond and affect therapy. It has beenseen that irradiated or damaged fibroblasts could better support tumour cellgrowth than non-irradiated fibroblasts. Stromal derived hepatocyte growthfactor ( HGF) is responsible for rendering tumour cells resistant to BRAFinhibition in cell lines  that harbourthe BRAF mutation.

Moreover, abundance of HGF expression in patients correlatedwith reduced responsiveness to drug treatment. These examples show theimportance of assessing potential stromal mediators of inherent resistance andhighlight the challenge of elucidating how therapeutic treatment could elicitresistance through unforeseen stromal changes. The response of the supportingstroma to treatment may show a more complicated picture in whichstress-response programs in these cells may be limiting treatment efficacy byproviding an effective  protectiveenvironment for tumour cells.VASCULAR-MEDIATED RESISTANCEItis speculated that tumour vasculature serves as a barrier to optimal drugdelivery. The blood vessels in the tumour are compressed as the dense nature oftumour  restrains the tumour vasculatureand disrupts the efficient blood flow, thus elevating the interstitial pressure. This may obstruct movement within and across tumour vessels . Recent studieshave shown that in order to reduce the interstitial pressure and improve vessel flow, cytoreduction of stroma  through the enzymatic destruction ofhyaluronan is a possible option.

Cytoreduction is literally the reduction inthe number of cells. Moreover, normalization of leaky vascular beds throughVEGFA pathway inhibition has also been suggested to transiently increase drugdelivery in solid tumours.  Paracrine signalling from endothelial cells withinthis niche has been shown to increase chemoresistance by inducing astem-cell-like phenotype in a subset of colorectal tumour cells. Similarly,hypoxic regions of tumours can harbour and support the survival of colon cancerstem cells during chemotherapy.  Thesestudies suggest that distinct niches within a tumour could support and instructtumour regrowth following treatment and highlight the unanticipated effectsthat therapeutic interventions can have on non-tumour cell components, whichcan then limit treatment efficacy.IMMUNE MEDIATED RESISTANCETheimmune system is an active component of the disease as it recognizes cancercells. However, tumour cells evade the immune system due to defects in antigenpresentation and loss in antigenicity.

This leads to malignancies and is one ofthe major reasons for patient to become refractory to treatment. Tumour cellsalso escape from the immune system by modifying tumour microenvironment in animmune-suppressive state. Infact, immunotherapy refers to the harnessing of thepatient’s immune surveillance to cure the cancer. Several of them have shownpromising results.However, there are some reports of resistance toimmunotherapy. Intrinsic resistanceis shown in patients who fail to evoke T cell responses and antitumouractivity.

Generally,patients with immunodeficient virus infection, who havereceived transplants, or  elderly peoplemay not have a strong systemic immune response because of a decrease in theirtotal T-cell pool . Moreover, many tumour antigens are also expressed inhealthy cells, which would lower the response of T cells to these antigens . Inthe tumour microenvironment, secretion of TGF-? and IL-10 could inhibit thefunction of T cells .  In the context ofanti-angiogenic therapy, tumours may be rendered refractory to anti-VEGFtherapy by a pro-inflammatory micro-environment that includes multiple celltypes such as myeloid cells and TAMs that secrete factors compensating for VEGFloss to support angiogenesis. Depletion of MDSC expansion and recruitment thatis mediated predominantly by secretion of G-CSF in anti-VEGF insensitiveexperimental models could rescue responsiveness to VEGF depletion, leading todecreased vessel density and tumour growth.

Expression of checkpoint molecules including lymphocyteactivation gene 3, T cell membrane protein 3, and B and T lymphocyte attenuatoris able to inhibit the activity of T cells in the tumour immature. The adoptive cell transfer (ACT) asthe name suggests is the transfer of cells into patients. The cells mayoriginate from the patient or a different individual. This is a way to therapeuticallyharness the anti-tumour effects of adaptive immunity in patients. The aim ofACT is to boost a patient’s anticancer immunity by transplanting T cells thatrecognize tumour-specific antigens, leading to elimination of cancer cells.  It is a very effective method but responsesare not always sustained.

Recent work  suggests that inflammation, especially thepresence of TNF (tumour necrosis factor-?) secreted by infiltrating macrophagesresulting from the initial tumour response leads to environmental changes thatinduce loss of the targeted tumour antigens. In summary, the immune system canbe implicated in both inherent, as well as acquired resistance to targetedtherapies.FUTURE PERSPECTIVES AND STRATEGIES TOOVERCOME THERAPEUTIC RESISTANCE The contribution of TME in the cancer therapeuticresistance has been discussed above . This discussion elaborates  on the different types of cells that  contribute to the induction of therapeuticresistance through their independent mechanisms .  Intercellular communication within the tumourand its heterogeneity both result in increased resistance to varioustherapeutic options . Moreover, tumour cells often utilize secreted moleculessuch as exosomes to communicate.

Thus, it would be promising to target intratumouralinteractions in anticancer therapies because they are largely responsible forthe therapeutic resistance of tumour cells. It was seen that ?-elemenetreatment  inhibits transfer of multidrugresistance-associated miRNAs and thus blocks intercellular communication in thetumour . Myeloid cells develop tumour therapeutic resistance through alterationof the characteristics of tumour cells, ECM remodelling and angiogenesis .

Co-cultureof MTLn3 cancer cells derived from primary bone marrow-derived macrophages and isolatedfrom cathepsin B- or S-deficient mice gave results of  impaired cancer cell invasion than co-culturewith macrophages from wild-type mice . When combined with sorafenib (aninhibitor of tyrosine protein kinases) treatment, TANs depletion suppressed cancergrowth and angiogenesis . However, this is not all as other pathways of myeloid cells-induced therapeuticresistance should be investigated.  Tumour microenvironment has beenconsidered to be of great importance in various therapeutic attempts includingone investigating the use of nanomedicine . Other parts of the tumourmicroenvironment also have been targets of treatment . Bevacizumab (Avastin)which is a variant of anti-VEGF antibody  was approved by the United States Food andDrug Administration as a therapy for metastatic colorectal cancer. CONCLUSIONTumour microenvironment has been implicated in tumourgrowth, invasion, and metastasis.

There has been a great deal of progress inunderstanding how myeloid cells and CAFs in TME can affect cancer itself . Inparticular, the mechanism by which tumours are generally resistant to conventional therapies has been a subject ofinterest in the recent days. We summarized some recent reports revealing signalcascades relevant to tumour therapeutic resistance. In addition to thecontribution of tumour microenvironments in causing therapeutic resistancedescribed in this review, other features such as interaction of cancer cellswith the ECM should be evaluated. Utilizing appropriate models that reflectcharacteristics of the microenvironment would help us treating cancereffectively. Moreover, it is desirable to develop therapeutic approachestargeting multiple signal pathways rather than those involved in sustaining tumourmicroenvironments. This will ultimately help improve cancer treatment and savemany lives. REFERENCES1.

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