Wolf H Fridman

Ian Miller

Catherine Sautès-Fridman

Annette T Byrne

Therapeutic Targeting of the Colorectal Tumor Stroma

Keywords: CRC, MSI, microenvironment, matrix

Colorectal tumors have been classified based on histologic factors, genetic factors, and consensus molecular subtypes, which all affect the tumor microenvironment (TME). Elements of the TME serve as therapeutic targets and might be used as prognostic factors. For example, immune checkpoint inhibitors are used to treat tumors with microsatellite instability, and anti-angiogenic agents may be used in combination with other drugs to slow or inhibit tumor growth. We review the features of the colorectal tumor stroma that associate with outcomes of patients and discuss potential therapeutic agents that target these features.

In patients with colorectal cancer (CRC), distinct molecular features of tumor cells alter the tumor's microenvironment (TME) to affect its growth and metastasis [START_REF] Fridman | The immune contexture in human tumours: impact on clinical outcome[END_REF][START_REF] Fridman | The immune contexture in cancer prognosis and treatment[END_REF] . The TME contains an extracellular matrix (ECM) made of collagen fibers, layers of fibroblasts, blood and lymphatic vessels, nerves, and cells of hematopoietic origin [START_REF] Fridman | The immune contexture in human tumours: impact on clinical outcome[END_REF][START_REF] Kobayashi | Cancer-associated fibroblasts in gastrointestinal cancer[END_REF] . Among the hematopoietic cells, lymphocytes and myeloid cells influence tumor development directly or via the mediators they produce. In general, colorectal tumors are most heavily infiltrated by macrophages, followed by T and B cells [START_REF] Bindea | Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer[END_REF] . These immune cells interact with tumor cells and other stromal cells. The tumor stroma determines interactions among lymphocytes, myeloid cells, fibroblasts, endothelial cells, lymphatics, and tumor cells. Different components of the TME can affect clinical outcome.

There are different types of colorectal tumors, which each have different features of the TME. For example, colorectal tumors with microsatellite instability (MSI) have defects in DNA repair enzymes and are highly infiltrated by lymphocytes [START_REF] Dolcetti | High prevalence of activated intraepithelial cytotoxic T lymphocytes and increased neoplastic cell apoptosis in colorectal carcinomas with microsatellite instability[END_REF] . Most colorectal tumors, however, have low levels of infiltration by lymphocytes and varying densities of myeloid cells, endothelial and lymphatic cells, and fibroblasts. [START_REF] Spranger | Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity[END_REF][START_REF] Luke | WNT/β-catenin pathway activation correlates with immune exclusion across human cancers[END_REF] . However, a subgroup of colorectal tumors is characterized by medium levels of infiltration by lymphocytes and high densities of endothelial cells and fibroblasts. This group, called mesenchymal colorectal tumors, has high metastatic potential and patients have poor prognoses [START_REF] Becht | Immune Contexture, Immunoscore, and Malignant Cell Molecular Subgroups for Prognostic and Theranostic Classifications of Cancers[END_REF] . These highly aggressive tumors have a complex stroma. Colorectal tumors with specific mutations in RAS are an intriguing subgroup that are resistant to inhibitors of epidermal growth factor receptor (EGFR) [START_REF] Lièvre | KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer[END_REF][START_REF] Van Emburgh | Acquired RAS or EGFR mutations and duration of response to EGFR blockade in colorectal cancer[END_REF][START_REF] Zdanov | Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells[END_REF] . The efficacy of antibodies against EGFR might depend on their interactions with immune cells [START_REF] Bibeau | Impact of Fc{gamma}RIIa-Fc{gamma}RIIIa polymorphisms and KRAS mutations on the clinical outcome of patients with metastatic colorectal cancer treated with cetuximab plus irinotecan[END_REF] , so learning more about the TME can lead to strategies to improve therapies. We review the major components of the colorectal tumor stroma and their potential for therapeutic targeting.

We discuss new therapeutic strategies to alter the colorectal tumor stroma.

The Microenvironment in Tumorigenesis

CRC develops via a multistep process that involves the sequential accumulation of mutations in colonic epithelial cells [START_REF] Vogelstein | Genetic Alterations during Colorectal-Tumor Development[END_REF] . However, colorectal tumor development also involves interactions between these cancer cells and their microenvironment. This environment includes immune cells, neurons, fibroblasts, blood vessels, and lymph tissues (see Table 1).

Inflammation

Inflammatory diseases of the colon, such as ulcerative colitis and Crohn's disease, [START_REF] Baker | Precancer in ulcerative colitis: the role of the field effect and its clinical implications[END_REF][START_REF] Dyson | Colorectal cancer in inflammatory bowel disease: what is the real magnitude of the risk?[END_REF][START_REF] Hovde | Mortality and causes of death in Crohn's disease: results from 20 years of follow-up in the IBSEN study[END_REF] promote tumorigenesis via alterations to immune cells, blood vessels, and the colon mucosa [START_REF] Parang | AOM/DSS Model of Colitis-Associated Cancer[END_REF] . Mice given dextran sulfate sodium (to induce colitis) followed by azoxymethane (an mutagenic alkylating agent) develop colitis-associated cancer [START_REF] Parang | AOM/DSS Model of Colitis-Associated Cancer[END_REF] similar to that in patients.

Studies of these mice have indicated the roles of toll-like receptors and inflammatory cytokines, as well as colonic microbiome, in the generation of CRC [START_REF] Uronis | Modulation of the intestinal microbiota alters colitis-associated colorectal cancer susceptibility[END_REF] .

Patients with inflammatory bowel diseases have a significant increase in risk of CRC, due to the neoplastic effects of chronic intestinal inflammation. Chronic inflammation can lead to chromosome abnormalities, MSI, and epigenetic changes such as DNA hypermethylation. Inflammation also involves changes in expression of cytokines, chemokines, cyclooxygenase enzymes, and transcription factors, as well as in production of reactive oxygen species and the composition of the intestinal microbiome. Loss of p53 from colorectal tumors is associated with increased intestinal permeability, causing formation of an NF-κB-dependent inflammatory microenvironment and the induction of epithelialmesenchymal transition (EMT) [START_REF] Schwitalla | Loss of p53 in Enterocytes Generates an Inflammatory Microenvironment Enabling Invasion and Lymph Node Metastasis of Carcinogen-Induced Colorectal Tumors[END_REF] .

T cells, myeloid cells, and blood and lymphatic vessels are found in the center of the tumor and its invasive margin. Natural killer (NK) cells are often anergic [START_REF] Platonova | Profound coordinated alterations of intratumoral NK cell phenotype and function in lung carcinoma[END_REF] and relegated to the invasive margin. B cells are primarily incorporated in lymphoid aggregates, called tertiary lymphoid structures (TLS), within the invasive margin [START_REF] Dieu-Nosjean | Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers[END_REF] . Macrophages are the most abundant hematopoietic cells in the colorectal TME and are distributed between the tumor center and the invasive margin, whereas T-helper 17 (Th17) cells, mast cells, and neutrophils are mostly present in the invasive margin [START_REF] Bindea | Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer[END_REF] . Although most mature dendritic cells are present in TLS, which are in close contact with T cells, immature dendric cells are also detected throughout the center of the tumor. Intestinal microbes can also induce production of inflammatory cytokines such as interleukin 17 (IL17) [START_REF] Grivennikov | Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth[END_REF] . Increased levels of IL17 in colorectal tumors have been associated with shorter survival times of patients [START_REF] Tosolini | Clinical impact of different classes of infiltrating T cytotoxic and helper cells (Th1, th2, treg, th17) in patients with colorectal cancer[END_REF] .

Neural cells

The colon contains millions of neurons, which interact with lymphoid tissue in the intestine.

Neural cells located in the tumor stroma facilitate migration of metastatic cells-neural invasion of a tumor is an early sign of its invasiveness [START_REF] Pagès | Effector memory T cells, early metastasis, and survival in colorectal cancer[END_REF] . In prostate tumors, neurogenesis is initiated from neural progenitors from the central nervous system and newly formed nerve fibers sustain tumor initiation and progression [START_REF] Mauffrey | Progenitors from the central nervous system drive neurogenesis in cancer[END_REF] . Signaling by the chemokine CXCL13 via its receptor CXCR5 mediates interactions between neural cells, cancer cells, and the TME. High levels of CXCL13 and CXCR5 correlate with neural invasion of the TME and shorter survival times for patients with advanced CRC [START_REF] Qi | Expression features of CXCR5 and its ligand, CXCL13 associated with poor prognosis of advanced colorectal cancer[END_REF] . CXCL13 can induce the migration of CXCR5-positive neural precursor cells across the endothelium in humans [START_REF] Weiss | IL8 and CXCL13 are potent chemokines for the recruitment of human neural precursor cells across brain endothelial cells[END_REF] . Moreover, CXCR5 expression is required for differentiation of neural precursor cells into neurons in adult zebrafish [START_REF] Kizil | The chemokine receptor cxcr5 regulates the regenerative neurogenesis response in the adult zebrafish brain[END_REF] . CXCR5 is expressed by many colon cancer cell lines [START_REF] Meijer | The CXCR5 chemokine receptor is expressed by carcinoma cells and promotes growth of colon carcinoma in the liver[END_REF] , by B cells, and a subset of T cells in the TME [START_REF] Cremonesi | Gut microbiota modulate T cell trafficking into human colorectal cancer[END_REF][START_REF] Sautès-Fridman | Tertiary lymphoid structures in the era of cancer immunotherapy[END_REF] . More studies are needed to investigate interactions among nerves, immune cells, and tumor cells.

Fibroblasts

Fibroblasts are major constituents of the invasive margin, where they provide a physical barrier and remodel the ECM [START_REF] Kobayashi | Cancer-associated fibroblasts in gastrointestinal cancer[END_REF] . The densities, the location, and the functional orientation of these different cell types, as well as the presence or absence of TLS are prognostic factors for patients with CRC 1 .

Cancer-associated fibroblasts (CAFs) originate from several cell types, including epithelial and endothelial cells, local fibroblasts, and mesenchymal cells from the bone marrow (reviewed in ref 3). CAFs are a heterogeneous population of cells that undergo epigenetic modifications during cancer development. CAFs that resemble quiescent fibroblasts facilitate tissue regeneration during the early steps of carcinogenesis. Activation of CAFs during tumor progression is regulated by factors including transforming growth factor beta (TGFB), platelet derived growth factor (PDGF), hedgehog, bone morphogenic protein, IL1, IL6, TNF, and reactive oxygen species [START_REF] Kobayashi | Cancer-associated fibroblasts in gastrointestinal cancer[END_REF] . Activated CAFs acquire a highly contractile and proliferative phenotype and produce ECM proteins (collagen, fibronectin, proteoglycan, periostin and tenascin C). They support tumor growth indirectly, via collagen fibers that form the stiff ECM that prevents the entry of lymphocytes and drugs in the tumor center, and through the production of immunosuppressive, angiogenic, and inflammatory factors (Table 1). For a review on CAFs in colorectal tumor development, see Kabayashi et al [START_REF] Kobayashi | Cancer-associated fibroblasts in gastrointestinal cancer[END_REF] . Better markers are needed to detect CAF subtypes and determine their prognostic value.

Vasculature

Blood and lymphatic vessels infiltrate the tumor core and the invasive margin [START_REF] Pagès | Effector memory T cells, early metastasis, and survival in colorectal cancer[END_REF] . Endothelial cells regulate angiogenesis, but pericytes, (peri-endothelial smooth muscle cells that express α smooth muscle actin) support endothelial cell function and are required for development of a tumor vascular network [START_REF] Volz | Genes involved in pericyte-driven tumor maturation predict treatment benefit of first-line FOLFIRI plus bevacizumab in patients with metastatic colorectal cancer[END_REF] (Table 1). Eberhard et al have shown that the percentage of endothelial cells covered with pericytes varies among tumor types (such as 65% pericyte coverage in colorectal tumors vs 13% in glioblastomas) [START_REF] Eberhard | Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies[END_REF] .

Endothelial cells within the tumor form new blood vessels. However, these cells are highly proliferative and prone to apoptosis, unlike the endothelium of normal tissue 120 .

Tumor vessels are disorganized, tortuous, and dysfunctional, whereas the normal vasculature has a hierarchical branching pattern of arteries, veins, and capillaries [START_REF] Schwitalla | Loss of p53 in Enterocytes Generates an Inflammatory Microenvironment Enabling Invasion and Lymph Node Metastasis of Carcinogen-Induced Colorectal Tumors[END_REF] . In the normal vasculature, expression of VEGF and angiogenic factors is tightly regulated, and levels decrease rapidly upon new vessel formation. However, during tumor growth, the balance in expression of angiogenic vs anti-angiogenic factors is shifted toward continuous neoangiogenesis 121 .

VEGF signaling is complex 122 , with VEGF gene expression upregulated by hypoxia, through activation of the HIF1 transcription factor, and by integrin or oncogene signaling 123 (such as EGFR signaling). VEGF receptors are expressed not only by vascular endothelial cells but also by other cells, including macrophages and monocytes 125 , providing evidence for a role in immune modulation, angiogenesis and metastatic progression . Other signaling pathways interact with VEGF signaling, such as the angiogenin, TIE1, and Notch signaling pathways 126 .

In a meta-analysis, Wang et al associated higher levels of VEGF with tumor metastasis to lymph nodes and blood vessels. 127 Tumor level of VEGF might therefore be a prognostic marker for patients with CRC. Similarly, the incidence of metastases was higher in patients whose tumors expressed high levels of VEGF, which might be used in prognosis. Sustained levels of angiogenesis, revealed by the tumor endothelial cell signature, correlate with reduced patient survival times [START_REF] Ikenoue | Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors[END_REF] . Mohamed et al showed that patients whose colorectal tumors expressed high levels of VEGF, CD105 (endoglin; glycoprotein involved in TGF receptor complex), and CD31 (endothelial cell marker) had poor outcomes 128 .

Lymphangiogenesis

Lymphatic vessels maintain fluid balance by draining interstitial fluid to regional lymph nodes. During metastasis, they provide a pathway for tumor cell dissemination. 145,146 Lymphangiogenesis (the process where new lymphatic vessels are formed) occurs in and around tumors 147 . In colorectal tumors, there is a correlation between lymphatic microvessel density and risk of metastasis 146 .

Lymphangiogenesis is mediated by VEGFC and VEGFD 148 , 149,150 . These factors bind to the receptor tyrosine kinase VEGFR3 expressed on lymphatic cells resulting in neolymphangiogenesis. Other factors such as HGF, PGDF, FGF2, IGF1, and IGF2 stimulate lymph vessel outgrowth 148 . Lymphangiogenesis is inhibited by TGFB1, which also regulates tumor development. In mice undergoing wound repair, addition of exogenous TGFB1 inhibited assembly of lymphatic vessels, reduced lymphatic endothelial cell proliferation, and inhibited lymphatic endothelial cell migration 151 .

Tumor Cell Mutations and the TME

Among patients with colorectal tumors with high levels of MSI, 16% were found to have Lynch syndrome [START_REF] Pino | The chromosomal instability pathway in colon cancer[END_REF] -an inherited cancer syndrome caused by mutations in genes that encode DNA repair enzymes. These tumors have a high mutation burden and are infiltrated by a large number of lymphocytes [START_REF] Grasso | Genetic Mechanisms of Immune Evasion in Colorectal Cancer[END_REF] . Patients with these tumors have better outcomes than tumors without MSI, because of the adaptive immune response mediated by T cells that recognize the tumor neo-antigens created by the high-frequency mutations [START_REF] Mlecnik | Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival Than Microsatellite Instability[END_REF] . This immune response slows tumor growth and metastasis. Microsatellite-stable (MSS) tumors [START_REF] Bindea | Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer[END_REF][START_REF] Pagès | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF] , alternatively, have less infiltration of by lymphocytes. MSS tumors often have mutations in oncogenes such as APC, KRAS, TP53, or PIK3CA. These tumors can acquire additional mutations due to mutations in the DNA polymerase epsilon gene (POLE) [START_REF] Palles | Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas[END_REF][START_REF] Domingo | Somatic POLE proofreading domain mutation, immune response, and prognosis in colorectal cancer: a retrospective, pooled biomarker study[END_REF] , which increases their activation of the anti-tumor immune response. Patients with these colorectal tumors have longer than average survival times [START_REF] Glaire | Cancer predisposition syndromes: lessons for truly precision medicine[END_REF] . COLOSSUS (www.colossusproject.eu), a multi-disciplinary European Commissionfunded research network, is studying the development, stromal composition, and resistance mechanisms of colorectal tumors with RAS mutations. Tumors with RAS mutations are resistant to treatment with antibodies against EGFR [START_REF] Lièvre | KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer[END_REF][START_REF] Van Emburgh | Acquired RAS or EGFR mutations and duration of response to EGFR blockade in colorectal cancer[END_REF] . Most mutations in KRAS occur in exon 2 (codon 12 and 3) [START_REF] Roock | PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer[END_REF] ; patients whose tumors have these mutations do not benefit from anti-EGFR therapy [START_REF] Amado | Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer[END_REF] , with the possible exception of patients with tumors with the KRAS.G13D mutation [START_REF] De Roock | Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab[END_REF][START_REF] Allegra | Extended RAS Gene Mutation Testing in Metastatic Colorectal Carcinoma to Predict Response to Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015[END_REF] . Other mutations in RAS (KRAS exons 3 and 4; NRAS exons 2, 3, and 4) are also associated with poor response to anti-EGFR treatments [START_REF] Van Cutsem | Addition of aflibercept to fluorouracil, leucovorin, and irinotecan improves survival in a phase III randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin-based regimen[END_REF] . BRAF is downstream of Ras in the EGFR signaling pathway and the BRAF V600E mutation is associated with resistance to EGFR therapy [START_REF] Ikenoue | Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors[END_REF] . It is not clear why antibodies that bind and activate effector cells in the TME do not induce tumor cell killing by macrophages or NK cells. Tumors with RAS mutations might become resistant to NK cell killing downregulate the anti-tumor immune response by unknown mechanisms [START_REF] Liao | KRAS-IRF2 Axis Drives Immune Suppression and Immune Therapy Resistance in Colorectal Cancer[END_REF][START_REF] Nakadate | KRAS mutation confers resistance to antibody-dependent cellular cytotoxicity of cetuximab against human colorectal cancer cells[END_REF] . For a review of tumor mechanisms of resistance to EGFR inhibitors, see ref [START_REF] Zhao | Mechanisms of resistance to anti-EGFR therapy in colorectal cancer[END_REF] .

Molecular Classifications and the TME

Transcriptome-based classifications of colorectal tumors have been proposed [START_REF]Comprehensive molecular characterization of human colon and rectal cancer[END_REF][START_REF] Roepman | Colorectal cancer intrinsic subtypes predict chemotherapy benefit, deficient mismatch repair and epithelial-to-mesenchymal transition[END_REF][START_REF] Budinska | Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer[END_REF][START_REF] Schlicker | Subtypes of primary colorectal tumors correlate with response to targeted treatment in colorectal cell lines[END_REF][START_REF] Sadanandam | A colorectal cancer classification system that associates cellular phenotype and responses to therapy[END_REF][START_REF] Marisa | Gene expression classification of colon cancer into molecular subtypes: characterization, validation, and prognostic value[END_REF][START_REF] Sousa | Poor-prognosis colon cancer is defined by a molecularly distinct subtype and develops from serrated precursor lesions[END_REF][START_REF] Perez-Villamil | Colon cancer molecular subtypes identified by expression profiling and associated to stroma, mucinous type and different clinical behavior[END_REF] . A Consensus Molecular Classification system [START_REF] Guinney | The consensus molecular subtypes of colorectal cancer[END_REF] has been developed an provides a useful tool to classify tumors and study their corresponding TMEs. There are 4 consensus molecular subtypes (CMSs). CMS1 (14% of colorectal tumors) contains most, although not all, hypermutated MSI tumors with BRAF mutations and the high CpG island methylator phenotype, resulting in the methylation and subsequent inhibition of transcription of the mismatch repair gene MLH1 [START_REF] Herman | Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma[END_REF] and few somatic copy number alterations. CMS2 (37% of colorectal tumors) is characterized by mutations in APC and activation of WNT and MYC.

CMS3 tumors (13% of colorectal tumors) have metabolic deregulation and have many tumor cells with KRAS mutations. CMS4 tumors (23% of colorectal tumors) upregulate genes involved in the EMT, TGFB signaling, angiogenesis, and ECM remodeling [START_REF] Guinney | The consensus molecular subtypes of colorectal cancer[END_REF] .

An in-depth analysis of the composition and activation states of the stromal components associated with each CMS [START_REF] Becht | Immune and Stromal Classification of Colorectal Cancer Is Associated with Molecular Subtypes and Relevant for Precision Immunotherapy[END_REF] revealed that CMS1 tumors had a high expression of genes that regulate T-cell trafficking and activation as well as differentiation of Th1 and cytotoxic T cells, and high expression of CXCL13. CMS1 tumors have a high density of infiltrating CD8 + T cells [START_REF] Becht | Immune and Stromal Classification of Colorectal Cancer Is Associated with Molecular Subtypes and Relevant for Precision Immunotherapy[END_REF] . Patients with CMS1 tumors have longer survival times than patients with other CMSs, supporting the concept that hypermutated tumors of this subtype induce specific T-and B-cell responses that control tumor dissemination and metastasis.

However, CMS1 tumors express high levels of immune check point molecules such as PD1 and cytotoxic T-lymphocyte associated protein 4 (CTLA4).

The TME of CMS2 tumors is characterized by low numbers of lymphocytes, macrophages endothelial, and fibroblastic cells. CMS3 tumors are heterogeneous but are characterized by low levels of immune cell infiltration [START_REF] Becht | Immune and Stromal Classification of Colorectal Cancer Is Associated with Molecular Subtypes and Relevant for Precision Immunotherapy[END_REF] . CMS4 tumors are the most aggressive subtype with the worst outcomes. CMS4 tumors express immune checkpoint molecules, and are highly infiltrated by macrophages, myeloid-derived suppressor cells (MDSCs), MDSCs, and fibroblasts. CMS4 tumors express high levels of the chemokines CCL2 and CXCL12, which recruit myeloid cells and promote neural migration. CMS4 tumors have low levels of CXCL13, which regulates formation of TLS, indicating a disorganized anti-tumor immune response and lack of T and B cells that recognize tumor antigens [START_REF] Sautès-Fridman | Tertiary lymphoid structures in the era of cancer immunotherapy[END_REF] . CMS4 tumors are characterized by an inflammatory gene expression signature, with high expression of genes encoding components of complement system. They also express high levels of genes that encode TGFB and LGALS1, which are immunesuppressive, and the angiogenic factors VEGF and PDGFC [START_REF] Becht | Immune and Stromal Classification of Colorectal Cancer Is Associated with Molecular Subtypes and Relevant for Precision Immunotherapy[END_REF][START_REF] Guinney | The consensus molecular subtypes of colorectal cancer[END_REF][START_REF] Dienstmann | Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer[END_REF] . The abundance of fibroblasts found in CMS4 tumors correlates with myeloid and endothelial cell abundance, indicating that fibroblasts might promote angiogenesis and recruitment of inflammatory cells [START_REF] Becht | Immune and Stromal Classification of Colorectal Cancer Is Associated with Molecular Subtypes and Relevant for Precision Immunotherapy[END_REF] .

In the stroma of CMS4 tumors, fibroblasts express high levels of VEGFB, VEGFC, PDGFC, LGALS1, CXCL12, PTGS1, and TGFB to promote angiogenesis, lymphangiogenesis, and immune suppression. Endothelial cells in these tumors express high levels of CCL2, PDGFB, and TGFB1 and TGFB2. Finally, monocytes in CMS4 tumors express complement components (C1QA, C1QC, C3, C3AR1, and C5AR1) and chemokines that attract macrophages (CCL19 and CCL23). These cell populations contribute to progression of CMS4 colorectal tumors by promoting inflammation, angiogenesis, and immunosuppression.

Although practical, the CMS classification system faces many hurdles. Integration of cancer cell and stromal gene expression signatures depends on the purity of the tumor sample analyzed. CMS1 and CMS4 are over-represented in samples containing a large proportion of stromal tissue [START_REF] Alderdice | Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies[END_REF] . In addition, the CMS system is based on average characteristics, and does not take into account tumor heterogeneity [START_REF] Alderdice | Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies[END_REF] . The cancer cell intrinsic subtype classification system, established from tumor patient-derived xenografts [START_REF] Lal | An immunogenomic stratification of colorectal cancer: Implications for development of targeted immunotherapy[END_REF] appears to be more robust, but does not integrate the TME. Other immune classification systems overlap imperfectly with the CMS system, so further molecular classifications are needed to guide TME targeted therapies.

Chromosome Instability and the TME

There have been many studies of gene copy number changes in colorectal tumors; [START_REF] Dijk E Van | Loss of Chromosome 18q11.2-q12.1 Is Predictive for Survival in Patients With Metastatic Colorectal Cancer Treated With Bevacizumab[END_REF] these have also been used to create a CRC classification system, based on chromosome instability. [START_REF] Smeets | Copy number load predicts outcome of metastatic colorectal cancer patients receiving bevacizumab combination therapy[END_REF] . Copy number load was initially studied as a potential biomarker of response to bevacizumab in patients with metastatic CRC. Specifically, 472 primary tumors that metastasized were classified into 3 subgroups (clusters 1-3), each characterized by different degrees of chromosome instability. Tumors with increasing cluster numbers (cluster 1-3) had an increasing number of chromosomal breakpoints and a higher proportion of the genome with copy number alterations (CNAs).

Researchers used publicly available TCGA datasets to correlate clusters of CNAs with

CMSs [START_REF] Remark | Characteristics and clinical impacts of the immune environments in colorectal and renal cell carcinoma lung metastases: influence of tumor origin[END_REF][START_REF] Mlecnik | Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction[END_REF][START_REF] Galon | The adaptive immunologic microenvironment in colorectal cancer: a novel perspective[END_REF][START_REF] Angelova | Evolution of Metastases in Space and Time under Immune Selection[END_REF][START_REF] Ijsselsteijn | Revisiting immune escape in colorectal cancer in the era of immunotherapy[END_REF][START_REF] Middha | Majority of B2M-Mutant and -Deficient Colorectal Carcinomas Achieve Clinical Benefit From Immune Checkpoint Inhibitor Therapy and Are Microsatellite Instability-High[END_REF][START_REF] Ozcan | Complex pattern of immune evasion in MSI colorectal cancer[END_REF][START_REF] Mlecnik | Biomolecular network reconstruction identifies T-cell homing factors associated with survival in colorectal cancer[END_REF] . Gene set enrichment analysis of 50 cancer-associated pathways applied to differentially expressed genes between clusters of CNAs revealed that cluster 1 tumors were characterized by a strong immune-activated microenvironment, whereas cluster 2 and 3 tumors were characterized by angiogenesis, EMT, and inflammatory response pathways.

Tumors from cluster 1 overlapped with CMS1 or CMS3 tumors, whereas cluster 2 and 3 tumors overlapped with CMS2 or CMS4 tumors, respectively.

Colon Side and the TME

Left-sided and right-sided colon tumors have distinct histologic and molecular characteristics.

Right-side colon tumors arise from the ascending colon and proximal two thirds of the transverse colon whereas left-side colon tumors arise from the descending or sigmoid colon, and distal third of the transverse colon [START_REF] Lee | Is right-sided colon cancer different to left-sided colorectal cancer? -a systematic review[END_REF] . Right-side stage III or IV colon tumors are generally associated with short survival times. These tumors are more commonly MSI, have BRAF mutations, and are hypermutators [START_REF] Kim | Differences Regarding the Molecular Features and Gut Microbiota Between Right and Left Colon Cancer[END_REF] , compared with left-side tumors, which have chromosome instability. High numbers of PD1+ CD8 + T cells, FOXP3 + T cells, CD20 + B cells, and CD138 + IGKC + plasma cells in tumor tissues has been associated with increased OS survival times of patients with right-side colon tumors [START_REF] Berntsson | Expression of programmed cell death protein 1 (PD-1) and its ligand PD-L1 in colorectal cancer: Relationship with sidedness and prognosis[END_REF] . Differences in immune cell features of the right vs left colon might account for the different outcomes of patients with right-side vs leftside colon tumors [START_REF] Lee | Is right-sided colon cancer different to left-sided colorectal cancer? -a systematic review[END_REF] .

The human colon contains complex and diverse microbial colonies of approximately 10 13 to 10 14 bacteria, [START_REF] Lee | Is right-sided colon cancer different to left-sided colorectal cancer? -a systematic review[END_REF] with colony numbers increasing from right to left. Therefore, the left colon, with the highest concentration of microbes, has a more tolerant immune environment.

Tumors that develop in the right colon face a more active immune environment than tumors in the left colon, [START_REF] Lee | Is right-sided colon cancer different to left-sided colorectal cancer? -a systematic review[END_REF] and are infiltrated by higher numbers of lymphocytes. Tumors in the left colon have a higher level of immune-suppressive cells than tumors of the right colon [START_REF] Baran | Difference Between Left-Sided and Right-Sided Colorectal Cancer: A Focused Review of Literature[END_REF] .

Clinical Effects of TME Composition

Primary tumors with no perineural infiltration and no vascular or lymphatic invasion have a higher density of memory T cells than tumors with early signs of metastasis [START_REF] Pagès | Effector memory T cells, early metastasis, and survival in colorectal cancer[END_REF] . High densities of memory and effector T cells, particularly CD8 + T cells, in the center and the invasive margins of tumors correlated with longer progression-free survival (PFS) and overall survival (OS) times of patients [START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF] . Analysis of hepatic [START_REF] Halama | Localization and density of immune cells in the invasive margin of human colorectal cancer liver metastases are prognostic for response to chemotherapy[END_REF] and lung [START_REF] Remark | Characteristics and clinical impacts of the immune environments in colorectal and renal cell carcinoma lung metastases: influence of tumor origin[END_REF] metastases also associated higher densities of CD8 + T cell with better outcome. Although infiltration by CD8 + T cells appears to have positive effects for tumors of all stages, more advanced tumors (stages III and IV) have lower densities of these cells than early-stage tumors [START_REF] Mlecnik | Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction[END_REF] . A reduced adaptive immune response might therefore promote tumor progression [START_REF] Mlecnik | Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction[END_REF][START_REF] Galon | The adaptive immunologic microenvironment in colorectal cancer: a novel perspective[END_REF] .

Analyses of primary tumors and metastases from the same patients provided evidence for immune selection of malignant cells [START_REF] Angelova | Evolution of Metastases in Space and Time under Immune Selection[END_REF] . This mechanism is prevalent in MSI tumor cells, which often lose membrane HLA molecules, [START_REF] Ijsselsteijn | Revisiting immune escape in colorectal cancer in the era of immunotherapy[END_REF][START_REF] Middha | Majority of B2M-Mutant and -Deficient Colorectal Carcinomas Achieve Clinical Benefit From Immune Checkpoint Inhibitor Therapy and Are Microsatellite Instability-High[END_REF][START_REF] Ozcan | Complex pattern of immune evasion in MSI colorectal cancer[END_REF] so they escape T-cell cytotoxicity but not NK cell cytotoxicity. This reduces their metastatic potential [START_REF] Liao | KRAS-IRF2 Axis Drives Immune Suppression and Immune Therapy Resistance in Colorectal Cancer[END_REF] . Analyses of tumor transcriptomes revealed that high expression of genes that regulate T-cell chemotaxis (CXCL9, CXCL10, CXCL11), T-and NK-cell activation (IL15), and Th1 cell development (IFNG) associated with longer survival times of patients [START_REF] Fridman | The immune contexture in human tumours: impact on clinical outcome[END_REF][START_REF] Mlecnik | Biomolecular network reconstruction identifies T-cell homing factors associated with survival in colorectal cancer[END_REF] . Tumors with mutations resulting in loss of expression of IL15 [START_REF] Mlecnik | The tumor microenvironment and Immunoscore are critical determinants of dissemination to distant metastasis[END_REF] or CXCL13 [START_REF] Bindea | Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer[END_REF] , which attracts B cells and is involved in TLS formation, resulted in shorter survival times of patients. Infiltration of colorectal tumors by Th2 [START_REF] Tosolini | Clinical impact of different classes of infiltrating T cytotoxic and helper cells (Th1, th2, treg, th17) in patients with colorectal cancer[END_REF] and Th17 4 cells has been associated with shorter survival times, whereas T follicular helper cells were associated with longer survival times, as were high levels of tumor infiltration by B cells [START_REF] Fridman | The immune contexture in cancer prognosis and treatment[END_REF][START_REF] Bindea | Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer[END_REF][START_REF] Becht | Estimating the population abundance of tissueinfiltrating immune and stromal cell populations using gene expression[END_REF] and the presence of TLS [START_REF] Fridman | The immune contexture in cancer prognosis and treatment[END_REF][START_REF] Caro | Occurrence of tertiary lymphoid tissue is associated with T-cell infiltration and predicts better prognosis in early-stage colorectal cancers[END_REF] . The overall positive effects of high Tcell density in colorectal tumors led to the establishment of an Immunoscore, based on quantification of CD3 + and CD8 + T cells in the center and the invasive margin, which was associated with increased survival times of patients with MSI or microsatellite stable (MSS) tumors [START_REF] Bindea | Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer[END_REF][START_REF] Pagès | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF] .

CRC tissues are enriched in commensal bacteria such as Bacteroides fragilis and Escherichia coli [START_REF] Castellarin | Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma[END_REF] . These bacteria produce stimuli that upregulate expression of genes encoding chemokines that attract T cells to tumors, associated with longer survival times of patients. The abundance of these bacteria in colorectal tumors also correlates with expression of chemokines that recruit T cells [START_REF] Cremonesi | Gut microbiota modulate T cell trafficking into human colorectal cancer[END_REF] . The mechanisms of bacterial species such as Fusobacterium nucleatum, which are associated with lower densities of T cells [START_REF] Mima | Fusobacterium nucleatum and T Cells in Colorectal Carcinoma[END_REF] , lymph node metastasis [START_REF] Castellarin | Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma[END_REF] , and poor outcomes, [START_REF] Mima | Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis[END_REF] require further study [START_REF] Zhou | Fusobacterium and Colorectal Cancer[END_REF] .

Tumor-associated macrophages (TAMs) form a heterogeneous and versatile population of cells, most of which are located in the stroma along the invasive front. The presence of CD68 + macrophages has been associated with increased survival times of patients with CRC 2 whereas CD163 + macrophages have a negative effect [START_REF] Fridman | The immune contexture in cancer prognosis and treatment[END_REF][START_REF] Herrera | Cancer-associated fibroblast and M2 macrophage markers together predict outcome in colorectal cancer patients[END_REF] . Whereas colorectal tumor cells express low levels of the immune checkpoint ligand CD274 molecule (also called PDL1), TAMs located in the invasive margin express high levels of PDL1 and are more abundant in MSI than MSS tumors, indicating a role in the CRC adaptive resistance phenomenon [START_REF] Llosa | The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints[END_REF] . Neutrophils are also present in the TME and correlate with improved outcomes and response to 5 FU-based chemotherapy [START_REF] Galdiero | Occurrence and significance of tumorassociated neutrophils in patients with colorectal cancer[END_REF] . Macrophages and neutrophils might derive from the local differentiation of MDSCs, a heterogenous population of immature myeloid cells that lack robust cell surface markers for detection by immunohistochemistry.

However, the prognostic value of MDSCs requires evaluation in large cohorts of patients.

Mast cells are associated with poor outcomes in 1 study [START_REF] Saadalla | Mast cells promote small bowel cancer in a tumor stage-specific and cytokine-dependent manner[END_REF] .

A gene expression pattern characteristic of fibroblasts associated with reduced survival time survival and decreases the positive effects of a cytotoxic cell signature [START_REF] Becht | Estimating the population abundance of tissueinfiltrating immune and stromal cell populations using gene expression[END_REF] This finding has been attributed mostly to the fact that CAFs produce immunosuppressive TGFB [START_REF] Tauriello | TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis[END_REF][START_REF] Turley | Immunological hallmarks of stromal cells in the tumour microenvironment[END_REF] and VEGF, [START_REF] Palma | Microenvironmental regulation of tumour angiogenesis[END_REF] which impair immune responses even when lymphocytes are able to cross the fibroblastic barrier [START_REF] Kobayashi | Cancer-associated fibroblasts in gastrointestinal cancer[END_REF] .

Immune-based Therapies

CRC was once considered to be resistant to immunotherapy. This changed in 2015, when the striking response of patients with metastatic MSI tumors to anti-PD-1 therapy 100 has opened new avenues and raised questions about therapies to target the tumor stroma. These agents re-activate T-cell anti-tumor responses by blocking checkpoint molecules such as programmed cell death 1 (PDCD1, also called PD1) 100 . Immunotherapy for CRC has become the paradigm for all types of tumors with MSI, and for tumors with a high mutation burden 101 . Trials are underway to determine whether the combination of anti-PD1 and anti-CTLA4 increases the response rate and survival times of patients with MSI tumors 102,103 (see Table 2).

MSI tumors have all characteristics required to respond to immune checkpoint inhibitors 104 , are surrounded by PD1+ CD8+ T cells, and express high levels of PDL1 [START_REF] Llosa | The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints[END_REF] . MSI tumors account for only 3%-5% of all metastatic colorectal tumors [START_REF] Latham | Microsatellite Instability Is Associated With the Presence of Lynch Syndrome Pan-Cancer[END_REF] . However, extension of immune checkpoint inhibitor therapy to treatment for primary colorectal tumors might increase the number of patients who benefit from these therapies. Computational strategies (deep residual learning) diminish the price to identify tumors with MSI based on histologic features that might result in immune checkpoint inhibitor therapy for a larger number of patients with CRC 105 .

Interestingly, the efficacy of PD1 inhibitors against MSI tumors raises questions that, if answered, might reveal new treatment options for MSS tumors. For example, due to immune-cell selection, many MSI tumors express few or no HLA molecules, which are required for antigen presentation to CD8 + T cells [START_REF] Grasso | Genetic Mechanisms of Immune Evasion in Colorectal Cancer[END_REF] . This might explain the growth of primary MSI tumors despite their infiltration by T cells. However, HLA defects are rare in liver metastases, in contrast to metastases in other organs [START_REF] Ijsselsteijn | Revisiting immune escape in colorectal cancer in the era of immunotherapy[END_REF] , which could account for the response of patients with metastatic CRC, which usually spreads to the liver, to PD1 inhibitors 100 .

Studies of responses in patients with primary MSI tumors that have lost HLA expression [START_REF] Middha | Majority of B2M-Mutant and -Deficient Colorectal Carcinomas Achieve Clinical Benefit From Immune Checkpoint Inhibitor Therapy and Are Microsatellite Instability-High[END_REF] should help answer this important question and provide additional information about mechanisms of immune checkpoint inhibitor therapy. In MSI tumors that have lost HLA expression, responses to PD1 inhibitors could resemble the sensitivity of Hodgkin disease, which despite a loss of HLA expression responds to PD1 inhibitors. 106 Other T-cell subsets or NK cells might act as effectors.

It is also important to learn why MSS colorectal tumors that are highly infiltrated by T cells do not respond to immune checkpoint inhibitors. It has been proposed that the Immunoscore (the density of CD3 + and CD8 + T cells) more accurately predicts survival times of patients with CRC than MSI 107 . In MSS tumors, CD8 + T cells might control tumor growth but still cannot promote tumor regression-other elements of the TME might continue to support tumor development. CMS4 tumors are characterized by high levels of myeloid cell infiltration, high levels of angiogenesis, and fibroblastic contents. [START_REF] Becht | Immune and Stromal Classification of Colorectal Cancer Is Associated with Molecular Subtypes and Relevant for Precision Immunotherapy[END_REF] These tumors might be a paradigmatic stroma-rich colorectal tumor sub-class that contains many different cell types and would be a good candidate for testing TME-targeted therapies. Studies are needed to determine whether immune checkpoint inhibitors can be used in combination with other strategies for treatment of MSS tumors. This question is further under consideration by the COLOSSUS CRC research network.

Most TAMs have an M2 phenotype, produce complement components, and have inflammatory and angiogenic activities 108 such as production of VEGF 109 and immunosuppressive cytokines (IL10) cytokines, 110 resulting in T-cell exhaustion 111 and angiogenesis 112 . Therefore, the combination of anti-angiogenic and immune checkpoint inhibitor therapy might result in reactivation of the anti-tumor immune response 112 . This combination is currently being tested in patients with MSI colorectal tumors (TECENTRIC, NCT02982694). Agents that block colony stimulating factor 1 receptor, or the CCL2 receptors CCR2 and CCR5, which are expressed by macrophages and MDSCs and induce macrophage repolarization, 113 are being tested in MSS in combination with anti-PD1 and anti-PDL1 in patients with advanced CRC (NCT02713529 with AMG 820, and MARACON, NCT03184870).

Neutralization of inflammatory complement components is a new strategy for treatment of CRC 108 . Agents that block C5aR reduced tumor growth in mice, alone [START_REF] Spranger | Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity[END_REF] or in combination with a checkpoint inhibitor. [START_REF] Meijer | The CXCR5 chemokine receptor is expressed by carcinoma cells and promotes growth of colon carcinoma in the liver[END_REF]109,110 A phase 1 study is underway to test the combination of a C5aR inhibitor and anti-PDL1 in patients (STELLAR-001, NCT03665129 (see Table 2). Agents that target different steps of the complement cascade might be adapted for cancer therapy 112 .

Fibroblasts produce TGFB and VEGF and mechanically prevent entry of therapeutic cells and agents into the tumor core [START_REF] Kobayashi | Cancer-associated fibroblasts in gastrointestinal cancer[END_REF] . It is a challenge to target fibroblasts therapeutically, given their heterogeneity, plasticity, and the role of ECM in maintenance of tissue stiffness.

Of the immune-suppressive cytokines present in CMS4 tumors, TGFB is a challenge to target, given its multiple functions 114 . It may be similarly challenging to target IL10, due to its dual effects on the immune response 115 Chemotherapies that include oxaliplatin have been reported increase immune cell cytotoxicity toward cancer cells and activation of the adaptive immune response 117 . Also, oncolytic viruses not only induce immune-cell killing of tumor cells, but also remodel the TME 118 . Inhibitors of beta-catenin or PAX4 might increase tumor infiltration by immune cells, and strategies are being developed to increase the immune response against MSS tumors (see Figure 3).

Other ways to increase the anti-tumor immune response would be to deliver T cells directly to the tumor core, using chemokines such as CXCL9 and CXCL10. It might be possible to increase interaction of tumor cells with immune cells using bi-specific antibodies, or by infusing effector T cells, tumor-infiltrating lymphocytes, or T cells with chimeric antigen receptors against tumor antigens (reviewed in 103 ). Stem cell features of cancer cells have been associated with a suppressed immune response, higher intra-tumor heterogeneity, and reduced survival times of patients 119 . Inhibitors of stem cell markers such as CD133 or the polycomb group protein BMI1, or agents that induce tumor cell differentiation, might slow tumor growth or progression and reduce immunosuppression.

Targeting the Tumor Vasculature

In tumor tissues, the most common method to evaluate angiogenesis is to measure microvessel density (MVD), based on endothelial markers such as CD31, CD34, or endoglin.

Nevertheless, studies have produced conflicting results on the prognostic value of MVD for patients with CRC. MVD was correlated with depth of invasion, metastasis to lymph nodes and distant sites, and tumor node metastasis stage; there was an inverse correlation between MVD and OS. 127,[129][130][131] In other studies, researchers found no correlation between MVD and PFS or OS [132][133][134][135] . Specifically, Prall et al 134 reported that patients with tumors with high MVD had longer times of cancer-specific survival. The conflicting results might be due to differences in the methods used to determine MVD 136 . For markers that are detected by immunohistochemistry (CD31, CD34, and Von Willebrand factor) and size of area examined varied among studies. MVD has also been studied in patients treated with bevacizumab, a humanized monoclonal antibody that binds VEGF and inhibits its binding to its receptor. However Jubb et al 137 did not associate MVD with efficacy of bevacizumab in a post-hoc analysis of data from a trial of bevacizumab in addition to the standard of care (irinotecan, 5fluorouracil, and leucovorin) in patients with previously untreated metastatic CRC 138 .

Angiogenesis inhibitors (antibodies or small molecules) are included in standard treatments for patients with CRC (for review, see ref 139 ). Bevacizumab was the first angiogenesis inhibitor approved by the Food and Drug Administration for treatment of renal cancer 140 . It was subsequently approved as a first-line treatment agent for metastatic CRC, in combination with irinotecan, 5-fluorouracil, and leucovorin 138 . Inclusion of bevacizumab in this combination increased the mean OS time of patients from 15.6 months to 20.3 months. A retrospective analysis found no association between tumor mutations in KRAS, BRAF, or TP53 and survival time after bevacizumab therapy 141 . Bevacizumab alone is approved for first-line therapy for CRC, whereas other anti-angiogenic agents have only been approved for treatment of patients with tumor progression. The efficacy of bevacizumab as a second-line agent has been evaluated in patients whose metastatic CRC progressed after they were given the standard bevacizumab-containing regimen as their first-line therapy. 142 This study found that that continued bevacizumab therapy prolonged OS (by 1.4 months) and PFS (by 1.6 months) (Table 2).

Drugs with a broader scope of inhibition have approved for treatment of CRC, such as aflibercept (VEGF Trap). Aflibercept is a high-affinity soluble decoy receptor for VEGF that was approved (in combination with FOLFIRI) for treatment of patients with metastatic CRC that progressed or is resistant to oxaliplatin-based therapies 143 . Ramucirumab is a monoclonal human IgG1 against the extracellular domain of VEGFR2 that prevents binding of VEGF A-E, and consequently, VEGFR2 activation 144 . The small molecule angiogenesis inhibitor regorafenib has been approved for treatment of metastatic CRC. It is an orally administrated inhibitor of the tyrosine kinases VEGFR1-3, TIE2, FGFR1, PDGFR beta, KIT, and RET, RAF, RAF1, BRAF, and BRAFV600E. Regorafenib has been approved for salvage monotherapy in patients with refractory metastatic CRC, but it has a significant toxicity profile and questionable efficacy. Nevertheless, the drug is being tested in combination with FOLFIRINOX (NCT03828799) (See Table 3).

Li et al found an inverse correlation between levels of SMAD4 and TGFB1 with lymphatic microvessel density in a study of 147 patients colorectal tumors. 152 Additionally, patients with SMAD4-positive tumors had significantly longer overall and tumor-free survival times than patients with SMAD4-negative tumors, indicating that TGFB1 signaling inhibits lymphatic outgrowth and reduces metastasis. Nevertheless, TGFB1 is a complex pleiotropic growth factor with paradoxical effects-it inhibits proliferation of normal epithelial cells and cells in early-stage tumors, but promotes proliferation of malignant and stroma cells in late-stage tumors. For a review of TGFB1 signaling in metastatic colorectal tumors and therapeutic strategies, see ref 153 Agents designed to block lymphangiogenesis are being tested for their ability to prevent colorectal tumor metastasis. Unfortunately, lymphangiogenesis has proven a difficult process to specifically target in patients with CRC 148 . Sorafenib, an inhibitor of multiple tyrosine kinases, blocks VEGFR3, which regulates lymph vessel outgrowth. 154 First-line treatment of patients with CRC with sorafenib in combination with FOLFOX did not increase patient survival time (RESPECT trial, NCT00865709) 155 . There is a large amount of redundancy in lymphangiogensis signaling, so if 1 pathway is blocked, another will compensate. No agent that interferes with lymphangiogenesis is being used in treatment of CRC 148 .

Biomarkers of Response to Treatment

Resistance of tumor cells to drugs (initial or acquired during treatment) poses an constant challenge 156 , and strategies are needed to determine which tumors are most likely to respond to which therapies. Genomic [START_REF] Van Cutsem | Addition of aflibercept to fluorouracil, leucovorin, and irinotecan improves survival in a phase III randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin-based regimen[END_REF][START_REF] Smeets | Copy number load predicts outcome of metastatic colorectal cancer patients receiving bevacizumab combination therapy[END_REF][157][158][159][160][161][162][163] and other classes of biomarkers of response have been proposed, 164 but there are no markers that can be used to predict response to anti-angiogenic agents.

Chromosome instability was reported to be a biomarker of response to bevacizumab in patients with metastatic CRC [START_REF] Smeets | Copy number load predicts outcome of metastatic colorectal cancer patients receiving bevacizumab combination therapy[END_REF] . Tumors with intermediate to high levels of chromosome instability (clusters 2 and 3) had better responses to chemotherapy with bevacizumab than to chemotherapy alone (prolonging PFS by 149 days for cluster 2 and 85 days for cluster 3).

Colorectal tumors with low levels of chromosome instability (cluster 1), which include those with mutations in POLE and MSI, did not have an increased to chemotherapy that included bevacizumab, nor did metastatic colorectal tumors in phase 2 MoMa study (NCT02271464).

A chromosome instability threshold in which ≥25% of chromosomal regions contained CNAs has been proposed for identification of tumors most likely to respond to bevacizumab.

Patients whose tumors were above this threshold who received bevacizumab therapy had significantly longer times of PFS than those given the standard of care chemotherapy (REF132). This difference was not observed in when patients with tumors with high levels of chromosome instability were compared with patients with tumors with low levels of chromosome instability given chemotherapy alone. These require confirmation in a prospective trial, but CNA might be a biomarker of response to certain therapies.

Tebbutt et al investigated the association between CMS and response of patients

with unresectable metastatic CRC to capecitabine; capecitabine and bevacizumab; and capecitabine, bevacizumab, and mitomycin (NCT00294359). 165 Patients with CMS2 tumors (and possibly CMS3 tumors) given the combination of capecitabine and bevacizumab or capecitabine with bevacizumab and mitomycin had longer PFS than patients given capecitabine alone, but this association was not observed in patients with CMS1 or CMS4 tumors 166 . A retrospective analysis of patients with colorectal tumors without mutations in RAS treated with either mFOLFOX6 or FOLFIRI, combined with bevacizumab or cetuximab as first-line therapy, reported equal survival times (CALGB/SWOG 80405 trial). 167 Interestingly, patients with CMS1 tumors had longer survival times after bevacizumab-based treatment than cetuximab-based treatment. This study compared the effects bevacizumab with those of different control groups (patients treated with cetuximab vs standard of care chemotherapy) than those included in the analyses of Smeets et al. [START_REF] Smeets | Copy number load predicts outcome of metastatic colorectal cancer patients receiving bevacizumab combination therapy[END_REF] Nevertheless, findings from all 3 studies indicate a need for additional analyses, using large and diverse patient cohorts to confirm the association between CMS and outcomes of patients treated with bevacizumab.

The side of the colon in which a tumor develops associates with response to therapy.

In the FIRE3 trial (NCT00433927) of patients with metastatic colorectal tumors without mutations in RAS, those with left-side colon tumors had a significantly better outcomes after first-line therapy with FOLFIRI and cetuximab (an antibody against EGFR) than with FOLFIRI and bevacizumab (OS time, 38.3 months vs 28 months) 168 . In contrast, in patients with right-side colon tumors, there was no significant difference in survival between patients given either combination (OS 18.3 months vs 23.0 months respectively). Yoshimo et al showed that addition of ramucirumab to FOLFIRI (in the RAISE trial, NCT01183780) as a second-line therapy increased survival times of patients with metastatic colon cancer, regardless of tumor side (or mutational status) 169 . These findings indicate that the side of the colon on which the tumor develops affects to some treatment regimens, but not all.

Future Directions

Colorectal tumors develop via many different pathways that result in many different TMEs.

Mutations in DNA repair genes, the DNA polymerase E gene, and BRAF, as well as the CpG island methylator phenotype, result in high mutation burden and tumor infiltration by lymphocytes. Conversely, APC mutations are associated with lack of lymphocyte infiltration, due to activation of beta-catenin. Tumors with mutations in RAS (and probably BRAF) are resistant to anti-EGFR therapies whereas MSI colorectal tumors often respond to immune checkpoint inhibitors.

Different colorectal tumors subtypes therefore respond differently to therapies that target the TME. Integration of data on TMEs with genome and transcriptome profiles might identify the best therapeutic combinations for each patient's tumor type, comprising chemotherapies, immunotherapies, anti-angiogenic therapies, and anti-stromal agents.

Immunogenic chemo-and radiotherapies, and oncolytic virus-based therapies, are in development. Cell-based therapies such as autologous tumor-infiltrating lymphocytes or T cells with chimeric antigen receptors are also being developed and might be effective against tumors that do not induce and immune response. These types of therapies will be selected based on specific features of each patient's tumor and TME.

Figure 2. The Consensus Molecular Subtypes

CMS1 and CMS4 tumors are highly infiltrated by immune cells, whereas CMS1 tumors are characterized by a Th1-cell response and activated and inflamed TME. These tumors can be treated with immune checkpoint inhibitors. CMS4 tumors have an inflamed, complementrich, suppressive and highly angiogenic TME that can be targeted with combination therapies. CMS2 do not activate an anti-tumor immune response, due to activation of the beta-catenin pathway, and CMS3 considered to be metabolic tumors.

Figure 3. Treatment of MSI and MSS Tumors

MSI tumors should be treated with immune checkpoint inhibitors, specifically with inhibitors of PD1, potentially combined with inhibitors of CTLA4. Among MSS tumors, patients with tumors without mutations in RAS respond to cetuximab or panitumumab in combination with chemotherapy. Tumors with mutations in APC and activation of WNT signaling to betacatenin might be treated with inhibitors of beta-catenin or PAX4. Mesenchymal-type tumors might be treated with a combination of immune checkpoint inhibitors and anti-angiogenic, anti-inflammatory, anti-complement, or anti-TGFB agents, in combination with chemotherapy. RAS mutant tumors might respond to anti-angiogenic therapies, T-cell-based, or T-cell activating immunotherapies.

.

  Indoleamine deoxygenase (IDO) is expressed by mesenchymal cells, myeloid dendritic cells, T-regulatory cells, and tumor cells. Trials of the IDO inhibitor, epadadostat, in combination with anti-PD1 blockade (ECHO-204, NCT02327078) are underway in patients with CRC. Oncolytic viruses replicate specifically in tumor cells and promote tumor infiltration by lymphocytes and induction of specific anti-tumor immune responses. These might be used to increase the response of MSS colorectal tumors to anti-PD1 therapy (NCT02963831 with ONCOS102). Tumor development results in epigenetic alterations that reduce antigen presentation and responses of T cells to tumor cells, allowing tumors to evade immune surveillance. Demethylating agents, which increase expression of genes including HLA genes, might increase antigen presentation 116 . These types of agents reduced tumor growth in mice and are being tested in combination with immune checkpoint inhibitors in patients with melanomas (Di Giacomo et al, Clin Cancer Res, in press).The histone deacetylase inhibitor entinostat is being tested in combination with anti-PD1 in patients with MSS colorectal tumors (NCT02437136).

Figure 1 .

 1 Figure 1. The Microenvironment of Colorectal TumorsDispersed immune cells are in the tumor center, mostly in the invasive margin that juxtaposes the non-tumor area of the colon, with some forming TLS. Blood vessels, high endothelial venules, and lymphatic vessels allow entry and/or egress of immune cells.

Table 1 . Cells the Tumor Microenvironment and Functions

 1

	Cell Type	STRUCTURES	FUNCTIONS
	Lymphocytes
	NK cells	MHC class I-negative cells	killing MHC class I-negative cells
		IgG bound to target cells	antibody-dependent cellular cytotoxicity of an
	B cells	native soluble or membrane	antibody production
		antigens	antigenic peptide presentation via MHC class
	CD8+ T CELLS
		peptides presented by MHC class	killing MHC class I-positive cells
		I	regulate responses of T and B cells
	CD4+ T CELLS		produce IFNG
		peptides presented by MHC class	produce IFNG IL2
	Th2 cells	II	activate CD8+ T cells to become cytotoxic
			produce IL4, IL13
	T-follicular helper cells		activate B cells to become antibody-producing
			produce CXCL13
	Th17 cells		recruit and activate B cells in TLS
			produce IL17
	T-regulatory cells		activate macrophages to produce IL6 and IL8
			produce IL10, TGFB
			suppress responses of T and B cells
	NK T Cells		killing target cells in an MHC-unrestricted ma
	Myeloid Cells
	Dendritic cells	danger signals (DAMP, PAMP	produce IL12, IL18
			present antigen to T cells via MHC I and II
	Macrophages	danger signals (DAMP, PAMP)
	M1	IgG-coated target cells	produce IL1, IL6
			phagocytosis of target cells
			antibody-dependent cytotoxicity of antibody-c
			produce VEGF, IL10, TGFB, and complemen
	M2		promote angiogenesis, fibroblast activation im
	Mast cells		produce inflammatory mediators (serotonin, le
	Polymorphonuclear		phagocytosis of target cells or inflammatory
	cells
	MDSC	danger signals (DAMP, PAMP)	immature cells of heterogeneous population fr
			suppress immune responses
	Stromal cells
	Follicular dendritic		present antigen to B cells via immune comple
	cells
	CAF		produce angiogenic factors (VEGF, CXCL12,
			produce immunosuppressive TGFB and M2 p
			CXCL18

Table 3 . Angiogenic Agents Approved for Treatment of CRC Name

 3

		Targets	Type	Use in CRC	Novel combinations in	Predicted response
					progress	per molecular
						subtype
	Bevacizumab	VEGFA	humanized	1 st line in	phase 2 trial of	debatable, CMS:
	(Avastin)		monoclonal	patients with	pembrolizumab,	predicted to
			antibody	tumors with	capecitabine, and	provide benefit to
			against	mutations in	bevacizumab in	CMS2 and CMS4
			VEGFA	RAS, given	patients with	tumors (Smeets et
				with	microsatellite-stable,	al) or CMS2 and
				leucovorin or	locally advanced, or	CMS3 tumors
				capecitabine a	metastatic tumors, or	(Mooi et al) or
					tumors that cannot be	patients with
					removed by surgery	tumors with more
					(NCT03396926).	than 25%
						chromosome
						instability b
	Aaflibercept	VEGFA,	Decoy	2 nd line in	phase 1:	unknown but
	(Zaltrap)	VEGFB,	receptor	combination	pembrolizumab and	predicted to work
		placental		with	ziv-aflibercept in	in CMS2 and
		growth		FOLFIRI a	treating patients with	CMS4 tumors,
		factor			advanced solid tumors	based on
					(NCT02298959)	bevacizumab
	Ramucirumab	VEGFR2	Fully human	2nd Line in	RAMTAS:	unknown,
	(Cyramza)		anti-	combination	Ramucirumab in	predicted to have
			VEGFR2	with	combination with	efficacy against
			antibody.	FOLFIRI a	TAS102 vs TAS102	CMS2 and CMS4
					alone in patients with	tumors, based on
					refractory metastatic	bevacizumab
					CRC (NCT03520946).	activity
	Regorafenib	PDGFRB,	Multi	Salvage	phase 1b,	inconclusive:
	(Stivarga)	TIE2, KIT,	receptor	Monotherapy	NCT01973868	predicted to
		Pan-	Tyrosine	in refractory	regorafenib and	provide benefit to
		VEGFR,	kinase	mCRC	cetuxumab in	CMS2, CMS3,
		RAF,	inhibitor		colorectal tumors with	and CMS4 tumors
		BRAF,			full-length RAS b	in PFS and CMS2
		BRAF v600E , FGFR1,				and CMS4 in OS c,d
		RET

Table 2 . Clinical Trials of Agents Designed to Target Tumor Stroma

 2 colorectal cancer; MMRP, DNA mismatch repair proficient; PLK1, polo-like kinase1;

	Trial	Drug combination	Patient population	Status
	anti-angiogenics
	bevacizumab	1st line tas 102 + bevacizumab vs	854 untreated patients with	recruiting. due
	(avastin)	capcitabine + bevacizumab	MCRC who were not candidates	for completion
	solstice		for irinotecan or oxaliplatin	September 2022
			therapy
	vitality	1 st line oxaliplatin, 5-fluorouracil,	428 previously untreated patients recruiting, due
		leucovorin +/-bevacizumab and		for completion
		vitamin C vs line oxaliplatin, 5-		December 2020
		fluorouracil, leucovorin +/-
		bevacizumab
	bevacizumab	bevacizumab + binimetinib (MEK	40 patients with MCRC without a	Recruiting, due
	(avastin)	inhibitor) + prembrolizumab	response to prior therapy	for completion
				August 2019
	bevacizumab	2 nd line bevacizumab + 5-	44 patients with metastatic	recruiting. due
	(avastin)	fluorouracil, leucovorin + irinotecan	colorectal tumors with a mutation	for completion
		+ onvansertib (an inhibitor of polo-	in KRAS failed by previous	May 2021
		like kinase 1)	treatment, or patients who are
			intolerant to, oxaliplatin
	aflibercept	aflibercept + prembrolizumab	78 patients with advanced solid	recruiting. due
	(zaltrap)		tumors	for completion
				December 2021
	ramucirumab	tas102 +/-ramucirumab	144 patients with advanced	Recruiting, due
	(cyramza)		MCRC that progressed on or	for completion
	ramtas		after, or patients who did not	June2021
			tolerate, fluoropyrimidines,
			oxaliplatin, irinotecan, or anti-
			angiogenic therapies
	donafenib (multi	2 nd line donafenib vs best supported	510, patients with MCRC that	active not
	tyrosine kinase	care	progressed during or within 3	recruiting, due
	inhibitor)		months of final dose of therapy	for completion
				April 2020
	regorafenib	salvage regorafenib + irinotecan vs	78 previously treated patients	recruiting, due
	(stivarga)	regorafenib only	with MCRC that progressed	for completion
	next-regiri		during or within 3 months of last	June 2023
			treatment of standard therapy
			bearing genotype a/a of CCND1
	remety	2 nd line tas 102 + regorafenib	18 patients with MCRC that	Recruiting, due
			progressed after standard therapy	for completion
				October 2019
	folfirinox-r	regorafenib + 5-fluorouracil,	87 patients with metastatic	recruiting. due
		leucovorin + irinotecan + oxaliplatin	colorectal tumors with mutant	for completion
		(folfirinox)	RAS	March 2022
	regorafenib	regorafenib + pembrolizumab	75 patients with MCRC failed	not yet
	(stivarga)		by, or patients who are intolerant	recruiting, due
			to, oxaliplatin, irinotecan, or	for completion
			fluorouracil	in July 2022
	checkpoint

Funding WHF, CSF and ATB receive funding from the European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 754923 " COLOSSUS" A.T.B. is further supported by Science Foundation Ireland under grant 13/CDA/2183 "COLOFORETELL' and by the Health Research Board under grant ILP-POR-2019-066 WHF, CSF receive funding from INSERM, Université de Paris, Sorbonne University, CARPEM T8, the Labex Immuno-Oncology Excellence Program, Institut du Cancer (INCa), HTE Plan Cancer (C1608DS), and the Cartes d'Identité des Tumeurs (CIT) Program from the Ligue Nationale Contre le Cancer Acknowledegments