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Colorectal cancer (CRC) has high mortality rates, especially in patients with advanced disease stages, who often do not respond to therapy. The cellular components of the tumor microenvironment are essentially responsible for dictating disease progression and response to therapy. Expansion of different myeloid cell subsets in CRC tumors has been reported previously. However, tumor-infiltrating myeloid cells have both pro- and anti-tumor roles in disease progression. In this study, we performed transcriptomic profiling of cells of myeloid lineage (CD33 + ) from bulk CRC tumors at varying disease stages. We identified differentially expressed genes and pathways between CRC patients with advanced stage and early stages. We found that pro-angiogenic and hypoxia-related genes were upregulated, while genes related to immune and inflammatory responses were downregulated in CD33 + myeloid cells from patients with advanced stages, implying that immune cell recruitment and activation could be compromised in advanced disease stages. Moreover, we identified a unique "poor prognosis CD33 + gene signature" by aligning top upregulated and downregulated genes in tumor-infiltrating myeloid cells from our analyses with data from The Cancer Genome Atlas. Our results showed that this gene signature is an independent prognostic indicator for disease-specific survival in CRC patients, potentially reflecting its clinical importance.

Aim:  To elucidate the epigenetic alterations behind the upregulation of immune checkpoints and T cell exhaustion markers in colorectal cancer (CRC) patients. Materials & methods: mRNA expressions of different immune checkpoint/exhaustion markers were analyzed by quantitative real-time reverse transcriptase PCR and epigenetic investigations were performed using bisulfite sequencing and chromatin immunoprecipitation quantitative PCR. Results: mRNA expressions of PD-1, TIM-3, CTLA-4, PD-L1 and TOX2 were significantly upregulated in CD4 + and CD8 + tumor-infiltrating lymphocytes and bulk CRC tumor tissues. Histone 3 lysine 9 trimethylation was downregulated and histone 3 lysine 4 trimethylation was upregulated in PD-L1 and TOX2 promoters in tumor tissues, suggesting that PD-L1 and TOX2 upregulation in CRC tumors could be mediated by activating histone 3 lysine 4 trimethylation. Conclusion: Epigenetic modifications in promoters of immune checkpoint and T cell exhaustion genes could induce their upregulation, and potentially implicate the use of epigenetic modifiers to enhance antitumor immunity in CRC patients.

Despite recent advances in colorectal cancer (CRC) treatment, a large proportion of patients show limited responses to therapies, especially in advanced stages. There is an urgent need to identify prognostic biomarkers and/or therapeutic targets in advanced stages, aiming to improve the efficacy of current treatments. We aimed to determine prognostic biomarkers in tumor tissue and circulation of CRC patients, with a special focus on T cell exhaustion markers. We found that mRNA levels of PD-1, TIM-3, CTLA-4, TIGIT, CD160, CD244, KLRG1, TOX2, TOX3, Ki-67, and PRDM1 were elevated in CRC tumor tissues. We also investigated differences in gene expression between early and advanced disease stages. We found that TOX and potentially TIM-3, CTLA-4, VISTA, TIGIT, KLRG1, TOX2, SIRT1, Ki-67, and Helios mRNA levels in tumor tissue were elevated in advanced disease stages, suggesting their potential roles in CRC progression. In contrast, PD-1 and CD160 levels in tumor tissue were downregulated in advanced stages. In the circulation of CRC patients, mRNA levels of PD-1, VISTA and LAG-3 were higher than those of healthy individuals. Moreover, in circulation, PD-1, CTLA-4 and TIGIT mRNA levels were reduced in advanced stages. Interestingly, levels of PD-1 in both tumor tissue and circulation were reduced in advanced stages, suggesting that targeting PD-1 in patients with advanced stages could be less effective. Altogether, these findings suggest some potential T cell exhaustion markers that could be utilized as prognostic biomarkers and/or therapeutic targets for CRC. However, further investigations and validations in larger cohorts are required to confirm these findings.

Tumor-infiltrating lymphocytes (TILs) play indispensable roles in the progression and response to treatment of solid tumors. However, the prognostic significance of CD4 + TILs is not fully disclosed in cancers generally and in CRC in particular, mainly due to the existence of different functional subsets of CD4 + T cells. We performed transcriptomic profiling of CD4 + TILs isolated from CRC patients in order to identify differentially expressed genes and their functional pathways in early versus advanced disease stages. We found that in advanced stages, genes related to immune and inflammatory responses, in particular Th1-mediated immune response and cytotoxicity-mediated genes, were downregulated; while epigenetic-mediated silencing genes were upregulated. Interestingly, we identified genes, which were steadily upregulated or downregulated in CD4 + TILs with CRC progression from stage I to IV. Additionally, of the top 200 deregulated genes, 43 upregulated and 64 downregulated genes showed similar deregulation trends in the cancer genome atlas CRC dataset. From these 97 deregulated genes, we identified a "poor prognosis CD4 gene signature (ppCD4sig)". Patients with high ppCD4sig score showed shorter disease-specific survival (DSS) and progression-free interval (PFI). The ppCD4sig was an independent prognostic indicator for DSS (HR = 1.73, 95% CI 1.32-2.27, P = 0.0001) and PFI (HR = 1.75, 95% CI 1.3-2.35, P = 0.0016). Additionally, patients at advanced stages and at a younger age (<55 years) were more likely to have a high ppCD4sig score. Altogether, our data provide novel insights and a unique prognostic gene signature of CD4 + TILs in the CRC microenvironment., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Cytotoxic CD8 + T cell-mediated response is the most important arm of adaptive immunity, which dictates the capacity of the host immune response in eradicating tumor cells. Due to tumor intrinsic and/or extrinsic factors, the density and function of CD8 + tumor-infiltrating lymphocytes (TILs) could be compromised, leading to poor prognosis and survival., Methods: Using RNA-Seq, transcriptomes of sorted CD3 + CD8 + TILs from treatment-naïve colorectal cancer (CRC) patients at advanced stages (III and IV) were compared with those from patients with early stages (I and II). A signature referred to as 'poor prognosis CD8 gene signature (ppCD8sig)' was identified and analyzed in The Cancer Genome Atlas CRC dataset. Scores for the ppCD8sig were calculated and classified as high, intermediate and low, and its prognostic significance was assessed using multivariate analysis and Cox proportional hazard model. Densities of CD3 + and CD8 + T cell infiltration in tumors from patients with high and low ppCD8sig scores were assessed by flow cytometry and immunostaining., Results: Genes related to epigenetic regulation and response to hypoxia were upregulated in CD8 + TILs from patients with advanced stages, while genes related to T cell activation, cell proliferation and cell cycle were downregulated. Patients with high ppCD8sig score had poorer disease-specific survival (DSS) and shorter progression-free interval (PFI). The ppCD8sig was an independent prognostic indicator for DSS (HR 1.83, 95% CI 1.40 to 2.38, p<0.0001) and PFI (HR 1.42, 95% CI 1.04 to 1.93, p=0.026). Additionally, patients with high ppCD8sig score were more likely to have advanced stages (χ 2 p<0.0001) and residual disease after primary therapy (χ 2 p=0.046). Patients with high ppCD8sig score had reduced levels of CD3 + and CD8 + TILs and low Immunoscores (IS), compared to patients with low ppCD8sig score., Conclusions: Our data provided insights into the altered regulation of biological mechanisms and signaling pathways in CD8 + TILs during CRC progression, and revealed a gene signature as an independent prognostic indicator. Patients with high ppCD8sig score had lower levels of TILs and low IS. These data further confirm the prognostic value of the identified ppCD8sig and potentially highlight its clinical relevance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Increased numbers of myeloid-derived suppressor cells (MDSCs) are positively correlated with poor prognosis and reduced survivals of cancer patients. They play central roles in tumor immune evasion and tumor metastasis. However, limited data are available on phenotypic/transcriptomic characteristics of the different MDSCs subsets in cancer. These cells include immature (I-MDSCs), monocytic (M-MDSCs), and polymorphonuclear/granulocytic (PMN-MDSCs)., Methods: Phenotypic characterization of myeloid subsets from 27 colorectal cancer (CRC) patients was assessed by flow cytometric analyses. RNA-sequencing of sorted I-MDSCs, PMN-MDSCs, and antigen-presenting cells (APCs) was also performed., Results: We found that the levels of I-MDSCs and PMN-MDSCs were increased in tumor tissues (TT), compared with normal tissues (NT) in colorectal cancer. Our functional annotation analyses showed that genes associated with histone deacetylase (HDAC) activation- and DNA methylation-mediated transcriptional silencing were upregulated, and histone acetyl transferase (HAT)-related genes were downregulated in tumor-infiltrating I-MDSCs. Moreover, pathways implicated in cell trafficking and immune suppression, including Wnt, interleukin-6 (IL-6), and mitogen-activated protein kinase (MAPK) signaling, were upregulated in I-MDSCs. Notably, PMN-MDSCs showed downregulation in genes related to DNA methylation and HDAC binding. Using an ex vivo model, we found that inhibition of HDAC activation or neutralization of IL-6 in CRC tumor tissues downregulates the expression of genes associated with immunosuppression and myeloid cell chemotaxis, confirming the importance of HDAC activation and IL-6 signaling pathway in MDSC function and chemotaxis., Conclusions: This study provides novel insights into the epigenetic regulations and other molecular pathways in different myeloid cell subsets within the CRC tumor microenvironment (TME), giving opportunities to potential targets for therapeutic benefits.

Background: Colorectal cancer (CRC) is the third most commonly diagnosed human malignancy worldwide. Upregulation of inhibitory immune checkpoints by tumor-infiltrating immune cells (TIICs) or their ligands by tumor cells leads to tumor evasion from host immunosurveillance. Changes in DNA methylation pattern and enrichment of methylated histone marks in the promoter regions could be major contributors to the upregulation of immune checkpoints (ICs) in the tumor microenvironment (TME)., Methods: Relative expressions of various immune checkpoints and ligands in colon normal tissues (NT) and colorectal tumor tissues (TT) were assessed by qRT-PCR. The epigenetic modifications behind this upregulation were determined by investigating the CpG methylation status of their promoter regions using bisulfite sequencing. Distributions of histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) in promoter regions of these genes were assessed by chromatin immunoprecipitation (ChIP) assay., Results: We found that the expression levels of PD-1, CTLA-4, TIM-3, TIGIT, PD-L1, and galectin-9 were significantly higher in colorectal tumor tissues, compared with colon normal tissues. To study the role of DNA methylation, we checked the promoter CpG methylation of ICs and ligands and found that only CTLA-4 and TIGIT, among other genes, were significantly hypomethylated in TT compared with NT. Next, we checked the abundance of repressive histones (H3K9me3 and H3K27me3) in the promoter regions of ICs/ligands. We found that bindings of H3K9me3 in PD-1 and TIGIT promoters and H3K27me3 in CTLA-4 promotor were significantly lower in TT compared with NT. Additionally, bindings of both H3K9me3 and H3K27me3 in the TIM-3 promoter were significantly lower in TT compared with NT., Conclusion: This study shows that both DNA hypomethylation and H3K9me3 and H3K27me3 repressive histones are involved in upregulation of CTLA-4 and TIGIT genes. However, repressive histones, but not DNA hypomethylation, are involved in upregulation of PD-1 and TIM-3 genes in CRC tumor tissue. These epigenetic modifications could be utilized as diagnostic biomarkers for CRC.