Prolyl hydroxylase inhibition protects against murine MC903-induced skin inflammation by downregulating TSLP.

Previously, we reported an anti-inflammatory effect of mTORC1 in a mouse model of type 2 skin inflammation. TSLP, one of the epithelial cell-derived cytokines, was upregulated by Raptor deficiency or rapamycin treatment, which was inhibited by dimethyloxalylglycine (DMOG). However, it remains unclear how DMOG regulates TSLP expression and type 2 skin inflammation. In this study, we investigated the protective effect of DMOG on MC903 (calcipotriol)-induced type 2 skin inflammation. Morphological and immunological changes were assessed by H-E staining, flow cytometry and RT-qPCR. DMOG treatment attenuated MC903-induced skin inflammation in a T cell-independent manner. The anti-inflammatory effect of DMOG was accompanied by downregulation of TSLP and IL-33, and supplementation with recombinant TSLP and IL-33 abolished the effect of DMOG. MC903 increased ROS levels in skin tissue, which was prevented by DMOG. Furthermore, the ROS scavenger N-acetylcysteine (NAC) downregulated TSLP and ameliorated MC903-induced skin inflammation, as did DMOG. Finally, the effect of DMOG on ROS and TSLP was reduced by HIF knockdown. These results suggest that DMOG downregulates TSLP and ROS through the HIF pathway, which reduces MC903-induced skin inflammation.

Impact of vitamin C on the development, differentiation and functional properties of T cells.

Vitamin C plays a multifaceted role in various biological processes and is well-known to facilitate pleiotropic activities in both innate and adaptive immune responses, where the antioxidant capacity of vitamin C is most likely highly relevant since immune responses mainly occur in reducing environments. Beyond its antioxidant properties, vitamin C can enhance the transcription potential of genes by promoting DNA demethylation through ten-eleven-translocation (Tet) methylcytosine dioxygenases, which have been recently demonstrated to be critical for the development and differentiation of T cells. In this minireview, we will provide a broader overview on the impact of vitamin C on signaling and regulatory activities in both innate and adaptive immune cells. Particularly, we will summarize recent findings on the decisive role of finely tuned vitamin C concentrations for T cell development, T helper cell differentiation, and optimal T cell-mediated immune responses.

The molecular mechanisms of apoptosis accompanied with the epigenetic regulation of the NY-ESO-1 antigen in non-small lung cancer cells treated with decitabine (5-aza-CdR).

Dysregulated epigenetic modifications are common in lung cancer but have been reversed using demethylating agent like 5-Aza-CdR. 5-Aza-CdR induces/upregulates the NY-ESO-1 antigen in lung cancer. Therefore, we investigated the molecular mechanisms accompanied with the epigenetic regulation of NY-ESO-1 in 5-Aza-CdR-treated NCI-H1975 cell line. We showed significant induction of the NY-ESO-1 protein (**p < 0.0097) using Cellular ELISA. Bisulfite-sequencing demonstrated 45.6% demethylation efficiency at the NY-ESO-1 gene promoter region and RT-qPCR analysis confirmed the significant induction of NY-ESO-1 at mRNA level (128-fold increase, *p < 0.050). We then investigated the mechanism by which 5-Aza-CdR inhibits cell proliferation in the NCI-H1975 cell line. Upregulation of the death receptors TRAIL (2.04-fold *p < 0.011) and FAS (2.1-fold *p < 0.011) indicate activation of the extrinsic apoptotic pathway. The upregulation of Voltage-dependent anion-selective channel protein 1 (1.9-fold), Major vault protein (1.8-fold), Bax (1.16-fold), and Cytochrome C (1.39-fold) indicate the activation of the intrinsic pathway. We also observed the differential expression of protein Complement C3 (3.3-fold), Destrin (-5.1-fold), Vimentin (-1.7-fold), Peroxiredoxin 4 (-1.6-fold), Fascin (-1.8-fold), Heme oxygenase-2 (-0.67-fold**p < 0.0055), Hsp27 (-0.57-fold**p < 0.004), and Hsp70 (-0.39-fold **p < 0.001), indicating reduced cell growth, cell migration, and metastasis. The upregulation of 40S ribosomal protein S9 (3-fold), 40S ribosomal protein S15 (4.2-fold), 40S ribosomal protein S18 (2.5-fold), and 60S ribosomal protein L22 (4.4-fold) implied the induction of translation machinery. These results reiterate the decisive role of 5-Aza-CdR in lung cancer treatment since it induces the epigenetic regulation of NY-ESO-1 antigen, inhibits cell proliferation, increases apoptosis, and decreases invasiveness.

CD4+ T cells play an essential role in chronic MC903-induced skin inflammation.

MC903 skin inflammation model is one of well-characterized murine models of atopic dermatitis and driven by TSLP-mediated type 2 inflammation. Since it can be prepared simply by repetitive applications of MC903 and shows consistent clinical results, this model has been widely used. However, in contrast to human atopic dermatitis which is chronic and closely related to TH2 cells, MC903 induces inflammations temporarily and even in the absence of T cells. Here, we modified the MC903 treatment schedule and developed a chronic MC903-induced skin inflammation model. Mice were sensitized with a high dose of MC903 and challenged with a low dose of MC903. Prior to challenge, mice were allowed to recover completely from the inflammation which occurred during the sensitization. The challenge of MC903 induced skin swelling and type 2 inflammations more rapidly, which was dependent on CD4+ T cells and IL-33. We expect that our mouse model will be beneficial for studying the late course of atopic dermatitis.

Impact of gut microenvironment on epigenetic signatures of intestinal T helper cell subsets.

The intestine, which is constantly exposed to an extensive range of antigens and immune stimuli, harbours a large number of highly diverse CD4+ T helper (Th) cells. Maintaining a balance between pro-inflammatory and tolerogenic intestinal Th cell subsets is crucial for the homeostasis and functionality of the gastrointestinal tract. Recent evidence suggests that epigenetic mechanisms play a vital role for Th cell differentiation and specialization, and dysregulation of these epigenetic regulations can result in an imbalance of Th cell subsets, causing the onset and progression of intestinal inflammatory diseases. Furthermore, epigenetic alterations might be orchestrated through microbiota-derived metabolites, which can function as epigenetic modifiers by catalyzing major enzymes involved in epigenetic modifications. In this review, we focus on the epigenetic control of Th cell differentiation and functional specialization in intestinal tissues in health and disease, and also discuss the role of microbiota in shaping intestinal Th cell epigenomes.

Transcriptome of Tumor-Infiltrating T Cells in Colorectal Cancer Patients Uncovered a Unique Gene Signature in CD4+ T Cells Associated with Poor Disease-Specific Survival.

Colorectal cancer (CRC) is influenced by infiltration of immune cell populations in the tumor microenvironment. While elevated levels of cytotoxic T cells are associated with improved prognosis, limited studies have reported associations between CD4+ T cells and disease outcomes. We recently performed transcriptomic profiling and comparative analyses of sorted CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) from bulk tumors of CRC patients with varying disease stages. In this study, we compared the transcriptomes of CD4+ with CD8+ TILs. Functional annotation pathway analyses revealed the downregulation of inflammatory response-related genes, while T cell activation and angiogenesis-related genes were upregulated in CD4+ TILs. The top 200 deregulated genes in CD4+ TILs were aligned with the cancer genome atlas (TCGA) CRC dataset to identify a unique gene signature associated with poor prognosis. Moreover, 69 upregulated and 20 downregulated genes showed similar trends of up/downregulation in the TCGA dataset and were used to calculate "poor prognosis score" (ppScore), which was significantly associated with disease-specific survival. High ppScore patients showed lower expression of Treg-, Th1-, and Th17-related genes, and higher expression of Th2-related genes. Our data highlight the significance of T cells within the TME and identify a unique candidate prognostic gene signature for CD4+ TILs in CRC patients.

Metabolic reprogramming of T regulatory cells in the hypoxic tumor microenvironment.

Metabolic dysregulation in the hypoxic tumor microenvironment (TME) is considered as a hallmark of solid tumors, leading to changes in biosynthetic pathways favoring onset, survival and proliferation of malignant cells. Within the TME, hypoxic milieu favors metabolic reprogramming of tumor cells, which subsequently affects biological properties of tumor-infiltrating immune cells. T regulatory cells (Tregs), including both circulating and tissue-resident cells, are particularly susceptible to hypoxic metabolic signaling that can reprogram their biological and physicochemical properties. Furthermore, metabolic reprogramming modifies Tregs to utilize alternative substrates and undergo a plethora of metabolic events to meet their energy demands. Major impact of this metabolic reprogramming can result in differentiation, survival, excessive secretion of immunosuppressive cytokines and proliferation of Tregs within the TME, which in turn dampen anti-tumor immune responses. Studies on fine-tuning of Treg metabolism are challenging due to heterogenicity of tissue-resident Tregs and their dynamic functions. In this review, we highlight tumor intrinsic and extrinsic factors, which can influence Treg metabolism in the hypoxic TME. Moreover, we focus on metabolic reprogramming of Tregs that could unveil potential regulatory networks favoring tumorigenesis/progression, and provide novel insights, including inhibitors against acetyl-coA carboxylase 1 and transforming growth factor beta into targeting Treg metabolism for therapeutic benefits.

Role of circular RNAs in colorectal tumor microenvironment.

Circular RNAs (circRNAs) are a class of endogenous noncoding RNA, which were previously considered as a byproduct of RNA splicing error. Numerous studies have demonstrated the altered expression of circRNAs in organ tissues during pathological conditions and their involvements in disease pathogenesis and progression, including cancers. In colorectal cancer (CRC), multiple circRNAs have been identified and characterized as "oncogenic", given their involvements in the downregulation of tumor suppressor genes and induction of tumor initiation, progression, invasion, and metastasis. Additionally, other circRNAs have been identified in CRC and characterized as "tumor suppressive" based on their ability of inhibiting the expression of oncogenic genes and suppressing tumor growth and proliferation. circRNAs could serve as potential diagnostic and prognostic biomarkers, and therapeutic targets or vectors to be utilized in cancer therapies. This review briefly describes the dynamic changes of the tumor microenvironment inducing immunosuppression and tumorigenesis, and outlines the biogenesis and characteristics of circRNAs and recent findings indicating their roles and functions in the CRC tumor microenvironment. It also discusses strategies and technologies, which could be employed in the future to overcome current cancer therapy challenges associated with circRNAs.

Transcriptome of CD8+ tumor-infiltrating T cells: a link between diabetes and colorectal cancer.

There is an increased risk of colorectal cancer (CRC) development in patients with non-insulin-dependent type 2 diabetes. CD8+ T cells have been implicated in diabetes and are crucial for anti-tumor immunity. However, transcriptomic profiling for CD8+ T cells from CRC diabetic patients has not been explored. We performed RNA sequencing and compared transcriptomic profiles of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in CRC diabetic patients with CRC nondiabetic patients. We found that genes associated with ribogenesis, epigenetic regulations, oxidative phosphorylation and cell cycle arrest were upregulated in CD8+ TILs from diabetic patients, while genes associated with PI3K signaling pathway, cytokine response and response to lipids were downregulated. Among the significantly deregulated 1009 genes, 342 (186 upregulated and 156 downregulated) genes were selected based on their link to diabetes, and their associations with the presence of specific CRC pathological parameters were assessed using GDC TCGA colon database. The 186 upregulated genes were associated with the presence of colon polyps history (P = 0.0007) and lymphatic invasion (P = 0.0025). Moreover, CRC patients with high expression of the 186 genes were more likely to have poorer disease-specific survival (DSS) (Mantel-Cox log-rank P = 0.024) than those with low score. Our data provide novel insights into molecular pathways and biological functions, which could be altered in CD8+ TILs from CRC diabetic versus nondiabetic patients, and reveal candidate genes linked to diabetes, which could predict DSS and pathological parameters associated with CRC progression. However, further investigations using larger patient cohorts and functional studies are required to validate these findings.

Tumor-Infiltrating Lymphoid Cells in Colorectal Cancer Patients with Varying Disease Stages and Microsatellite Instability-High/Stable Tumors.

Immune checkpoint inhibition is an effective anti-cancer therapeutic approach but has shown limited efficacy in treating colorectal cancer (CRC) patients. Importantly, immune constituents of the tumor microenvironment (TME) can influence therapy response and cancer progression. We investigated the expression of immune checkpoints (ICs) on lymphoid populations within the CRC TME and compared with cells from normal colon tissues using samples from 50 patients with varying disease stages. We found that the levels of B cells, T cells, and NK cells were similar, IC-expressing CD4+ and CD4+CD8+ double positive T cells were higher, while CD8+ T cells and CD4-CD8- double negative T cells were significantly lower in CRC tumors. Notably, patients with mismatch-repair deficiency/microsatellite instability-high tumors had higher levels of IC-expressing CD4+ and CD8+ T cells than patients with proficient MMR and microsatellite stable tumors. Lastly, The Cancer Genome Atlas Colon Adenocarcinoma datasets showed associations between low expression of selective genes and poorer progression-free interval. Our findings highlight differential expression of ICs on lymphoid cells in CRC tumors in the era of cancer immunotherapy, which at present is solely approved for anti-PD-1 therapy in patients with dMMR/MSI-H tumors. Further investigations into their functionality have potentials for deciphering resistance mechanisms to IC inhibition.

Transcriptomic Profiling of Circulating HLA-DR- Myeloid Cells, Compared with HLA-DR+ Myeloid Antigen-presenting Cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells with potent immunosuppressive functions, which can inhibit the activation of immune responses under a steady-state condition and pathological conditions. We performed transcriptomic profiling of circulating CD33+HLA-DR+ myeloid antigen-presenting cells (APCs) and CD33+HLA-DR- myeloid cells (potentially MDSCs) in healthy individuals. We sorted both subpopulations from peripheral blood mononuclear cells (PBMCs) of 10 healthy donors and performed RNA sequencing (RNA-Seq). We found that several signaling pathways associated with the positive regulation of immune responses, such as antigen presentation/processing, FcγR-mediated phagocytosis and immune cell trafficking, phosphoinositide 3-kinase (PI3K)/Akt signaling, DC maturation, triggering receptor expressed on myeloid cells 1 (TREM1) signaling, nuclear factor of activated T cells (NFAT) and IL-8 signaling were downregulated in CD33+HLA-DR- myeloid cells. In contrast, pathways implicated in tumor suppression and anti-inflammation, including peroxisome proliferator-activated receptor (PPAR) and phosphatase and tensin homolog (PTEN), were upregulated in CD33+HLA-DR- myeloid cells. These data indicate that PPAR/PTEN axis could be upregulated in myeloid cells to keep the immune system in check in normal physiological conditions. Our data reveal some of the molecular and functional differences between CD33+HLA-DR+ APCs and CD33+HLA-DR- myeloid cells in a steady-state condition, reflecting the potential suppressive function of CD33+HLA-DR- myeloid cells to maintain immune tolerance. For future studies, the same methodological approach could be applied to perform transcriptomic profiling of myeloid subsets in pathological conditions.

T-cell responses and therapies against SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, a novel coronavirus strain. Some studies suggest that COVID-19 could be an immune-related disease, and failure of effective immune responses in initial stages of viral infection could contribute to systemic inflammation and tissue damage, leading to worse disease outcomes. T cells can act as a double-edge sword with both pro- and anti-roles in the progression of COVID-19. Thus, better understanding of their roles in immune responses to SARS-CoV-2 infection is crucial. T cells primarily react to the spike protein on the coronavirus to initiate antiviral immunity; however, T-cell responses can be suboptimal, impaired or excessive in severe COVID-19 patients. This review focuses on the multifaceted roles of T cells in COVID-19 pathogenesis and rationalizes their significance in eliciting appropriate antiviral immune responses in COVID-19 patients and unexposed individuals. In addition, we summarize the potential therapeutic approaches related to T cells to treat COVID-19 patients. These include adoptive T-cell therapies, vaccines activating T-cell responses, recombinant cytokines, Th1 activators and Th17 blockers, and potential utilization of immune checkpoint inhibitors alone or in combination with anti-inflammatory drugs to improve antiviral T-cell responses against SARS-CoV-2.

Differential gene expression of tumor-infiltrating CD33+ myeloid cells in advanced- versus early-stage colorectal cancer.

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.

RNA-Seq Analysis of Colorectal Tumor-Infiltrating Myeloid-Derived Suppressor Cell Subsets Revealed Gene Signatures of Poor Prognosis.

Elevated levels of myeloid-derived suppressor cells (MDSCs), including polymorphonuclear MDSCs (PMN-MDSCs) and immature MDSCs (I-MDSCs), are usually associated with disease progression in cancer patients, including colorectal cancer (CRC). However, biological mechanisms and molecular pathways regulated by MDSC subpopulations in the CRC tumor microenvironment (TME) have not been fully investigated. In this study, we performed transcriptomic analysis of tumor-infiltrating I-MDSCs and PMN-MDSCs isolated from tumor tissues of six CRC patients, compared to antigen-presenting cells (APCs). We also compared the transcriptomic profiles of tumor-infiltrating PMN-MDSCs to I-MDSCs. Our results showed different molecular pathways regulated by each MDSC subset, potentially reflecting their phenotypical/molecular/functional characteristics in the CRC TME. Moreover, we identified gene signatures in PMN-MDSC and I-MDSC of poor overall survival (OS) and disease-free survival (DFS) using the Cancer Genome Atlas (TCGA) dataset from patients with colon adenocarcinoma (COAD). However, functional studies are required to validate these findings.

Epigenetic regulation of immune checkpoints and T cell exhaustion markers in tumor-infiltrating T cells of colorectal cancer patients.

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.

Differential gene expression of tumor-infiltrating CD4+ T cells in advanced versus early stage colorectal cancer and identification of a gene signature of poor prognosis.

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.

An evaluation of sorter induced cell stress (SICS) on peripheral blood mononuclear cells (PBMCs) after different sort conditions - Are your sorted cells getting SICS?

Flow cytometry and fluorescence-activated cell sorting have become invaluable tools to analyze and isolate specific cell populations in a wide range of biomedical research and clinical applications. In countless approaches worldwide, scientists are using single cell analyses to better understand the significance and variation within different cellular populations, and fluorescence-activated cell sorting has become a major technique for cell isolation in both basic and clinical research. However, majority of available cell sorters are pressurized, droplet-based systems, which apply significant environmental pressure and shear stress to cells during sorting. Recently, the flow cytometry community has become increasingly aware about the potential negative effects this could have on sorted cells and the term "sorter induced cell stress" (SICS) has been proposed. However, up to date only a limited number of studies have investigated the effects of cell sorting on cell viability and function. Therefore, solid data on the effects of sheath pressure and nozzle size on survival and function of sorted cells are surprisingly rare. With this in mind, we sorted "CD4+" T-cells and "live" cells from human peripheral blood mononuclear cells (PBMCs) at different sort conditions and analyzed their quality before and after sorting in a series of assays. Here we present our findings in reference to cell viability and cell proliferation following sorting on different instruments (BD FACSAria III SORP and BD FACSJazz), utilizing different nozzle sizes (70 to 100 µm) and sheath pressure settings (20 to 70 psi). The results show no significant differences in cell viability and proliferation after the different tested sort conditions, but rather differences between individual experiments. These findings are evaluated and their potential significance in cell sorting experiments is discussed.

Transcriptomic Analyses of Myeloid-Derived Suppressor Cell Subsets in the Circulation of Colorectal Cancer Patients.

Myeloid-derived suppressor cells (MDSCs) promote tumor immune evasion and favor tumorigenesis by activating various tumor-promoting downstream signals. MDSC expansion is evident in the circulation and tumor microenvironment of many solid tumors including colorectal cancer (CRC). We have recently reported the transcriptomic profiles of tumor-infiltrating MDSCs in CRC patients and uncovered pathways, which could potentially assist tumor progression. In this study, we sorted different subsets of circulating MDSCs in CRC patients and investigated their transcriptomic profiles in order to disclose pathways, which could potentially contribute to disease progression. The sorted subsets included polymorphonuclear/granulocytic MDSCs (PMN-MDSCs), immature MDSCs (I-MDSCs), and monocytic MDSCs (M-MDSCs). Our functional annotation analyses revealed that multiple pathways including DNA damage-, chemotaxis-, apoptosis-, mitogen-activated protein kinase-, transforming growth factor ß-, and myeloid differentiation-related transcripts were higher in PMN-MDSCs, compared with monocytic antigen-presenting cells (APCs) or I-MDSCs. Furthermore, genes related to Janus kinase (JAK)-signal transducer and activator of transcription (STAT) were also elevated in PMN-MDSCs. These data suggest that upregulation of JAK-STAT pathway could trigger multiple downstream targets in PMN-MDSCs, which favor tumor progression. Additionally, we found that pathways including phosphatidyl inositol 3-kinase (PI3K), interleukin 6, and TGF-ß in M-MDSCs and cell cycle-related pathways in I-MDSCs were upregulated, compared with monocytic APCs. Moreover, acetylation-related genes were upregulated in both PMN-MDSCs and M-MDSCs. This latter finding implicates that epigenetic modifications could also play a role in the regulation of multiple tumor-promoting genes in PMN-MDSCs and M-MDSCs. Taken together, this study reveals various signaling pathways, which regulate the function of MDSC subsets in the circulation of CRC patients. However, functional studies are warranted to support these findings.