M6A regulator-based methylation modification patterns characterized by distinct tumor microenvironment immune profiles in colon cancer.
ABSTRACT: Recent studies have highlighted the biological significance of RNA N6-methyladenosine (m6A) modification in tumorigenicity and progression. However, it remains unclear whether m6A modifications also have potential roles in immune regulation and tumor microenvironment (TME) formation. Methods: In this study, we curated 23 m6A regulators and performed consensus molecular subtyping with NMF algorithm to determine m6A modification patterns and the m6A-related gene signature in colon cancer (CC). The ssGSEA and CIBERSORT algorithms were employed to quantify the relative infiltration levels of various immune cell subsets. An PCA algorithm based m6Sig scoring scheme was used to evaluate the m6A modification patterns of individual tumors with an immune response. Results: Three distinct m6A modification patterns were identified among 1307 CC samples, which were also associated with different clinical outcomes and biological pathways. The TME characterization revealed that the identified m6A patterns were highly consistent with three known immune profiles: immune-inflamed, immune-excluded, and immune-desert, respectively. Based on the m6Sig score, which was extracted from the m6A-related signature genes, CC patients can be divided into high and low score subgroups. Patients with lower m6Sig score was characterized by prolonged survival time and enhanced immune infiltration. Further analysis indicated that lower m6Sig score also correlated with greater tumor mutation loads, PD-L1 expression, and higher mutation rates in SMGs (e.g., PIK3CA and SMAD4). In addition, patients with lower m6Sig scores showed a better immune responses and durable clinical benefits in three independent immunotherapy cohorts. Conclusions: This study highlights that m6A modification is significantly associated with TME diversity and complexity. Quantitatively evaluating the m6A modification patterns of individual tumors will strengthen our understanding of TME characteristics and promote more effective immunotherapy strategies.
Project description:Sustaining efficacious T cell-mediated antitumor immune responses in the tumor tissues is the key to the success of cancer immunotherapy. Current strategies leverage altering the signals T cells sense in the tumor microenvironment (TME). Checkpoint inhibitor-based approaches block inhibitory signals such as PD-1 whereas cytokine-based therapies increase the level of immune-stimulatory cytokines such as IL-2. Besides extrinsic signals, the genetic circuit within T cells also participates in determining the nature and trajectory of antitumor immune responses. Here, we showed that efficacy of the IL33-based tumor immunotherapy was greatly enhanced in mice with T cell-specific Eomes deficiency. Mechanistically, we demonstrated that Eomes deficient mice had diminished proportions of exhausted/dysfunctional CD8<sup>+</sup> T cells but increased percentages of tissue resident and stem-like CD8<sup>+</sup> T cells in the TME. In addition, the IFN?<sup>+</sup>TCF1<sup>+</sup> CD8<sup>+</sup> T cell subset was markedly increased in the Eomes deficient mice. We further demonstrated that Eomes bound directly to the transcription regulatory regions of exhaustion and tissue residency genes. In contrast to its role in inhibiting T cell immune responses at the tumor site, Eomes promoted generation of central memory T cells in the peripheral lymphoid system and memory recall responses against tumor growth at a distal tissue site. Finally, we showed that Eomes deficiency in T cells also resulted in increased efficacy of PD-1-blockade tumor immunotherapy. In all, our study indicates that Eomes plays a critical role in restricting prolonged T cell-mediated antitumor immune responses in the TME whereas promoting adaptive immunity in peripheral lymphoid organs.
Project description:BACKGROUND:The epigenetic regulation of immune response has been demonstrated in recent studies. Nonetheless, potential roles of RNA N6-methyladenosine (m6A) modification in tumor microenvironment (TME) cell infiltration remain unknown. METHODS:We comprehensively evaluated the m6A modification patterns of 1938 gastric cancer samples based on 21 m6A regulators, and systematically correlated these modification patterns with TME cell-infiltrating characteristics. The m6Ascore was constructed to quantify m6A modification patterns of individual tumors using principal component analysis algorithms. RESULTS:Three distinct m6A modification patterns were determined. The TME cell-infiltrating characteristics under these three patterns were highly consistent with the three immune phenotypes of tumors including immune-excluded, immune-inflamed and immune-desert phenotypes. We demonstrated the evaluation of m6A modification patterns within individual tumors could predict stages of tumor inflammation, subtypes, TME stromal activity, genetic variation, and patient prognosis. Low m6Ascore, characterized by increased mutation burden and activation of immunity, indicated an inflamed TME phenotype, with 69.4% 5-year survival. Activation of stroma and lack of effective immune infiltration were observed in the high m6Ascore subtype, indicating a non-inflamed and immune-exclusion TME phenotype, with poorer survival. Low m6Ascore was also linked to increased neoantigen load and enhanced response to anti-PD-1/L1 immunotherapy. Two immunotherapy cohorts confirmed patients with lower m6Ascore demonstrated significant therapeutic advantages and clinical benefits. CONCLUSIONS:This work revealed the m6A modification played a nonnegligible role in formation of TME diversity and complexity. Evaluating the m6A modification pattern of individual tumor will contribute to enhancing our cognition of TME infiltration characterization and guiding more effective immunotherapy strategies.
Project description:Sigma-1 receptors (Sig-1Rs) are integral ER membrane proteins. They bind diverse ligands, including psychoactive drugs, and regulate many signaling proteins, including the inositol 1,4,5-trisphosphate receptors (IP<sub>3</sub>Rs) that release Ca<sup>2+</sup> from the ER. The endogenous ligands of Sig-1Rs are unknown. Phospholipase D (PLD) cleaves phosphatidylcholine to choline and phosphatidic acid (PA), with PA assumed to mediate all downstream signaling. We show that choline is also an intracellular messenger. Choline binds to Sig-1Rs, it mimics other Sig-1R agonists by potentiating Ca<sup>2+</sup> signals evoked by IP<sub>3</sub>Rs, and it is deactivated by metabolism. Receptors, by stimulating PLC and PLD, deliver two signals to IP<sub>3</sub>Rs: IP<sub>3</sub> activates IP<sub>3</sub>Rs, and choline potentiates their activity through Sig-1Rs. Choline is also produced at synapses by degradation of acetylcholine. Choline uptake by transporters activates Sig-1Rs and potentiates Ca<sup>2+</sup> signals. We conclude that choline is an endogenous agonist of Sig-1Rs linking extracellular stimuli, and perhaps synaptic activity, to Ca<sup>2+</sup> signals.
Project description:The tumor microenvironment (TME) constitutes a complex milieu of cells and cytokines that maintain equilibrium between tumor progression and prognosis. However, comprehensive analysis of the TME and its clinical significance in head and neck squamous cell carcinoma (HNSCC) remains to be unreported. In this study, based on large-scale RNA sequencing data pertaining to single nucleotide variants (SNVs) and copy number variations (CNVs) in HNSCC patients from The Cancer Genome Atlas database, we analysed subpopulations of infiltrating immune cells and evaluated the role of TME infiltration pattern (TME score) in assessing immunotherapy outcome. TME signature genes involved in several inflammation and immunity signalling pathways were observed in the TME score subtype, which were considered immunosuppressive and potentially responsible for significantly worse prognosis. In comparison with SNV- and CNV-mediated tumor mutation burden, TME score can significantly differentiate between high- and low-risk HNSCC and predict immunotherapy outcome. Our data provide clarity on the comprehensive landscape of interactions between clinical characteristics of HNSCC and tumor-infiltrating immune cells. TME score seems to be a useful biomarker that can predict immunotherapy outcome in HNSCC patients.
Project description:The tumor microenvironment (TME) chiefly consists of tumor cells and tumor-infiltrating immune cells admixed with the stromal component. A recent clinical trial has shown that the tumor immune cell infiltration (ICI) is correlated with the sensitivity to immunotherapy and the head and neck squamous cell carcinoma (HNSC) prognosis. However, to date, the immune infiltrative landscape of HNSC has not yet been elucidated. Herein, we proposed two computational algorithms to unravel the ICI landscape of 1,029 HNSC patients. Three ICI patterns were defined, and the ICI scores were determined by using principal-component analysis. A high ICI score was characterized by an increased tumor mutation burden (TMB) and the immune-activating signaling pathways. Activation of transforming growth factor-? (TGF-?) and WNT signaling pathways were observed in low ICI score subtypes, indicating T cell suppression, and may be responsible for poor prognosis. Two immunotherapy cohorts confirmed patients with higher ICI scores demonstrated significant therapeutic advantages and clinical benefits. This study demonstrated that the ICI scores serve as an effective prognostic biomarker and predictive indicator for immunotherapy. Evaluating the ICI patterns of a larger cohort of samples will extend our understanding of TME, and it may provide directions to the current research investigations on immunotherapeutic strategies for HNSC. Graphical Abstract Understanding the tumor-immune microenvironment is critical for improving the efficacy of current immunotherapies. Zhang et al. comprehensively evaluated the cellular, molecular, and genetic factors associated with immune cell infiltration patterns. These have important implications for how tumors respond to immunotherapies and may guide more effective immunotherapy strategies.
Project description:Long noncoding RNA (lncRNA) is emerging as an essential regulator in the development and progression of cancer, including cervical cancer (CC). In this study, we found a CC-related lncRNA, KCNMB2-AS1, which was significantly overexpressed in CC and linked to poor outcomes. Depletion of KCNMB2-AS1 remarkably inhibited CC cell proliferation and induced apoptosis. <i>In vivo</i> xenograft models revealed that knockdown of KCNMB2-AS1 evidently delayed tumor growth. Mechanistically, KCNMB2-AS1 was predominantly located in the cytoplasm and served as a competing endogenous RNA to abundantly sponge miR-130b-5p and miR-4294, resulting in the upregulation of IGF2BP3, a well-documented oncogene in CC. Moreover, IGF2BP3 was able to bind KCNMB2-AS1 by three N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) modification sites on KCNMB2-AS1, in which IGF2BP3 acted as an m<sup>6</sup>A "reader" and stabilized KCNMB2-AS1. Thus, KCNMB2-AS1 and IGF2BP3 formed a positive regulatory circuit that enlarged the tumorigenic effect of KCNMB2-AS1 in CC. Together, our data clearly suggest that KCNMB2-AS1 is a novel oncogenic m<sup>6</sup>A-modified lncRNA in CC, targeting KCNMB2-AS1 and its related molecules implicate the therapeutic possibility for CC patients.
Project description:The gut microbiota produces metabolites such as short-chain fatty acids (SCFAs) that regulate the energy homeostasis and impact on immune cell function of the host. Recently, innovative approaches based on the oral administration of SCFAs have been discussed for therapeutic modification of inflammatory immune responses in autoimmune diseases. So far, most studies have investigated the SCFA-mediated effects on CD4<sup>+</sup> T cells and antigen presenting cells. Here we show that butyrate and, to a lesser degree, propionate directly modulate the gene expression of CD8<sup>+</sup> cytotoxic T lymphocytes (CTLs) and Tc17 cells. Increased IFN-? and granzyme B expression by CTLs as well as the molecular switch of Tc17 cells towards the CTL phenotype was mediated by butyrate independently of its interaction with specific SCFA-receptors GPR41 and GPR43. Our results indicate that butyrate strongly inhibited histone-deacetylases (HDACs) in CD8<sup>+</sup> T cells thereby affecting the gene expression of effector molecules. Accordingly, the pan-HDAC inhibitors trichostatin A (TSA) and sodium valproate exerted similar influence on CD8<sup>+</sup> T cells. Furthermore, higher acetate concentrations were also able to increase IFN-? production in CD8<sup>+</sup> T lymphocytes by modulating cellular metabolism and mTOR activity. These findings might have significant implications in adoptive immunotherapy of cancers and in anti-viral immunity.
Project description:Chemotaxis is an important aspect of immune cell behavior within the tumor microenvironment (TME). One prominent example of chemotaxis within the TME is the migration of regulatory T cells (Tregs) in response to the chemokine ligands CCL17 and CCL22. Tregs within the TME cause the suppression of anti-tumor immunity and inhibition of the effect of immunotherapeutic treatments. Therefore, the ability to screen for therapeutic antibodies that can inhibit or stimulate the chemotaxis of various immune cell types is crucial. Traditionally, chemotaxis is studied by determining the number of cells in the bottom reservoir of a Transwell microplate using flow cytometry; however, this method is time-consuming and thus not appropriate for high-throughput screening purposes. The Celigo Image Cytometer has been employed to perform high-throughput cell-based assays and was used to develop a new detection method for chemotaxis measurement. The image-based detection method was developed using chemokine ligands CCL17 and CCL22 to induce the migration of CCR4<sup>+</sup> T cells and directly count them on the bottom of the Transwell plates. Finally, the method was applied to measure the inhibitory effects of commercially available anti-CCL17 and anti-CCL22 antibodies, which caused a dose-dependent decrease in the number of migrated T cells. The proposed image cytometry method allowed screening of multiple antibodies at various concentrations, simultaneously, which can improve the efficiency for discovering potential antibody candidates that can induce or inhibit recruitment of immune cells to the tumor microenvironment.
Project description:Adoptive transfer of regulatory T cells (FOXP3<sup>+</sup> Tregs) has been developed as a potential curative immune therapy to prevent and treat autoimmune and graft-versus-host diseases (GVHD). A major limitation that has hindered the use of Treg immunotherapy in humans is the difficulty of consistently isolating and obtaining highly purified Tregs after <i>ex vivo</i> expansion. <b>Methods</b>: We isolated <i>bona fide</i> Tregs from expansion cultures based on their selective surface expression of latency-associated peptide (LAP). The TCR V? diversity and intracellular cytokine production of Tregs were determined by flow cytometer. The TSDR methylation was determined by epigenetic human FOXP3 qPCR Assay. Their <i>in vitro</i> and <i>in vivo</i> potency was confirmed with suppression assay and humanized xenogeneic GVHD (xGVHD) murine model, respectively. <b>Results</b>: LAP<sup>+</sup> repurification results in >90% LAP<sup>+</sup>FOXP3<sup>+</sup> Tregs, leaving behind FOXP3<sup>-</sup> and FOXP3<sup>+</sup> nonTregs within the LAP<sup>-</sup> population. After 4-week expansion, the LAP<sup>+</sup> Tregs were >1 billion cells, highly suppressive and anergic <i>in vitro</i>, >90% demethylated in the TSDR and able to maintain TCR V? diversity. In the xGVHD model, exogenous CD25<sup>-</sup>PBMC administered alone results in a median survival of 32 days. The co-transfer of LAP<sup>+</sup> Tregs increased median survival to 47 days, while the LAP parent (CD25<sup>+</sup>) and LAP<sup>-</sup> nonTregs had median survival of 39 and 31 days, respectively. <b>Conclusions</b>: These preclinical data together provide evidence that LAP<sup>+</sup> Tregs are highly purified with fully suppressive function for cell therapy. This population results in a more effective and safer product for immunotherapy to treat GVHD and provides the necessary preclinical data for transition into a clinical trial with LAP<sup>+</sup> Tregs to prevent or treat GVHD and other autoimmune diseases.
Project description:The drivers and the specification of CD4<sup>+</sup> T cell differentiation in the tumor microenvironment and their contributions to tumor immunity or tolerance are incompletely understood. Using models of pancreatic ductal adenocarcinoma (PDA), we show that a distinct subset of tumor-infiltrating dendritic cells (DC) promotes PDA growth by directing a unique T<sub>H</sub>-program. Specifically, CD11b<sup>+</sup>CD103<sup>-</sup> DC predominate in PDA, express high IL-23 and TGF-?, and induce FoxP3<sup>neg</sup> tumor-promoting IL-10<sup>+</sup>IL-17<sup>+</sup>IFN?<sup>+ </sup>regulatory CD4<sup>+</sup> T cells. The balance between this distinctive T<sub>H</sub> program and canonical FoxP3<sup>+ </sup>T<sub>REGS</sub> is unaffected by pattern recognition receptor ligation and is modulated by DC expression of retinoic acid. This T<sub>H</sub>-signature is mimicked in human PDA where it is associated with immune-tolerance and diminished patient survival. Our data suggest that CD11b<sup>+</sup>CD103<sup>-</sup> DC promote CD4<sup>+</sup> T cell tolerance in PDA which may underscore its resistance to immunotherapy.