TIM-3 Expression on Dendritic Cells in Colorectal Cancer.

TIM-3 was originally identified as a negative regulator of helper T cells and is expressed on dendritic cells (DCs). Since the inhibition of TIM-3 on DCs has been suggested to enhance T cell-mediated anti-tumor immunity, we examined its expression on DCs within the tumor microenvironment (TME) in colorectal cancer (CRC) using transcriptomic data from a public database (n = 592) and immunohistochemical evaluations from our cohorts of CRC (n = 115). The expression of TIM-3 on DCs in vitro was examined by flow cytometry, while the expression of its related molecules, cGAS and STING, on immature and mature DCs was assessed by Western blotting. The expression of HAVCR2 (TIM-3) was strongly associated with the infiltration of DCs within the TME of CRC. Immunohistochemical staining of clinical tissue samples revealed that tumor-infiltrating DCs expressed TIM-3; however, their number at the tumor-invasive front significantly decreased with stage progression. TIM-3 expression was higher on immature DCs than on mature DCs from several different donors (n = 6). Western blot analyses showed that the expression of STING was higher on mature DCs than on immature DCs, which was opposite to that of TIM-3. We demonstrated that TIM-3 was highly expressed on tumor-infiltrating DCs of CRC and that its expression was higher on immature DCs than on mature DCs.

TGFß-Responsive Stromal Activation Occurs Early in Serrated Colorectal Carcinogenesis.

Activated TGFß signaling in the tumor microenvironment, which occurs independently of epithelial cancer cells, has emerged as a key driver of tumor progression in late-stage colorectal cancer (CRC). This study aimed to elucidate the contribution of TGFß-activated stroma to serrated carcinogenesis, representing approximately 25% of CRCs and often characterized by oncogenic BRAF mutations. We used a transcriptional signature developed based on TGFß-responsive, stroma-specific genes to infer TGFß-dependent stromal activation and conducted in silico analyses in 3 single-cell RNA-seq datasets from a total of 39 CRC samples and 12 bulk transcriptomic datasets consisting of 2014 CRC and 416 precursor samples, of which 33 were serrated lesions. Single-cell analyses validated that the signature was expressed specifically by stromal cells, effectively excluding transcriptional signals derived from epithelial cells. We found that the signature was upregulated during malignant transformation and cancer progression, and it was particularly enriched in CRCs with mutant BRAF compared to wild-type counterparts. Furthermore, across four independent precursor datasets, serrated lesions exhibited significantly higher levels of TGFß-responsive stromal activation compared to conventional adenomas. This large-scale analysis suggests that TGFß-dependent stromal activation occurs early in serrated carcinogenesis. Our study provides novel insights into the molecular mechanisms underlying CRC development via the serrated pathway.

Biomarker-oriented chemo-immunotherapy for advanced gastric cancer.

The biomarker-oriented chemo-immunotherapy is useful and promising in the development of new anticancer agents, since the responders can be enriched by selecting patients with biomarkers. Compared to colorectal and lung cancers, the development of biomarker-driven molecular-targeted therapeutics for gastric cancers has been straggled. However, several new biomarkers in gastric cancers have been discovered and clinical trials in enrichment design with certain biomarkers have been conducted. Therefore, there are currently several treatment options to treat gastric cancer patients based on individual biomarker-oriented strategies. In the present review, we describe the useful biomarkers in gastric cancer, with focusing on HER2, PD-L1, and Claudin18.2, in relation to their clinical significance and associated targeted agents.

The tumor cell-intrinsic cGAS-STING pathway is associated with the high density of CD8+ T cells after chemotherapy in esophageal squamous cell carcinoma.

BACKGROUND: Chemotherapy has the potential to induce CD8+ T-cell infiltration in the tumor microenvironment (TME) and activate the anti-tumor immune response in several cancers including esophageal squamous cell carcinoma (ESCC). The tumor cell-intrinsic cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has been known as a critical component for regulating immune cell activation in the TME. However, its effect on the infiltration of immune cells induced by chemotherapy in the ESCC TME has not been investigated. METHODS: We examined the effect of the tumor-cell intrinsic cGAS-STING pathway on the infiltration of CD8+ T cells induced by chemotherapy in ESCC using ESCC cell lines and surgically resected ESCC specimens from patients who received neoadjuvant chemotherapy (NAC). RESULTS: We found that chemotherapeutic agents, including 5-fluorouracil (5-FU) and cisplatin (CDDP), activated the cGAS-STING pathway, consequently inducing the expression of type I interferon and T-cell-attracting chemokines in ESCC cells. Moreover, the tumor cell-intrinsic expression of cGAS-STING was significantly and positively associated with the density of CD8+ T cells in ESCC after NAC. However, the tumor cell-intrinsic expression of cGAS-STING did not significantly impact clinical outcomes in patients with ESCC after NAC. CONCLUSION: Our findings suggest that the tumor cell-intrinsic cGAS-STING pathway might contribute to chemotherapy-induced immune cell activation in the ESCC TME.

Combination of oligo-fractionated irradiation with nivolumab can induce immune modulation in gastric cancer.

BACKGROUND: Tumor-associated antigen (TAA)-specific CD8(+) T cells are essential for nivolumab therapy, and irradiation has been reported to have the potential to generate and activate TAA-specific CD8(+) T cells. However, mechanistic insights of T-cell response during combinatorial immunotherapy using radiotherapy and nivolumab are still largely unknown. METHODS: Twenty patients included in this study were registered in the CIRCUIT trial (ClinicalTrials.gov, NCT03453164). All patients had multiple distant metastases and were intolerance or had progressed after primary and secondary chemotherapy without any immune checkpoint inhibitor. In the CIRCUIT trial, eligible patients were treated with a total of 22.5 Gy/5 fractions/5 days of radiotherapy to the largest or symptomatic lesion prior to receiving nivolumab every 2 weeks. In these 20 patients, T-cell responses during the combinatorial immunotherapy were monitored longitudinally by high-dimensional flow cytometry-based, multiplexed major histocompatibility complex multimer analysis using a total of 46 TAAs and 10 virus epitopes, repertoire analysis of T-cell receptor ß-chain (TCRß), together with circulating tumor DNA analysis to evaluate tumor mutational burden (TMB). RESULTS: Although most TAA-specific CD8(+) T cells could be tracked longitudinally, several TAA-specific CD8(+) T cells were detected de novo after irradiation, but viral-specific CD8(+) T cells did not show obvious changes during treatment, indicating potential irradiation-driven antigen spreading. Irradiation was associated with phenotypical changes of TAA-specific CD8(+) T cells towards higher expression of killer cell lectin-like receptor subfamily G, member 1, human leukocyte antigen D-related antigen, T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain, CD160, and CD45RO together with lower expression of CD27 and CD127. Of importance, TAA-specific CD8(+) T cells in non-progressors frequently showed a phenotype of CD45RO(+)CD27(+)CD127(+) central memory T cells compared with those in progressors. TCRß clonality (inverted Pielou's evenness) increased and TCRß diversity (Pielou's evenness and Diversity Evenness score) decreased during treatment in progressors (p=0.029, p=0.029, p=0.012, respectively). TMB score was significantly lower in non-progressors after irradiation (p=0.023). CONCLUSION: Oligo-fractionated irradiation induces an immune-modulating effect with potential antigen spreading and the combination of radiotherapy and nivolumab may be effective in a subset of patients with gastric cancer.

[Remodeling of the Tumor Microenvironment by Radiotherapy through the cGAS-STING Pathway in Esophageal Squamous Cell Carcinoma].

It has been reported that tumor cell-intrinsic cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING) pathway is essential for radiotherapy(RT)-induced activation of anti-tumor immune responses. However, its role in the RT- induced remodeling of the tumor microenvironment(TME)in esophageal squamous cell carcinoma(ESCC), is largely unknown. In this study, we found that the tumor cell-intrinsic cGAS-STING pathway is a critical component for RT-induced activation of immune cells in the TME through the induction of type Ⅰ interferon and C-X-C motif chemokine ligand 10 in tumor cells in ESCC. However, at the same time, the tumor cell-intrinsic cGAS-STING pathway is also involved in RT-triggered infiltration and polarization of immunosuppressive CD163+ tumor-associated macrophages (TAM) through the induction of interleukin 34 (IL-34) in tumor cells in ESCC. Our findings suggest that targeting IL-34 to impede the infiltration and polarization of CD163+ TAM could potentially enhance the efficacy of RT-induced immune cell activation in ESCC TME.

TIM-3 Expression and M2 Polarization of Macrophages in the TGFß-Activated Tumor Microenvironment in Colorectal Cancer.

TGFß signaling in the tumor microenvironment (TME) drives immune evasion and is a negative predictor of immune checkpoint inhibitor (ICI) efficacy in colorectal cancer (CRC). TIM-3, an inhibitory receptor implicated in anti-tumor immune responses and ICI resistance, has emerged as an immunotherapeutic target. This study investigated TIM-3, M2 macrophages and the TGFß-activated TME, in association with microsatellite instability (MSI) status and consensus molecular subtypes (CMSs). Transcriptomic cohorts of CRC tissues, organoids and xenografts were examined (n = 2240). TIM-3 and a TGFß-inducible stromal protein, VCAN, were evaluated in CRC specimens using immunohistochemistry (n = 45). TIM-3 expression on monocytes and generated M2 macrophages was examined by flow cytometry. We found that the expression of HAVCR2 (TIM-3) significantly correlated with the transcriptional signatures of TGFß, TGFß-dependent stromal activation and M2 macrophage, each of which were co-upregulated in CMS4, CMS1 and MSI CRCs across all datasets. Tumor-infiltrating TIM-3+ immune cells accumulated in TGFß-responsive cancer stroma. TIM-3 was increased on M2-polarized macrophages, and on monocytes in response to TGFß treatment. In conclusion, we identified a close association between TIM-3 and M2-like polarization of macrophages in the TGFß-rich TME. Our findings provide new insights into personalized immunotherapeutic strategies based on the TME for CRCs.

[The Potential of a Novel Therapeutic Strategy for Colorectal Cancer Targeting the cGAS-STING Pathway].

The cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)pathway is one of the important intracellular signaling pathways responsible for the recognition of exogenous DNA and subsequent induction of type Ⅰ interferon responses. Interestingly, in recent years, the importance of the cGAS-STING pathway in promoting anti-tumor immune responses has been highlighted. Decreased expression of cGAS-STING in tumor cells was reported in various cancers, including colorectal cancer(CRC), and it has been found to be involved in inhibiting the anti-tumor immune responses. In our recent investigation, we specifically examined the impact of tumor cell-intrinsic cGAS-STING pathway on the activation of immune cells within the CRC tumor microenvironment, focusing on mismatch repair deficient/microsatellite instability-high (dMMR/MSI-H)and mismatch repair proficient/microsatellite stable(pMMR/MSS)CRCs. We revealed that cGAS-STING expression in tumor cells was decreased in pMMR/MSS CRC compared to dMMR/MSI-H CRC, which correlated with the decreased infiltration of cytotoxic T cells. Here, we discuss the possibility of a novel therapeutic strategy for CRC targeting the tumor cell-intrinsic cGAS-STING pathway based on the findings from recent studies.

Down-regulation of stimulator of interferon genes (STING) expression and CD8+ T-cell infiltration depending on HER2 heterogeneity in HER2-positive gastric cancer.

BACKGROUND: HER2 signaling might be involved in the regulation of immune cell activation in the tumor microenvironment (TME) of gastric cancer (GC). However, the relationship between HER2 status and immune cell condition in the HER2-positive GC TME is not clearly understood. METHODS: To investigate the effect of HER2 signaling on the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which contributes to immune cell activation in the GC TME, we evaluated the associations among the expressions of HER2, cGAS-STING, and the number of CD8+ tumor-infiltrating lymphocytes (TIL) by considering HER2 heterogeneity in HER2-positive GC tissues. We also examined the effect of HER2 signaling on the activation of STING signaling in vitro using human HER2-positive GC cell lines. RESULTS: The expression of HER2 is highly heterogeneous in HER2-positive GC tissues, and we found that the number of CD8+ TIL in HER2 high areas was significantly lower than that in HER2 low areas in HER2-positive GC tissues. Intriguingly, the tumor cell-intrinsic expression of STING, but not cGAS, was also significantly lower in the HER2 high areas than the HER2 low areas in HER2-positive GC tissues. Moreover, in vitro experiments, we demonstrated that the blockade of HER2 signaling increased the expression of STING and its target genes, including IFNB1, CXCL9/10/11, and CCL5, in HER2-positive GC cell lines. CONCLUSIONS: Our results suggest that HER2 signaling might suppress immune cell activation in the GC TME by inhibiting STING signaling in tumor cells in HER2-positive GC.

Phase I/II clinical trial of nivolumab in combination with oligo-fractionated irradiation for unresectable advanced or recurrent gastric cancer.

BACKGROUND: Although immune checkpoint inhibitors (ICI) targeting for PD-1 axis is a promising approach for advanced gastric cancer (GC) patients, the response rate is still limited. Induction of synergistic effect of irradiation with ICI targeting for the PD-1 axis can be an attractive strategy. The aim of this study was to assess the effect of the combination of irradiation with anti-PD-1 therapy for advanced GC. METHODS: We conducted a single-arm, phase I/II trial in GC patients treated with a combination of nivolumab and oligo-fractionated irradiation (22.5 Gy/5 fractions/5 days) (NCT03453164). Eligible patients (n = 40) had unresectable advanced or recurrent GC which progressed after primary and secondary chemotherapy with more than one lesion. The primary endpoint is the disease control rate (DCR) of non-irradiated target lesions and the secondary endpoints are the median survival time (MST), safety, and DCR of irradiated lesions. RESULTS: We observe that the DCR for the non-irradiated target as the abscopal effect is 22.5% (90% confidence interval (CI), 12.3-36.0), and the DCR for the irradiated lesion is 40.0% (90% CI, 26.9-54.2). The median survival time is 230 days (95% CI, 157-330), and grade 3 and higher adverse events (AEs) are observed in 16 patients (39 %) with no obvious additional AEs when adding irradiation. CONCLUSIONS: The present study suggests that the combination of nivolumab with oligo-fractionated irradiation has the potential to induce a promising anti-tumor effect for advanced GC.

Immunotherapy is a type of treatment that triggers the immune system to kill cancers. Combining immunotherapy with radiotherapy may enhance its effects. We evaluated this in a clinical trial in which we treated patients with advanced or recurrent cancers of the stomach (gastric cancer) with a combination of immunotherapy and radiotherapy. The combination was able to control disease in a subset of patients and was safe, with no obvious additional adverse effects when adding radiotherapy. The median survival time­at which point half of the patients treated are still alive­was 230 days. While these results are promising, larger, more rigorous studies are needed to determine whether this combination therapy is better than alternative approaches to treating advanced or recurrent gastric cancers.

A Potential Biomarker of Dynamic Change in Peripheral CD45RA-CD27+CD127+ Central Memory T Cells for Anti-PD-1 Therapy in Patients with Esophageal Squamous Cell Carcinoma.

In order to develop a biomarker predicting the efficacy of treatments for patients with esophageal squamous cell carcinoma (ESCC), we evaluated the subpopulation of T cells in ESCC patients treated with chemotherapy (CT), chemoradiotherapy (CRT), and nivolumab therapy (NT). Fifty-five ESCC patients were enrolled in this study, and peripheral blood samples were collected before and after CT or CRT and during NT. Frequencies of memory, differentiated, and exhausted T cells were evaluated using flow cytometry among cStages, treatment strategies, pathological responses of CT/CRT, and during NT. The frequencies of PD-1+ or TIM-3+CD4+ T cells were significantly higher in patients with cStage IV. PD-1+CD4+ and TIM-3+CD8+ T-cell populations were significantly higher in patients treated with CRT but were not associated with treatment response. The frequencies of both CD4+ and CD8+ CD45RA-CD27+CD127+ central memory T cells (TCM) were significantly decreased during the course of NT in the progressive disease group. Taken together, the alteration in frequency of CD45RA-CD27+CD127+ TCM during NT may be a biomarker to predict its therapeutic response in ESCC patients.

The Impact of Tumor Cell-Intrinsic Expression of Cyclic GMP-AMP Synthase (cGAS)-Stimulator of Interferon Genes (STING) on the Infiltration of CD8+ T Cells and Clinical Outcomes in Mismatch Repair Proficient/Microsatellite Stable Colorectal Cancer.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a crucial role in activating immune cells in the tumor microenvironment, thereby contributing to a more favorable response to immune checkpoint inhibitors (ICI) in colorectal cancer (CRC). However, the impact of the expression of cGAS-STING in tumor cells on the infiltration of CD8+ T cells and clinical outcomes in mismatch repair proficient/microsatellite stable (pMMR/MSS) CRC remains largely unknown. Our findings reveal that 56.8% of all pMMR CRC cases were cGAS-negative/STING-negative expressions (cGAS-/STING-) in tumor cells, whereas only 9.9% of all pMMR CRC showed cGAS-positive/STING-positive expression (cGAS+/STING+) in tumor cells. The frequency of cGAS+/STING+ cases was reduced in the advanced stages of pMMR/MSS CRC, and histone methylation might be involved in the down-regulation of STING expression in tumor cells. Since the expression level of cGAS-STING in tumor cells has been associated with the infiltration of CD8+ and/or CD4+ T cells and the frequency of recurrence in pMMR/MSS CRC, decreased expression of cGAS-STING in tumor cells might lead to poor immune cell infiltration and worse prognosis in most pMMR/MSS CRC patients. Our current findings provide a novel insight for the treatment of patients with pMMR/MSS CRC.

M2 tumor-associated macrophages resist to oxidative stress through heme oxygenase-1 in the colorectal cancer tumor microenvironment.

M2 tumor-associated macrophages (M2-TAMs) promote cancer cell proliferation and metastasis in the TME. Our study aimed to elucidate the mechanism of increased frequency of M2-TAMs infiltration in the colorectal cancer (CRC)-TME, focusing on the resistance to oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. In this study, we evaluated the correlation between M2-TAM signature and mRNA expression of antioxidant related genes using public datasets, and the expression level of antioxidants in M2-TAMs by flow cytometry and the prevalence of M2-TAMs expressing antioxidants by immunofluorescence staining using surgically resected specimens of CRC (n = 34). Moreover, we generated M0 and M2 macrophages from peripheral blood monocytes and evaluated their resistance to oxidative stress using the in vitro viability assay. Analysis of GSE33113, GSE39582, and The Cancer Genome Atlas (TCGA) datasets indicated that mRNA expression of HMOX1 (heme oxygenase-1 (HO-1)) was significantly positively correlated with M2-TAM signature (r = 0.5283, r = 0.5826, r = 0.5833, respectively). The expression level of both Nrf2 and HO-1 significantly increased in M2-TAMs compared to M1- and M1/M2-TAMs in the tumor margin, and the number of Nrf2+ or HO-1+M2-TAMs in the tumor stroma significantly increased more than those in the normal mucosa stroma. Finally, generated M2 macrophages expressing HO-1 significantly resisted to oxidative stress induced by H2O2 in comparison with generated M0 macrophages. Taken together, our results suggested that an increased frequency of M2-TAMs infiltration in the CRC-TME is related to Nrf2-HO-1 axis mediated resistance to oxidative stress.

ARID1A deficiency is targetable by AKT inhibitors in HER2-negative gastric cancer.

BACKGROUND: The PI3K/AKT signaling pathway is frequently activated in gastric cancer (GC); however, AKT inhibitors are not effective in unselected GC patients in clinical trials. Mutations in AT-rich interactive domain 1A (ARID1A), which are found in approximately 30% of GC patients, activate PI3K/AKT signaling, suggesting that targeting the ARID1A deficiency-activated PI3K/AKT pathway is a therapeutic candidate for ARID1A-deficient GC. METHODS: The effect of AKT inhibitors was evaluated using cell viability and colony formation assays in ARID1A-deficient and ARID1A knockdown ARID1A-WT GC cells as well as in HER2-positive and HER2-negative GC. The Cancer Genome Atlas cBioPortal and Gene Expression Omnibus microarray databases were accessed to determine the extent of dependence of GC cell growth on the PI3K/AKT signaling pathway. RESULTS: AKT inhibitors decreased the viability of ARID1A-deficient cells and the inhibitory effect was greater in ARID1A-deficient/HER2-negative GC cells. Bioinformatics data suggested that PI3K/AKT signaling plays a greater role in proliferation and survival in ARID1A-deficient/HER2-negative GC cells than in ARID1A-deficient/HER2-positive cells, supporting the higher therapeutic efficacy of AKT inhibitors. CONCLUSIONS: The effect of AKT inhibitors on cell proliferation and survival is affected by HER2 status, providing a rationale for exploring targeted therapy using AKT inhibitors in ARID1A-deficient/HER2-negative GC.

Radiation-Induced Remodeling of the Tumor Microenvironment Through Tumor Cell-Intrinsic Expression of cGAS-STING in Esophageal Squamous Cell Carcinoma.

PURPOSE: Radiation therapy (RT) has the potential to activate the tumor-microenvironment (TME) and promote the efficacy of immune checkpoint blockade therapy. Tumor cell-intrinsic expression of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) plays an important role in regulations of radiation-induced activation of immune cells in the TME. However, the role of tumor cell-intrinsic cGAS-STING in radiation-mediated remodeling of the TME in esophageal squamous cell carcinoma (ESCC) is not completely understood; thus, we investigated its effect on the radiation-mediated remodeling of the TME in ESCC. METHODS: We assessed the effect of tumor cell-intrinsic cGAS-STING on the expression of mediators of the immune system, including type I interferon, T-cell chemo-attractants, colony-stimulating factor-1, and interleukin 34 (IL-34), induced by radiation in ESCC cell lines. We also quantified the association between tumor cell-intrinsic expression of cGAS-STING and infiltrations of immune cells, including CD8+ T cells and CD163+ M2-tumor-associated macrophages (TAMs), in ESCC tissues before and after neoadjuvant chemo-RT (n = 47). RESULTS: We found that tumor cell-intrinsic expression of cGAS-STING was involved in radiation-induced infiltration of CD8+ T cells and expression of type I interferon and T-cell chemo-attractants in ESCC cells. Surprisingly, tumor cell-intrinsic cGAS-STING was also involved in radiation-triggered infiltration and/or M2-polarization of CD163+ TAMs and expression of IL-34, an important cytokine for recruitment and M2-polarization of TAMs, in ESCC cells. The number of CD163+ M2-TAMs was significantly associated with IL-34 expression in tumor cells in irradiated ESCC tissues. CONCLUSIONS: The tumor cell-intrinsic expression of cGAS-STING is essential for radiation-induced activation of immune cells in the TME, but it is also involved in the recruitment of tumor-promoting M2-TAMs in ESCC. Therefore, blocking of M2-TAM infiltration by targeting IL-34 might improve the efficacy of RT and combination therapy of RT with immune checkpoint inhibitors in ESCC.

[Expression of the cGAS-STING Pathway in dMMR/MSI-H in Colorectal Cancer].

Deficient mismatch repair (dMMR)/microsatellite instability (MSI)-H colorectal cancer (CRC) has high immunogenicity. Although the cyclic GMP-AMP synthase( cGAS)-stimulator of interferon genes( STING) pathway activation has considerably contributed to the high number of CD8+ tumor-infiltrating lymphocytes (TILs), its role in dMMR/MSI-H CRC is largely unknown. In this study, we investigated the association between cGAS-STING expression and CD8+ TILs in CRC. Data analysis using the TCGA dataset CRC cohort showed that cGAS, STING, and CD8 gene expression levels were significantly higher in the MSI group. Immunohistochemistry examination of resected clinical CRC samples showed that cGAS-STING expression in tumor cells was high in the MSI CRC, and CD8+ TILs was also significantly infiltrated in the MSI group. Moreover, significant CD8+ TILs infiltration was observed in CRC with high cGAS and STING expression levels. The results suggest that dMMR/MSI -H CRC has maintained a high cGAS-STING expression, which may contribute to abundant CD8+ TILs.

Role of the cGAS-STING pathway in regulating the tumor-immune microenvironment in dMMR/MSI colorectal cancer.

Deficient mismatch repair (dMMR)/microsatellite instability (MSI) colorectal cancer (CRC) has high immunogenicity and better prognosis compared with proficient MMR (pMMR)/microsatellite stable (MSS) CRC. Although the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has been considered to contribute to the high number of CD8+ TILs, its role in dMMR/MSI CRC is largely unknown. In this study, to examine the role of the cGAS-STING pathway on the recruitment of CD8+ TILs in dMMR/MSI CRC, we used public datasets and clinical tissue samples in our cohorts to evaluate the expression of cGAS, STING, and CD8+ TILs in pMMR/MSS and dMMR/MSI CRCs. According to the analysis of public datasets, the expression of cGAS-STING, CD8 effector gene signature, and CXCL10-CCL5, chemoattractants for CD8+ TILs which regulated by the cGAS-STING pathway, was significantly upregulated in dMMR/MSI CRC, and the expression of cGAS-STING was significantly associated with the expression of CD8 effector gene signature. Immunohistochemistry staining of the clinical tissue samples (n = 283) revealed that cGAS-STING was highly expressed in tumor cells of dMMR CRC, and higher expression of cGAS-STING in tumor cells was significantly associated with the increased number of CD8+ TILs. Moreover, we demonstrated that the downregulation of MMR gene in human CRC cell lines enhanced the activation of the cGAS-STING pathway. Taken together, for the first time, we found that dMMR/MSI CRC has maintained a high level of cGAS-STING expression in tumor cells, which might contribute to abundant CD8+ TILs and immune-active TME.

Immune escape mechanism behind resistance to anti-PD-1 therapy in gastrointestinal tract metastasis in malignant melanoma patients with multiple metastases.

Immunotherapy targeting the PD-1 axis has recently become a standard treatment for patients with malignant melanoma. However, approximately 25% of reported malignant melanoma patients who initially responded to immunotherapy with anti-PD-1 mAb had progressive disease, and the immune escape mechanism behind resistance to anti-PD-1 therapy is not yet fully understood in the clinical setting. In the present study, we included four malignant melanoma patients, in whom multiple metastases other than gastrointestinal tract metastasis had disappeared or were controlled under multidisciplinary treatment that included anti-PD-1 therapy. Using IHC, we evaluated the immune status of surgically resected specimens of gastrointestinal tract metastases as acquired resistant lesion to anti-PD-1 therapy. We herein report that the down-regulated expression of HLA class I and up-regulated expression of inhibitory immune checkpoint ligands, CD155 (ligand for T cell immunoglobulin and ITIM domain, TIGIT) and carcinoembryonic antigen-related adhesion molecule-1 (ligand for TIM-3), were observed on the tumor cells in the metastatic gastrointestinal tract tumors. Moreover, our results also suggest that stromal TGF-ß may be related to this down-regulation of HLA class I expression on the tumor cells. In conclusion, it is likely that the down-regulated expression of HLA class I and additional expression of inhibitory immune checkpoint ligands other than PD-L1 on the tumor cells were acquired in the gastrointestinal tract metastasis during anti-PD-1 therapy in the malignant melanoma patients.

SH2D4A downregulation due to loss of chromosome 8p is associated with poor prognosis and low T cell infiltration in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) develops through chromosomal instability (CIN) or microsatellite instability (MSI) due to deficient mismatch-repair (dMMR). We aimed to characterise novel cancer-associated genes that are downregulated upon malignant transformation in microsatellite stable (MSS) CRCs, which typically exhibit CIN with proficient mismatch-repair (pMMR). METHODS: Comprehensive screening was conducted on adenomas, MSI/MSS CRCs and cell lines, followed by copy number analysis, and their genetic and prognostic relevance was confirmed in microarray and RNA-seq cohorts (n = 3262, in total). Immunohistochemistry for SH2D4A was performed in 524 specimens of adenoma, carcinoma in situ and dMMR/pMMR CRC. The functional role of SH2D4A was investigated using CRC cell lines. RESULTS: A set of 11 genes, including SH2D4A, was downregulated during the adenoma-carcinoma sequence in MSS/CIN CRCs, mainly due to chromosome 8p deletions, and their negative prognostic impact was validated in independent cohorts. All adenomas were SH2D4A positive, but a subset of CRCs (5.3%) lacked SH2D4A immunohistochemical staining, correlating with poor prognosis and scarce T cell infiltration. SH2D4A depletion did not affect cell proliferation or IL-6-induced STAT3 phosphorylation. CONCLUSIONS: Our findings suggest that downregulation of multiple genes on chromosome 8p, including SH2D4A, cooperatively contribute to tumorigenesis, resulting in the immune cold tumour microenvironment and poor prognosis.

[Immunotherapy Targeting Tumor-Associated Carbohydrate Antigens in Deficient Mismatch Repair Colorectal Cancer].

The Tn antigen is the most prevalent tumor-associated carbohydrate antigen. It interacts with macrophage galactose-specific lectin(MGL)on dendric cells and macrophages, driving immune inhibitory signals. Colorectal cancer(CRC)exhibiting deficient mismatch repair(dMMR)is characterized by tumor-infiltrating lymphocytes(TILs), the expression of immune checkpoint molecules, and immune evasion. We recently reported that Tn antigen expression was associated with dMMR and that dMMR CRCs with strong Tn antigen expression demonstrated CD8+ T cell exclusion and a lack of PD-L1 expression. Our findings suggest that the immune cold subset of dMMR CRCs with strong Tn antigen may be effectively treated with immune checkpoint blockade therapy or cellular immunotherapy targeting Tn antigens.