TGFβ 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 TGFβ-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 TGFβ-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 TGFβ, TGFβ-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 TGFβ-responsive cancer stroma. TIM-3 was increased on M2-polarized macrophages, and on monocytes in response to TGFβ treatment. In conclusion, we identified a close association between TIM-3 and M2-like polarization of macrophages in the TGFβ-rich TME. Our findings provide new insights into personalized immunotherapeutic strategies based on the TME for CRCs.

Lynch syndrome, is an autosomal dominantly inherited disease that predisposes individuals to a high risk of colorectal cancers, and some mismatch-repair genes have been identified as causative genes. The purpose of this study was to investigate the genomic rearrangement of the gene in a family with Lynch syndrome followed for more than 45 years. The family with Lynch syndrome is family N, who received colorectal cancer treatment for 45 years. The proband of family N had multiple colorectal and uterine cancers. Because the proband met the diagnostic Amsterdam criteria and was Microsatellite instability (MSI) - positive, we performed genetic testing several times. However, germline mutations in MLH1 and MSH2 genes were not found by long-distance PCR or RT-PCR/direct sequencing analysis within the 45-year follow-up. MLPA analysis showed that the genomes of the proband and proband’s daughter contained a deletion from exon 4 through exon 19 in the MLH1 gene. Her son’s son and her daughter’s son were found to be carriers of the mutation. For carriers of mismatch-repair gene mutation among families with Lynch syndrome, the onset risk of associated cancers such as uterine cancer is particularly high, including colorectal cancer. The diagnosis of carriers among non-onset relatives is important for disease surveillance.

Background: Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is widely used for patients with locally advanced rectal cancer. However, response to nCRT varies substantially among patients, highlighting the need for predictive biomarkers that can distinguish non-responsive from responsive patients before nCRT. This study aimed to build novel multi-gene assays for predicting nCRT response, and to validate our signature and previously-reported signatures in multiple independent cohorts. Methods: Three microarray datasets of pre-therapeutic biopsies containing a total of 61 non-responders and 53 responders were used as the discovery cohorts to screen for genes that were consistently associated with nCRT response. The predictive values of signatures were tested in a meta-analysis using six independent datasets as the validation cohorts, consisted of a total of 176 non-responders and 99 responders. Results: We identified four genes, including BRCA1, GPR110, TNIK, and WDR4 in the discovery cohorts. Although our 4-gene signature and nine published signatures were evaluated, they were unable to predict nCRT response in the validation cohorts. Conclusions: Although this is one of the largest studies addressing the validity of gene expression-based classifiers using pre-treatment biopsies from patients with rectal cancer, our findings do not support their clinically meaningful values to be predictive of nCRT response.

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.

Colorectal cancer (CRC) cells often express Tn antigen, a tumor-associated truncated immature O-glycan (GalNAcα-O-Ser/Thr) that can promote tumor progression. Immunotherapies against Tn antigen have been developed and are being evaluated in clinical trials. Tn antigen can also be considered a novel immune checkpoint that induces immunosuppressive signaling through glycan-biding lectins to lead effector T cell apoptosis. We evaluated the correlation of Tn antigen expression by immunohistochemistry with mismatch-repair (MMR) status, tumor-infiltrating lymphocytes, tumor cell PD-L1 expression, and clinicopathological characteristics in 507 CRC patients. Although 91.9% of CRCs showed negative or weak Tn antigen staining (Tn-negative/weak), we identified a small subset of CRCs (8.1%) that displayed particularly intense and diffuse distribution of Tn antigen immunoreactivity (Tn-strong) that closely related to deficient MMR (dMMR). Moreover, 40 dMMR CRCs were stratified into 24 Tn-negative/weak dMMR tumors (60.0%) exhibiting dense CD8+ lymphocyte infiltrate concomitant with a high rate of PD-L1 positivity, and 16 Tn-strong dMMR tumors (40.0%) that demonstrated CD8+ T cell exclusion and a lack of PD-L1 expression, which was comparable to those of proficient MMR. Our finding suggests that the immune cold subset of patients with Tn-strong dMMR CRC may be effectively treated with immune checkpoint blockade therapy or cellular immunotherapy targeting Tn antigen.

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.