Microenvironmental changes in familial adenomatous polyposis during colorectal cancer carcinogenesis.

Familial adenomatous polyposis (FAP) is a heritable disease that increases the risk of colorectal cancer (CRC) development because of heterozygous mutations in APC. Little is known about the microenvironment of FAP. Here, single-cell RNA sequencing was performed on matched normal tissues, adenomas, and carcinomas from four patients with FAP. We analyzed the transcriptomes of 56,225 unsorted single cells, revealing the heterogeneity of each cell type, and compared gene expression among tissues. Then we compared the gene expression with that of sporadic CRC. Furthermore, we analyzed specimens of 26 FAP patients and 40 sporadic CRC patients by immunohistochemistry. Immunosuppressiveness of myeloid cells, fibroblasts, and regulatory T cells was upregulated even in the early stages of carcinogenesis. CD8+ T cells became exhausted only in carcinoma, although the cytotoxicity of CD8+ T cells was gradually increased according to the carcinogenic step. When compared with those in the sporadic CRC microenvironment, the composition and function of each cell type in the FAP-derived CRC microenvironment had differences. Our findings indicate that an immunosuppressive microenvironment is constructed from a precancerous stage in FAP.

Tumor-infiltrating monocytic myeloid-derived suppressor cells contribute to the development of an immunosuppressive tumor microenvironment in gastric cancer.

BACKGROUND: Gastric cancer (GC) is characterized by an immunosuppressive and treatment-resistant tumor immune microenvironment (TIME). Here, we investigated the roles of different immunosuppressive cell types in the development of the GC TIME. METHODS: Single-cell RNA sequencing (scRNA-seq) and multiplex immunostaining of samples from untreated or immune checkpoint inhibitor (ICI)-resistant GC patients were used to examine the correlation between certain immunosuppressive cells and the prognosis of GC patients. RESULTS: The results of the scRNA-seq analysis revealed that tumor-infiltrating monocytic myeloid-derived suppressor cells (TI-M-MDSCs) expressed higher levels of genes with immunosuppressive functions than other immunosuppressive cell types. Additionally, M-MDSCs in GC tissues expressed significantly higher levels of these markers than adjacent normal tissues. The M-MDSCs were most enriched in GC tissues relative to adjacent normal tissues. Among the immunosuppressive cell types assessed, the M-MDSCs were most enriched in GC tissues relative to adjacent normal tissues; moreover, their presence was most strongly associated with a poor prognosis. Immediate early response 3 (IER3), which we identified as a differentially expressed gene between M-MDSCs of GC and adjacent normal tissues, was an independent poor prognostic factor in GC patients (P = 0.0003). IER3+ M-MDSCs expressed higher levels of genes with immunosuppressive functions than IER3- M-MDSCs and were abundant in treatment-resistant GC patients. CONCLUSIONS: The present study suggests that TI-M-MDSCs, especially IER3+ ones, may play a predominant role in the development of the immunosuppressive and ICI-resistant GC TIME.

Tertiary lymphoid structures correlate with enhancement of antitumor immunity in esophageal squamous cell carcinoma.

BACKGROUND: Tertiary lymphoid structures (TLSs) are associated with a favorable prognosis in several cancers. However, the correlation between TLSs and outcomes of esophageal squamous cell carcinoma (ESCC) and the impact of TLSs on the tumor immune microenvironment (TIME) remain unknown. METHODS: We pathologically evaluated the significance of TLSs in ESCC focusing on TLS maturation using 180 ESCC specimens and performed single-cell RNA sequencing (scRNA-seq) using 14 ESCC tissues to investigate functional differences of immune cells according to TLS presence. RESULTS: TLS+ cases had better recurrence-free-survival (RFS) (p < 0.0001) and overall survival (OS) (p = 0.0016) compared with TLS- cases. Additionally, mature TLS+ cases had better RFS and OS compared with immature TLS+ cases (p = 0.019 and p = 0.015) and TLS- cases (p < 0.0001 and p = 0.0002). The scRNA-seq showed that CD8+ T cells in TLS+ tumors expressed high levels of cytotoxic signatures and antigen-presentation of dendritic cells (DCs) was enhanced in TLS+ tumors. Immunohistochemistry showed that the densities of tumor-infiltrating CD8+ T cells and DCs were significantly higher in TLS+ tumors than those in TLS- tumors. CONCLUSIONS: These data suggest the prognostic and functional significance of TLSs in ESCC and provides new insights into TLSs on the TIME.

Neoadjuvant chemotherapy enhances anti-tumor immune response of tumor microenvironment in human esophageal squamous cell carcinoma.

Although chemotherapy has been an essential treatment for cancer, the development of immune checkpoint blockade therapy was revolutionary, and a comprehensive understanding of the immunological tumor microenvironment (TME) has become crucial. Here, we investigated the impact of neoadjuvant chemotherapy (NAC) on immune cells in the TME of human esophageal squamous cell carcinoma using single cell RNA-sequencing. Analysis of 30 fresh samples revealed that CD8+/CD4+ T cells, dendritic cells (DCs), and macrophages in the TME of human esophageal squamous cell carcinoma showed higher levels of an anti-tumor immune response in the NAC(+) group than in the NAC(-) group. Furthermore, the immune cells of the NAC(+) group interacted with each other resulting in enhanced anti-tumor immune response via various cytokines, including IFNG in CD8+/CD4+ T cells, EBI3 in DCs, and NAMPT in macrophages. Our results suggest that NAC potentially enhances the anti-tumor immune response of immune cells in the TME.

Single-cell transcriptome analysis reveals functional changes in tumour-infiltrating B lymphocytes after chemotherapy in oesophageal squamous cell carcinoma.

BACKGROUND: Tumour immune microenvironment is related with carcinogenesis and efficacy of immunotherapy. B cells play major roles in humoral immunity, but detailed functions of tumour-infiltrating B lymphocytes (TIL-Bs) are unknown. Therefore, our aim was to investigate the functional heterogeneity of TIL-Bs in oesophageal squamous cell carcinoma (ESCC) and lymph nodes (LNs) during chemotherapy. METHODS: Single-cell transcriptome analysis was performed on 23 specimens. We also performed immunohistochemical analysis of immunoglobulin κ C (IGKC), an antibody-secreting cell (ASC) marker, in 166 ESCC samples and evaluated the implication of IGKC in 2-year recurrence free survival (RFS) and 3-year overall survival (OS). RESULTS: A total of 81,246 cells were grouped into 24 clusters. We extracted B cell clusters based on canonical markers and identified 12 TIL-B subtypes in ESCC. We found that several functions, such as co-stimulation and CD40 signalling, were enhanced in TIL-Bs after chemotherapy. The proportion of naive B cells (NBCs) decreased and B cell activation genes were up-regulated in NBCs after chemotherapy. The proportion of ASCs in tumours increased with the loss of migratory abilities and antibody production in ASCs was promoted after chemotherapy. Differentially expressed genes up-regulated with chemotherapy in ASCs correlated with prolonged survival with oesophageal cancer (p = .028). In a metastatic LN, the ASC proportion increased and B cell differentiation was enhanced. In immunohistochemical analysis, RFS and OS of high IGKC expression cases were significantly better than those of low IGKC expression cases (RFS: p < .0001, OS: p < .0001). And in multivariable analysis, the expression of IGKC was an independent favourable prognostic factor for RFS (hazard ratio (HR): 0.23, 95% confidence interval (CI): 0.12-0.45, p < .0001) and OS (HR: 0.20, 95% CI: 0.086-0.47, p = .0002) in ESCC. CONCLUSIONS: Our findings provide novel insights for the heterogeneity of TIL-Bs during chemotherapy and will be useful to understand the clinical importance of TIL-Bs.

Targeted intra-arterial carboplatin chemoradiotherapy and tegafur/uracil for oral and oropharyngeal cancer.

The aim of this study was to evaluate the usefulness of targeted intra-arterial carboplatin chemoradiotherapy in allowing less invasive surgery for patients with oral and oropharyngeal squamous cell carcinoma. Twenty patients with previously untreated squamous cell carcinoma of the oral cavity and oropharynx (T4; 8, T2; 12 patients) were treated with targeted transfemoral intra-arterial carboplatin infusion with concurrent hyperfractionated radiotherapy and the administration of tegafur/uracil (UFT). Of 20 patients, 15 underwent surgery after completion of one course of targeted chemoradiotherapy, and five were given another course or radiotherapy only. Eighteen (90%) of 20 patients had a clinically complete response at the primary site and two (10%) had a partial response. Of the 15 patients who underwent tumor resection, 11 (73%) showed histopathological disappearance of cancer cells at the primary site. Sixteen (80 %) of 20 tumors were controlled at the primary site within a mean follow-up of 30 months. Adverse effects were relatively mild. Targeted intra-arterial chemoradiotherapy caused a down-staging of tumors and facilitated the use of less invasive surgery in patients with squamous cell carcinoma of the oral cavity and oropharynx as a result of its favorable anti-tumor effect.