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Fibroblast activation protein (FAP) generally shows low or undetectable expression in most normal tissues but is highly expressed in fibroblasts in almost all carcinomas. FAP is one of the potential molecules to detect activated fibroblasts and has multiple roles in tumour progression. We generated transgenic mice that specifically expressed tdTomato along with FAP promoter activity. Coculturing a mouse gastric cancer cell line and FAP-tdTomato transgenic mouse-derived fibroblasts showed that tdTomato expression was elevated in the cocultured fibroblasts. Moreover, stomach wall transplanted tumours in mice also showed FAP-tdTomato expression in fibroblasts of the stomach and each metastatic legion. These results indicated that FAP-tdTomato expression in fibroblasts was elevated by stimulation through the interaction with cancer cells. Functionally, collagen production was increased in FAP/tdTomato-positive fibroblasts cocultured with mouse cancer cells. These FAP-tdTomato transgenic mice have the potential to be used to investigate real-time FAP dynamics and the importance of FAP expression in tumour development.

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti–PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti–PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.Significance:Stromal targeting with PDGFRα/β dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer.See related commentary by Tauriello, p. 655

Supplementary Figure8

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti–PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti–PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.Significance:Stromal targeting with PDGFRα/β dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer.See related commentary by Tauriello, p. 655

Supplementary Information

Immune checkpoint inhibitors have shown efficacy in various cancers. Although programmed death ligand 1/2 (PD-L1/L2) expressions have been demonstrated as predictive biomarkers of response to immune checkpoint inhibitors and prognostic markers, whether PD-L1/L2 expression is altered in esophageal squamous cell carcinoma during the therapeutic course is unclear. Whether PD-L1/L2 expression in metastatic or recurrent lesions is consistent with that in primary tumors is also unknown. This study included 561 surgically resected esophageal squamous cell carcinomas and PD-L1/L2 expression was evaluated by immunohistochemistry. We investigated the influence of chemotherapeutic drugs (cisplatin and fluorouracil) on PD-L1/L2 expression and PD-L1/L2-related pathways in vitro. We also examined PD-L1/L2 expression in 18 surgically resected lymph node metastases and 10 recurrent lesions compared with primary lesions. The positive rate of PD-L1 was significantly higher in patients with preoperative chemotherapy than in those without preoperative therapy. The positive rate of PD-L2 expression showed no significant difference between patient groups. Cisplatin increased PD-L1 expression in cancer cell lines in vitro, but decreased PD-L2 in some cell lines. The effects of cisplatin on phosphorylated signal transducer and activator of transcription 1/3 (pSTAT1/3) also differed depending on cell lines. Fluorouracil increased PD-L1 and PD-L2 expression. PD-L1/L2 expression in lymph node metastases and recurrent lesions did not always match expression in primary lesions. PD-L1/L2 expression may be altered by preoperative chemotherapy, and PD-L1 /L2 expression in primary lesions does not always match that of metastatic/recurrent lesions. Thus, one-time evaluation is not sufficient to evaluate PD-L1/L2 expression as a biomarker in esophageal cancer.

Remodeling the tumor microenvironment (TME) to benefit cancer cells is crucial for tumor progression. Although diffuse-type gastric cancer (DGC) preferentially interacts with the TME, the precise mechanism of the complicated network remains unknown. This study aimed to investigate the mutual activation mechanism underlying DGC progression.Mass cytometry analysis of co-cultured macrophages, noncancerous fibroblasts (NFs), and DGC cells was performed. RNA sequencing was applied to examine gene expression in fibroblasts. DGC cells were treated with cytokines to examine their effect on characteristic changes. The TCGA and Kumamoto University cohorts were used to evaluate the clinical relevance of the in vitro findings.Cohort analysis revealed that DGC patients had a poor prognosis. The fibroblasts and macrophages interacted with DGC cells to form a cell cluster in the invasive front of DGC tissue. The original 3D triple co-culture system determined the promotional effects of nonmalignant cells on DGC invasive growth. We notably identified a mixed-polarized macrophage cell type with M1/M2 cell surface markers in a triple co-culture system. IL-1β from mixed-polarized macrophages induced the conversion of NFs to cancer-associated fibroblast-like (CAF-like) cells, promoting the malignant phenotype of DGC cells by inducing the secretion of IL-6, IL-24, and leukemia inhibitory factor (LIF). Moreover, IL-6 and colony stimulating factor 2 (GM-CSF) cooperated to maintain the stable state of mixed-polarized macrophages. Finally, we found that mixed-polarized macrophages were frequently detected in DGC tissues.These findings demonstrated that mixed-polarized macrophages exist as a novel subtype through the reciprocal interaction between DGC cells and nonmalignant cells.

The arachidonic acid cascade is a major inflammatory pathway that produces prostaglandin E

Some reports showed the immune tolerance of soluble human leukocyte antigen E (HLA-E), but the role that soluble HLA-E plays in gastric cancer (GC) is unknown. We aimed to clarify the molecular mechanism and clinical significance of soluble HLA-E in GC.We examined the expression of HLA-E on GC cells and soluble HLA-E under co-culture with natural killer (NK) cells in a time-dependent manner. Changes in NK cell activity were investigated using anti-NK group 2 member A (NKG2A) antibodies in the presence of soluble HLA-E. Expression of soluble HLA-E in the serum of GC patients was determined.Whereas HLA-E expression on GC cells peaked with interferon (IFN)-γ secretion by NK cells in a time-dependent manner, soluble HLA-E was upregulated in conditioned medium. Pre-incubation with anti-NKG2A antibodies increased the activation of NKG2AHLA-E expression dynamically changes on GC cells and in conditioned medium. Furthermore, soluble HLA-E can contribute to immune escape in GC cell lines, which may have significance in clinical practice. Moreover, soluble HLA-E may be a potential prognostic biomarker.

Pancreatic cancer has an extremely poor prognosis, even after curative resection. Treatment options for pancreatic cancer remain limited, therefore new therapeutic targets are urgently needed. We searched for genes predictive of poor prognosis in pancreatic cancer using a public database and validated the survival impact of the selected gene in a patient cohort. We used a public database to search for genes associated with early pancreatic cancer recurrence. As a validation cohort, 201 patients who underwent radical resection in our institution were enrolled. Expression of the target gene was evaluated using immunohistochemistry (IHC). We evaluated growth and invasiveness using small interfering RNAs, then performed pathway analysis using gene set enrichment analysis. We extracted ARHGEF2 from GSE21501 as a gene with a high hazard ratio (HR) for early recurrence within 1 year. The high ARHGEF2 expression group had significantly poorer recurrence-free survival (RFS) and poorer overall survival (OS) than the low ARHGEF2 expression group. Multivariate analysis demonstrated that high ARHGEF2 expression was an independent poor prognostic factor for RFS (HR 1.92) and OS (HR 1.63). In vitro, ARHGEF2 suppression resulted in reduced cell growth and invasiveness. Bioinformatic analysis revealed that ARHGEF2 expression was associated with MYC, G2M, E2F, and CDC25A expression, suggesting that c-Myc and cell cycle genes are associated with high ARHGEF2 expression. IHC revealed a positive correlation between ARHGEF2 and c-Myc expression. High ARHGEF2 expression is associated with cell cycle progression, and predicts early recurrence and poor survival in patients with pancreatic cancer.

Background The predictive significance of programmed death ligand 1 (PD-L1) for programmed death 1 (PD-1) inhibitors remains unclear in gastric cancer (GC) due to the dynamic alteration by treatments. We aimed to elucidate the effects of trastuzumab (Tmab) on PD-L1 expression in GC. Methods PD-L1 expression was evaluated by multicolour flow cytometry analysis after co-culturing GG cell lines and immune cells with Tmab. IFN-γ in the co-culture experiments was quantified. Immunohistochemistry (IHC) for PD-L1 expression using clinical samples was also performed to confirm PD-L1 alteration by Tmab. Results PD-L1 expression was significantly upregulated by Tmab in HER2-amplified GC cell lines co-cultured with peripheral blood mononuclear cells (PBMCs). PD-L1 upregulation by Tmab was also observed in the GC cells co-cultured with NK cells in time-dependent manner, but not with monocytes. IFN-γ concentration in conditioned media from co-cultured PBMCs and NK cells with Tmab was significantly higher and anti-IFN-γ significantly suppress the Tmab-induced PD-L1 upregulation. IHC also suggested PD-L1 upregulation after Tmab treatment. Conclusions Tmab can upregulate PD-L1 expression on GC cells through interaction with NK cells. These results suggest clinical implications in the assessment of the predictive significance of PD-L1 expression for PD-1 inhibitors.

Summary: In the tumor microenvironment, senescent non-malignant cells, including cancer-associated fibroblasts (CAFs), exhibit a secretory profile under stress conditions; this senescence-associated secretory phenotype (SASP) leads to cancer progression and chemoresistance. However, the role of senescent CAFs in metastatic lesions and the molecular mechanism of inflammation-related SASP induction are not well understood. We show that pro-inflammatory cytokine-driven EZH2 downregulation maintains the SASP by demethylating H3K27me3 marks in CAFs and enhances peritoneal tumor formation of gastric cancer (GC) through JAK/STAT3 signaling in a mouse model. A JAK/STAT3 inhibitor blocks the increase in GC cell viability induced by senescent CAFs and peritoneal tumor formation. Single-cell mass cytometry revealed that fibroblasts exist in the ascites of GC patients with peritoneal dissemination, and the fibroblast population shows p16 expression and SASP factors at high levels. These findings provide insights into the inflammation-related SASP maintenance by histone modification and the role of senescent CAFs in GC peritoneal dissemination.