Regulatory B cells drive immune evasion in the tumor microenvironment and are involved peritoneal metastasis in gastric cancer

Abstract

Peritoneal metastasis (PM) of gastric cancer (GC) has an immune escape environment. Regulatory B cells (Bregs), characterized by IL-10 production, play an important role in the tumor immunity; however, the function of Bregs in PM remains unclear. We investigated the frequency and effects of Bregs on other immune cells in the PM using clinical specimens and mouse models of PM. In the peripheral blood and ascites, Breg frequency was significantly higher in patients with GC with PM than in those without PM. In clinical PM samples, Breg frequency was an independent prognostic factor. In the mouse PM model, peritoneal tumors showed higher Breg infiltration than subcutaneous tumors. In the PTEN-deficient PM model, activation of Bregs promoted ascites and peritoneal tumor growth, decreased the infiltration of CD8⁺ T cells, and increased the infiltration of M2 macrophages. In contrast, treatment with wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, suppressed Breg infiltration, resulting in decreased M2 macrophage infiltration and increased CD8⁺ T cell infiltration. Bregs are indicated to be involved in immunosuppression of PM and are promising targets for improving the efficacy of immunotherapy against PM.

Keywords: Gastric cancer; PI3K/Akt; Peritoneal metastasis; Regulatory B cell; Wortmannin.

Fig. 1

Regulatory B cell (Breg) frequency in the peripheral blood and ascites of patients with gastric cancer (GC). (A) Bregs among B cells in the peripheral blood samples from healthy control subjects, patients with early GC, advanced GC, and peritoneal metastases (PM) (n = 10 for each group).The Bregs ratio in the peripheral blood of patients with PM was higher than other groups. (B) Bregs among B cells in ascites from patients with early GC, advanced GC, and PM (n = 10 for each group). The Bregs ratio in ascites of patients with PM was higher than that of patients with early and advanced GC (*p < 0.05, **p < 0.01). The results are provided in terms of means ± standard deviation values.

Fig. 2

Association between regulatory B cells (Bregs) and immune cell infiltration in peritoneal metastases (PM) of patients with gastric cancer (GC). (A) Representative microscopic images of Bregs expression in primary tumors (n = 60), and peritoneal (n = 60), lymph mode (n = 36), liver (n = 10), and ovary (n = 10) metastases. B cells were stained brown and IL-10 was stained pink. Each cell was counted and the percentage of Bregs (yellow triangle) among the B cells were analyzed under 200 × magnification. (B) The Breg frequency in PM was higher than in primary tumors and other metastases (***p < 0.001) (C) Representative microscopic images of CD8, CD163 positive cells in PM in GC (magnification ×100). The number of CD8, CD163 positive cells was measured and shown as the average count in five non-overlapping intratumoral fields. (***p < 0.001). The results are provided in terms of means ± standard deviation values.

Fig. 3

Kaplan–Meier survival curve for patients with peritoneal metastases according to regulatory B cell (Breg) frequency (p = 0.005).

Fig. 4

Analysis of the localization of regulatory B cells (Bregs) in a mouse peritoneal metastasis (PM) model. (A) Flow cytometry analysis of Bregs from the spleen and ascites in the control mouse and PM mouse model (n = 6 for each group). (B) Flow cytometry analysis of Breg frequency in peritoneal tumors and subcutaneous tumors (n = 6). (**p < 0.01, ***p < 0.001). The results are provided in terms of means ± standard deviation values.

Fig. 5

Analysis of regulatory B cells (Bregs) and immune cells in a B cell-specific Pten-deficient mouse model of peritoneal metastasis (PM). (A) Comparison of ascites volume and peritoneal tumor weight between Pten-deficient mice (n = 6) and control mice (n = 6) (**p < 0.01, ***p < 0.001). (B) Flow cytometry analysis of Breg frequency from the spleen, ascites, and peritoneal tumors in Pten-deficient mice and control mice (**p < 0.01, ***p < 0.001). Each group consisted of 6 mice. (C) Comparison of CD8⁺ T cells and M2 macrophages from the spleen, ascites, and peritoneal tumors between Pten-deficient mice and control mice (*p < 0.05, **p < 0.01). Each group consisted of 6 mice. The results are provided in terms of means ± standard deviation values.

Fig. 6

Effect of regulatory B cells (Bregs) on CD8⁺ T cell proliferation and M2 macrophages polarization in vitro. (A) Antiproliferative effects of wartmannin in YTN16 cells. Cell viability was assessed by MTT assay after 48-h exposure to a single-dose of wartmannin (0–10 µM) (*p < 0.05, **p < 0.01). (B) IL-10 concentration in the supernatant of Bregs and other B cell subsets excluding Bregs cultured with or without 0.5µM wortmannin measured by ELISA (**p < 0.01, ***p < 0.001). (C) Flow cytometry analysis of M2 macrophage polarization by co-culturing bone marrow-derived macrophages (BMDMs) and Bregs with or without 0.5µM wortmannin (**p < 0.01). (D) The proliferation of CD8⁺ T cells cultured with Bregs and other B cell subsets with or without 0.5µM wortmannin, assessed using the CFSE assay (*p < 0.05). The results are provided in terms of means ± standard deviation values.

Fig. 7

Effect of the PI3K inhibitor wortmannin on regulatory B cells (Bregs), tumor-infiltrating immune cells, and peritoneal tumor growth. (A) Comparison of ascites volume and peritoneal tumor weight between the control and wortmannin-treated model (**p < 0.01, ***p < 0.001). Each group consisted of 6 mice. (B) Flow cytometry analysis of Bregs from the spleen, ascites, and peritoneal tumors in the control and wortmannin-treated model (*p < 0.05, **p < 0.01). (C) Infiltration of CD8⁺ T cells and M2 macrophages in the spleen, ascites, and peritoneal tumors in control and wortmannin-treated models (*p < 0.05, **p < 0.01, ***p < 0.001). The results are provided in terms of means ± standard deviation values.