Cancer-associated fibroblasts promote an immunosuppressive microenvironment through the induction and accumulation of protumoral macrophages

Abstract

Stromal cells in the tumor microenvironment (TME) closely interact with tumor cells and affect tumor cell behavior in diverse manners. We herein investigated the mechanisms by which cancer-associated fibroblasts (CAFs) affect the functional polarization of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC) in vitro and in human cancer samples. The expression of CD68, CD14, CD163, CD200R, CD206, HLA-G, CD80, and CD86 was higher in CD14-positive cells co-cultured with the culture supernatants of CAFs established from OSCC specimens (CAF-educated cells) than in control cells. The gene expression level of ARG1, IL10, and TGFB1 was increased in CAF-educated cells. CAF-educated cells suppressed T cell proliferation more strongly than control cells, and the neutralization of TGF-β IL-10, or arginase I significantly restored T cell proliferation. We then investigated the relationship between the infiltration of CAFs and TAMs using tissue samples obtained from patients with OSCC. The infiltration of CAFs was associated with the numbers of CD68-positive and CD163-positive macrophages. It also correlated with lymphatic invasion, vascular invasion, lymph node involvement, and the TNM stage. The infiltration of CAFs was identified as an independent prognostic factor in OSCC. Our results indicate that CAFs play important roles in shaping the tumor immunosuppressive microenvironment in OSCC by inducing the protumoral phenotype of TAMs. Therapeutic strategies to reverse CAF-mediated immunosuppression need to be considered.

Keywords: cancer-associated fibroblast (CAF); immunomodulation; immunosuppression; tumor microenvironment (TME); tumor-associated macrophage (TAM).

Figure 1. Representative photomicrographs of CAF3 established from resected tumor samples of patients with OSCC

CAFs were cultured in a chamber slide for 48 hours, then stained for a. fibroblast activation protein (FAP) or b. α-smooth muscle actin (α-SMA). CAFs were positive for FAP and α-SMA.

Figure 2. The culture supernatants of CAFs induced the protumoral phenotype of macrophages

CD14-positive PBMCs prepared from healthy donors were cultured with culture supernatants from CAFs (CAF-educated cells) or DMEM (control cells) for 48 hours, then analyzed using flow cytometry or real-time quantitative RT-PCR. a. The expression levels of CD68, CD14, CD163, CD206, CD200R, HLA-G, CD80, and CD86 were significantly higher in CAF-educated cells than in control cells. Data are presented as relative fold changes to control IgG in mean fluorescent intensity (MFI). b. Representative histograms of each molecule are shown. c. The gene expression levels of ARG1, IL10, and TGFB1 were higher in CAF-educated cells than in control cells. *P < 0.05, **P < 0.01.

Figure 3. CAF-educated cells were potent suppressors of autologous T cells

CAF-educated cells or control cells were co-cultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous CD14-negative cells for 96 hours with an anti-CD3/anti-CD28 stimulus. a. The proliferation of T cells was suppressed by CAF-educated cells more strongly than control cells in a dose-dependent manner. b. Representative histograms of the proliferation of T cells. c. The proliferation of both CD4+ T cells and CD8+ T cells was equally suppressed by CAF-educated cells compared with control cells. d. Neutralizing mAbs were added during the co-cultivation of CAF-educated cells and CFSE-labeled cells. The addition of anti-TGF-β mAb or anti-IL-10 mAb significantly restored the proliferation of T cells. e. The addition of the arginase I inhibitor Noha or L-arginine during co-cultivation significantly restored T cell proliferation. *P < 0.05.

Figure 4. Immunohistochemical staining of 73 oral squamous cell carcinoma (OSCC) surgical specimens

Representative photomicrographs of α-SMA staining are shown in (a)-(e): a. CAFs grade 0 case (×100 magnification). Blood vessel walls were used as an internal positive control for α-SMA; b. CAFs grade 1 case (×100 magnification); c. CAFs grade 2 case (×100 magnification); d. CAFs grade 3 case (×100 magnification); e. CAFs grade 3 case (×400 magnification). f. A representative photomicrograph of CD68 staining (×200 magnification). g. A representative photomicrograph of CD163 staining (×200 magnification). h. The number of CD68-positive macrophages was significantly higher than that of CD163-positive macrophages (P < 0.0001).

Figure 5. Kaplan-Meier survival analysis of 73 patients with OSCC

Patients were divided into two groups: CAFs grades 0-2 cases and CAFs grade 3 cases. a. CAFs grade 3 cases showed significantly shorter progression-free survival. b. CAFs grade 3 cases showed significantly shorter overall survival.