Cancer associated fibroblasts sculpt tumour microenvironment by recruiting monocytes and inducing immunosuppressive PD-1+ TAMs

Abstract

Fibroblasts turn into cancer associated fibroblasts (CAFs) in the tumour microenvironment. CAFs have recently attracted attention for their function as a regulator of immune cell recruitment and function in addition to their tumour-promoting roles. In this study, we aimed to determine the role of CAFs on monocyte recruitment and macrophage polarization in breast cancer. CAFs, which were α-SMA expressing fibroblasts in contrast to normal fibroblasts (NFs), effectively recruited monocytes. Recruitment of monocytes by CAFs might be mediated by monocyte chemotactic protein-1 (MCP-1) as well as stromal cell-derived factor-1 (SDF-1) cytokines. CAFs differentiated the recruited monocytes into M2-like macrophages which are capable of exerting their immunosuppressive roles via the PD-1 axis. CAF-educated monocytes exhibited strong immune suppression unlike NF-educated monocytes and enhanced the motility/invasion of breast cancer cells in addition to increasing the expressions of epithelial-mesenchymal transition (EMT)-related genes and vimentin protein in cancer cells. CAF-educated M1 macrophages displayed increased expression of M2 markers and production of anti-inflammatory cytokine IL-10 in contrast to decreased production of pro-inflammatory cytokine IL-12 compared with control M1 macrophages; suggesting that CAFs were also able to induce the trans-differentiation of M1 macrophages to M2 macrophages. We then investigated the relationship between the infiltration of CAFs and tumour associated macrophages (TAMs) using tissue samples obtained from breast cancer patients. High grade of CAFs significantly correlated with the number of TAMs in human breast cancer tissue samples. It was also associated with higher Ki-67 proliferation index, and higher tumour volume. This result is in line with our finding of increased breast cancer cell proliferation due to the effects of CAF-educated monocytes in vitro. Our results concluded that CAFs play pivotal roles in sculpturing the tumour microenvironment in breast cancer, and therapeutic strategies to reverse the CAF-mediated immunosuppressive microenvironment should be taken into consideration.

Figure 1

CAFs are α-SMA expressing fibroblasts in contrast to NFs. CAFs and NFs were cultured in 8-well chamber slides, then stained for vimentin (A,D), pan-cytokeratin (B,E) and α-SMA (C,F). Both CAF and NF cells were positive for vimentin and negative for pan-cytokeratin. However, CAFs were positive for α-SMA, unlike NFs. Sup. Figure 1 displays immunocytochemical stainings for fibroblast activation protein (FAP). One representative figure for each cell type is shown (×100).

Figure 2

CAF and MDA-MB-231 cells effectively recruit monocytes. Monocytes isolated from healthy donors were serum starved and then were allowed to migrate for 5 h toward CMs from CAFs, NFs or MDA-MB-231 cells. (A) Representative microscopic views of each condition are shown (i: Standard Medium, ii: CM from NFs, iii: CM from CAFs, iv: CM from MDA-MB-231 Cells). (B) CAFs and MDA-MB-231 cells resulted in monocyte migration more than NFs did. The presence of CXCR4 (10 µg/ml) or MCP-1 (130 µg/ml) blocking antibodies diminished monocyte recruitment. ^*p < 0.05 vs. standard medium, ^#p < 0.05 vs. each corresponding control. Arrows demonstrate migrated/counted monocytes.

Figure 3

CAF and MDA-MB-231 cells induce monocytes to differentiate into a M2 phenotype similar to TAMs. CD14⁺ PBMCs isolated from healthy donors were cultured with CMs from NFs (NF-educated monocytes), CAFs (CAF-educated monocytes), MDA-MB-231 cells (MDA-MB-231-educated monocytes) or standard culture medium DMEM (control monocytes) for 7 days, then were analysed using flow cytometry. (A) The expression levels of PD-1, CD163, and CD206 were significantly higher in CAF-educated monocytes than in NF-educated monocytes. The expression levels of HLA-DR and CD14 were significantly lower in CAF-educated monocytes than in NF-educated monocytes. Representative histograms of each molecule are shown. (B) Data are presented as relative fold changes to control IgG in mean fluorescent intensity (MFI). *p < 0.05, **p < 0.01.

Figure 4

CAF-educated monocytes suppressed CD4⁺ T cell proliferation more than NF-educated monocytes did. CAF-educated monocytes, NF-educated monocytes, MDA-MB-231-educated monocytes, control monocytes were co-cultured with carboxyfluorescein succinimidyl ester (CFSE) labelled and autologous CD4⁺ T cells that were activated by CD3/CD28 magnetic beads for 96 hours. (A) The proliferation of T cells was suppressed by CAF-educated monocytes more strongly than control and NF-educated monocytes. Suppression of T-cell proliferation by CAF-educated monocytes is E:T dependent (normalized percentage of suppression of T-cell proliferation considering respective control monocytes are 40.1% and 53.3% for 1:4 and 1:2 E:Ts, respectively). (B) Representative histograms of the proliferation of T cells (monocytes:CD4⁺ T cells, 1:4). *p < 0.05, **p < 0.01.

Figure 5

CAF-educated monocytes induce increased motility of MDA-MB-231 cells. Monocytes were cultured for 7 days with CMs from NF, CAF or MDA-MB-231 cells as well as in standard medium. All of the differentiated monocytes were then serum starved for 48 hours before obtaining the corresponding CMs. MDA-MB-231 cells were incubated with those CMs from differentiated monocytes for 24 hours. (A) Representative microscopic views of tumour cells that show invasion are shown (i: Standard Medium, ii: CM from Control Monocytes, iii: CM from CAF-educated Monocytes, iv: CM from NF-educated Monocytes, v: CM from MDA-MB-231-educated Monocytes). (B) Tumour cells that show invasion were counted and presented in bar graphs. *p < 0.05 vs. standard medium. Arrows demonstrate counted tumour cells that show invasion.

Figure 6

CAF-educated monocytes modify the expressions of EMT-related proteins and genes. Monocytes were cultured for 7 days with CMs from NF, CAF or MDA-MB-231 cells as well as in standard medium. All of the differentiated monocytes were then serum starved for 48 hours before obtaining the corresponding CMs. MDA-MB-231 cells were incubated with those CMs from differentiated monocytes for 24 hours. (A–D) The effects of NF-, CAF- or MDA-MB-231-educated monocyte CMs on the EMT of MDA-MB-231 breast cancer cells were analysed by Western blot analysis of vimentin and E-cadherin proteins. Densitometry shows relative protein expression normalized for GAPDH (n = 3). (E–G) The effects of NF-, CAF- or MDA-MB-231-educated monocyte CMs on the EMT of MDA-MB-231 breast cancer cells were analysed by quantitative real-time PCR of Snail, Slug and Twist gene expressions (n = 3). *p < 0.05. **p < 0.01. ***p < 0.001. ns: not significant.

Figure 7

CAF and MDA-MB-231 cells induce M1 macrophages to differentiate into M2-like macrophages. (A) M1 macrophages were subsequently cultured with CMs from CAFs or MDA-MB-231 cells. After 48 hours, cells were analysed by flow cytometry. The expression levels of CD163 and CD206 were significantly higher in CAF- and MDA-MB-231-educated M1 macrophages than in M1 macrophages alone. Representative histograms of each molecule are shown. (B) Data are presented as relative fold changes to control IgG in mean fluorescent intensity (MFI). *p < 0.05. (C) M1 macrophages were cultured alone or with CMs from CAFs, or MDA-MB-231 cells for 48 hours and the levels of IL-12 or IL-10 were measured by ELISA. Levels of IL-12 was decreased in CAF- and MDA-MB-231-educated M1 macrophages compared with that in M1 macrophages alone. In contrast, levels of IL-10 increased in CAF- and MDA-MB-231-educated M1 macrophages compared with that in M1 macrophages alone. *p < 0.05.

Figure 8

High grade of CAFs significantly correlated with the number of TAMs in breast cancer tissue samples. Immunohistochemical stainings for CD163, CD206, α-SMA, and Haematoxylin & Eosin (H&E) in breast cancer samples are shown. α-SMA (A), CD163 (B), CD206 (C), and H&E (D) were stained in a triple-negative breast cancer (TNBC) tissue sample. α-SMA (E), CD163 (F), CD206 (G), and H&E (H) were stained in a HER2⁺ breast cancer tissue sample. α-SMA (I), CD163 (J), CD206 (K), and H&E (L) were stained in an ER⁺PR⁺ breast cancer tissue sample (×400; scale bar, 50 µm). Sup. Figure 2 displays immunohistochemical stainings for laminin, CD34, and FAP. Three representative cases are shown.

Figure 9

CAF-educated monocytes induce increased proliferation of MDA-MB-231 cells. Monocytes were cultured for 7 days with CMs from NF or CAF cells. All of the differentiated monocytes were then serum starved for 48 hours before obtaining the corresponding CMs. (A) Proliferation of MDA-MB-231 breast cancer cells were evaluated with MTT assay after 48 hours of treatment with corresponding CMs and normalized cell numbers are shown (mean ± SD, n = 3). (B) MDA-MB-231 cells were seeded in an ACEA E-plate. After a day of incubation, they were incubated with those CMs from differentiated monocytes for an additional 48 hours. Cell index was measured by electrical impedance (n = 3). Normalized cell index (22:26:25, mean ± SD) as a measure for cell proliferation of MDA-MB-231 cells that were treated with NF- (blue) or CAF-educated (red) monocyte CMs is shown. Vertical line demonstrates the time of normalization. **p < 0.01. ***p < 0.001. ns: not significant.