Cancer-associated fibroblasts promote PD-L1 expression in mice cancer cells via secreting CXCL5

Abstract

Cancer-associated fibroblasts (CAFs) play a key role in orchestrating the tumor malignant biological properties within tumor microenvironment and evidences demonstrate that CAFs are a critical regulator of tumoral immunosuppression of the T cell response. However, the functions and regulation of CAFs in the expression of programmed death-ligand 1 (PD-L1) in melanoma and colorectal carcinoma (CRC) are not completely understood. Herein, by scrutinizing the expression of α-SMA and PD-L1 in melanoma and CRC tissues, we found that CAFs was positive correlated with PD-L1 expression. Further analyses showed that CAFs promoted PD-L1 expression in mice tumor cells. By detecting a majority of cytokines expression in normal mice fibroblasts and CAFs, we determined that CXCL5 was abnormal high expression in CAFs and the immunohistochemistry and in situ hybridization confirmed that were CAFs which were expressing CXCL5. In addition, CXCL5 promoted PD-L1 expression in B16, CT26, A375 and HCT116. The silencing of CXCR2, the receptor of CXCL5, inhibited the PD-L1 expression induced by CAFs in turn. Functionally, CXCL5 derived by CAFs promoted PD-L1 expression in mice tumor cells through activating PI3K/AKT signaling. LY294002, the inhibitor of PI3K, confirmed that CXCL5 forested an immunosuppression microenvironment by promoting PD-L1 expression via PI3K/AKT signaling. Meanwhile, the B16/CT26 xenograft tumor models were used and both CXCR2 and p-AKT were found to be positively correlated with PD-L1 in the xenograft tumor tissues. The immunosuppressive action of CAFs on tumor cells is probably reflective of them being a potential therapeutic biomarker for melanoma and CRC.

Keywords: CAFs; CXCL5; PD-L1; colorectal cancer; melanoma.

Figure 1

The expression of α‐SMA correlates with PD‐L1 in human melanoma and CRC tissues. (a) The expression level of α‐SMA and PD‐L1 in human melanoma and CRC tissues were detected by IHC and evaluated. (b) Representative immunostaining images of α‐SMA and PD‐L1 in melanoma and CRC tissues. Scale bar, 200 μm.

Figure 2

The CAFs isolated and enriched from the transplantation tumor promote PD‐L1 expression in cancer cells. (a) The protein levels of α‐SMA in 3T3 and CAFs were detected by western blot. (b) The expression of α‐SMA in 3T3 and CAFs was detected by flow cytometry. (c) The diagram was showed the cocultured model and the expression of PD‐L1 in tumor cells was detected by flow cytometry. (d) The expression of PD‐L1 in tumor cells cocultured with fibroblasts directly was detected by flow cytometry.

Figure 3

CXCL5 is significantly increased in CAFs compared to normal fibroblasts. (a) The differentially expressed genes between 3T3 and CAFs were showed in the heatmap by OmicShare Tool. (b) The protein level of CXCL5 in 3T3 and CAFs was showed by western blot analysis. (c) Immunofluorescence images showed CXCL5 expression in 3T3 and CAFs. (d) Representative immunostaining of CXCL5 and Masson's staining in human CRC tissues. (e) Representative images of immunofluorescence staining for α‐SMA and CXCL5 in human CRC tissues. (f) Representative images of ISH staining for CXCL5 in human CRC tissues (Scale bar, 200 μm). The figure in the top right corner was the enlargement of the red rectangle square (Scale bar, 50 μm).

Figure 4

CAFs‐derived CXCL5 promotes PD‐L1 expression in cancer cells. (a) The expression of PD‐L1 in B16, CT26, A375 and HCT116 was determined by flow cytometry after treated with different concentrations of recombined CXCL5. (b) The mRNA expression of PD‐L1 in B16, CT26, A375 and HCT116 was detected by RT‐PCR. *p < 0.05. (c) Western blot showed the protein levels of CXCR2 in B16 and CT26 after transfected with siCXCR2. (d) Flow cytometry showed the expression of PD‐L1 in B16 and CT26 cocultured with CAFs after transfected with siCXCR2. (e) The protein levels of CXCR2 in A375 and HCT116 after transfected with siCXCR2 were analyzed by western blotting. (f) The expression of PD‐L1 in A375 and HCT116 treated with CXCL5 after transfected with siCXCR2 was determined by flow cytometry. (g) CXCL5 and α‐SMA expression in 3T3 cells were detected by western after transfected with CXCL5‐overexpression plasmid. (h) PD‐L1 protein expression in B16 and CT26 cocultured with/without 3T3‐CXCL5 cells was analyzed by western blot and the normalized protein expression was analyzed by Image J. *p < 0.05.

Figure 5

CXCL5 promotes PD‐L1 expression through PI3K/AKT signaling pathway. (a) B16 and CT26 were treated with CXCL5 for different times in 2 hr and the key proteins of PI3K/AKT signaling pathway were detected via western blot. The normalized protein expression was analyzed by Image J. (b) B16, CT26, A375 and HCT116 were pretreated with or without LY294002 (10 μM) for 2 hr and treated with CXCL5 for 1 hr. The protein expression of p‐AKT, AKT, p‐PI3K and PI3K were detected by western and the corresponding graph was showed. Cells were pretreated with or without LY294002 for 2 hr and treated with CXCL5 for 48 hr. (c) The protein expressions and the corresponding graphs of PD‐L1 were detected by western. (d) The PD‐L1 expressions were determined by flow cytometry. *p < 0.05.

Figure 6

The correlation of CXCR2, PD‐L1 and p‐AKT in tumor tissues in vivo. (a) The expression level of CXCR2 and PD‐L1 in human melanoma and CRC tissues were detected by IHC and evaluated. (b) The correlation of CXCR2 and PD‐L1 in xenograft tumor tissues was analyzed by IHC and evaluated. (c) The correlation of p‐AKT and PD‐L1 in xenograft tumor tissues was detected by IHC and quantized. (d) Representative immunostaining images of PD‐L1, CXCR2 and p‐AKT in B16 and CT26 xenograft tumor tissues. Scale bar, 200 μm.

Figure 7

The crosstalk between PD‐L1 and PI3K/AKT signaling in melanoma and CRC cells in response to CXCL5.