Cancer-Associated Fibroblast-Derived FGF7 Promotes Clear Cell Renal Cell Carcinoma Progression and Macrophage Infiltration

Abstract

As the predominant stromal cells in the ccRCC surrounding environment, cancer-associated fibroblasts (CAFs) have been established as supportive of tumor growth. However, the detailed molecular mechanisms underlying the supporting role of CAFs in ccRCC have not been well characterized. Evidence from the clustering consensus analysis, single-cell analysis, and the experimental results illustrate that CAF-derived FGF7 plays a crucial role as a signaling mediator between CAFs and ccRCC tumor cells. Mechanistically, CAF-derived FGF7 triggers AKT activation to promote cell growth and cell invasion of ccRCC tumor cells. As a response, ccRCC tumor cells stimulate STAT3-mediated transcriptional regulation, directly increasing FGF7 expression at the chromatin level in CAFs. Moreover, there exists a positive clinical correlation between the abundance of CAFs, FGF7 expression, and the infiltration of M2 type macrophages. The RENCA in vivo mouse model also confirmed that FGF7 depletion could impede RCC development by reducing the recruitment of M2 type macrophages. Overall, this study delineates a key signaling axis governing the crosstalk between CAFs and ccRCC tumor cells, highlighting FGF7 as a promising therapeutic target of ccRCC.

Keywords: CAFs; FGF7; ccRCC.

Figure 1

Computational identification of FGF7 as a potential driver during ccRCC development. (A) The heatmap of CAF biomarkers in cluster 1 and cluster 2. (B) CAF-high (cluster 1) ccRCC have poor OS compared to CAF-low (cluster 2) control. (C) The heatmap of top 50 DEGs between cluster 1 and cluster 2. (D) FGF7 as a significant DEG according to the volcano plot analysis. The black arrow marks the location of FGF7. (E) Single cell analysis of FGF7 is ccRCC (ACTA2 as positive control). CAF population is marked by the red circle and the expression levels of FGF7 and ACTA2 in fibroblast are indicated within the red box. (F) The ccRCC patients with high FGF7 expression have shorter OS (Left) and DSS (Right). (G) FGF7 expression in different pathological stages of ccRCC.

Figure 2

FGF7 promotes ccRCC progression. (A) A cartoon of co-culture. (B) Western blotting analysis showed that FGF7 protein level in NIH/3T3 cells was increased in the co-culture system. The b-actin was utilized as the loading control. (C) qPCR assay revealed that FGF7 mRNA levels in NIH/3T3 cells was increased in the co-culture system. (D) CCK8 assay showed that knockdown of FGF7 attenuated co-culture induced cell viability of RENCA cells. (E) EdU assay indicated that knockdown of FGF7-attenuated co-culture induced cell proliferation of RENCA cells. Left, representative images of EdU staining. Right, a statistical analysis. (F) Transwell invasion assay demonstrated that knockdown of FGF7 attenuated co-culture-induced cell invasion of RENCA cells. Left, representative images of invading cells. Right, a statistical analysis. (G) CCK8 assay illustrated that FGF7 treatment (20 ng/mL) accelerated the cell proliferation of both A498 (top) and OSRC-2 cells (bottom). (H) EdU assay showed that FGF7 (20 ng/mL) treatment promoted cell proliferation of both A498 (top) and OSRC-2 cells (bottom). Left, representative images of EdU staining. Right, the statistical analyses of EdU staining. (I) Transwell invasion assay demonstrated that FGF7 treatment (20 ng/mL) promoted cell invasion of both A498 and OSRC-2 cells. Left, representative images of invading cells. Right, the statistical analyses of invading cells. Scale bar = 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. NC = negative control.

Figure 3

FGF7 activates AKT signaling to promote ccRCC progression. (A) KEGG analysis of the top 500 DEGs between FGF7-high and FGF7-low ccRCC patients. (B) CM and FGF7 treatment (20 ng/mL) activated renal AKT signaling, as monitored by the phosphorylation levels of AKT-S473 and AKT-T308. The β-actin was the loading control. (C) CCK8 assay showed that 10 μM AKT inhibitor LY294002 (LY) significantly blocked FGF7-induced cell viability of A498 (top) and OSRC-2 (bottom) cells. (D) EdU assay revealed that 10 μM LY dramatically attenuated FGF7-induced cell proliferation of A498. Left, representative images of EdU staining. Right, a statistical analysis. (E) Transwell invasion assay demonstrated that 10 μM LY treatment reversed FGF7-induced cell invasion of both A498. Left, representative images of invading cells. Right, a statistical analysis. (F) EdU assay revealed that 10 μM LY treatment significantly blocked CM-induced cell proliferation of RENCA cells. Left, representative images of EdU staining. Right, a statistical analysis. (G) Transwell invasion assay showed that 10 μM LY treatment significantly blocked CM-induced cell proliferation of RENCA cells. Left, representative images of invading cells. Right, a statistical analysis. Scale bar = 100 μm. ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. = no significance.

Figure 4

STAT3 transcriptionally regulates FGF7 expression in CAFs. (A) H3K4me3 peak analysis of FGF7 gene locus in fibroblasts according to the ChIP-seq Peak dataset (ChIP-Atlas: Peak Browser). The FGF7 gene locus at the chromosome is marked by the red box and the H3K4me3 peak in the promoter region of FGF7 gene locus is marked by the green box. (B) H3K4me3 ChIP-qPCR revealed that co-culture promoted H3K4me3 enrichment in the promoter region of FGF7. (C) JASPAR software predicted a conserved STAT3 binding sequence in the promoter region of FGF7. (D) Western blotting analysis of STAT3-Y705 level in NIH/3T3 cells before and after the co-culture with RENCA cells. The b-actin served as internal control. (E) STAT3-Y705 ChIP-qPCR revealed that co-culture increased the STAT3 enrichment in the promoter region of FGF7, which was attenuated by 5 μM STAT3-IN-11 treatment. (F) A qPCR assay revealed that 5 μM STAT3-IN-11 treatment reduced co-culture-induced FGF7 mRNA levels in NIH/3T3 cells. (G) Western blotting analysis showed that 5 μM STAT3-IN-11 blocked co-culture-induced FGF7 protein expression in NIH/3T3 cells. The β-actin was used as loading control. ** p < 0.01, **** p < 0.0001, n.s. = no significance.

Figure 5

FGF7 has a close relationship with M2 macrophage infiltration. (A) The infiltration of immune cells between CAF-high and CAF-low ccRCC patients. (B,C) TIMER analyses showed that both ACTA2 (B) and FGF7 expression (C) were positively correlated with the infiltration level of M2 type macrophages. (D) ACTA2 has a positive correlation with CD163+ macrophage population. Left, representative images. Right, a statistical analysis. (E) FGF7 expression had a positive correlation with CD163+ macrophage population. Left, representative images. Right, a statistical analysis. Scale bar = 100 μm. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 6

Depletion of FGF7 in CAFs hinders ccRCC growth. (A) Representative images of RENCA tumors. (B) The tumor volume of RENCA tumors. (C) IHC staining of Ki67, CD163, and FGF7 in RENCA tumors. (D) The working model of this study. Scale bar = 100 μm. ** p < 0.01, *** p < 0.001. NC = negative control.