Spatial expression of fibroblast activation protein-α in clear cell renal cell carcinomas revealed by multiplex immunoprofiling analysis of the tumor microenvironment

Abstract

Clear cell renal cell carcinoma (ccRCC) is one of the most challenging neoplasms because of its phenotypic variability and intratumoral heterogeneity. Because of its variability, ccRCC is a good test bench for the application of new technological approaches to unveiling its intricacies. Multiplex immunofluorescence (mIF) is an emerging method that enables the simultaneous and detailed assessment of tumor and stromal cell subpopulations in a single tissue section. This novel approach represents a promising step forward for analyzing the microenvironmental cell composition and distribution across the tumor and understanding its possible interactions with tumor cells. This study provides the first characterization of the spatial distribution of fibroblast activation protein-α (FAP)-expressing cancer-associated fibroblasts (FAP + CAFs) in conjunction with lymphoid (CD4 + , CD8 + , CD4 + FOXP3 + , and CD20 +) and myeloid (CD68 +) cells in tissue sections from ccRCC in their early phases of evolution (n = 88). Both the tumor center and periphery were analyzed with mIF. FAP + CAFs and tumor-infiltrating lymphocytes (TILs) were significantly concentrated at the tumor periphery. Additionally, elevated percentages of FAP + CAFs were correlated with larger tumors and synchronous metastases. Increased levels of CD68 + and CD4 + FOXP3 + cells (above the 75th percentile) were linked to worse cancer-specific survival (CSS) in patients with ccRCC. Furthermore, significant correlations emerged among FAP + CAFs, TILs, and CD68 + cells, and the co-occurrence of elevated FAP + CAFs, T-cytotoxic (CD8 +), T-regulatory (CD4 + FOXP3 +) cells, and macrophages (CD68 +) at the tumor center were independently associated with worse CSS. These findings suggest that FAP + CAFs contribute to the aggressiveness of ccRCC, and their role is potentially mediated by their ability to foster an immunosuppressive environment within the renal tumor microenvironment.

Keywords: Cancer-associated fibroblasts; Fibroblast activation protein; Immune cells; Multiplex immunofluorescence; Spatial imaging.

Fig. 1

Hematoxylin and eosin (A, C) and Multiplex immunofluorescence analysis (mIF) of CAFs (FAP +), T-helper (CD4 +), T-cytotoxic (CD8 +) and T-regulatory (CD4 + FOXP3 +) lymphocytes, macrophages (CD68 +), and tumor cells (pan-CK +) at the center (A and B) and the periphery (C and D) of clear cell renal cell carcinomas (ccRCCs). Percentages of different stromal and tumor cells obtained through mIF are displayed (E, F)

Fig. 2

Quantitative expression of FAP + , CD4 + , CD8 + , CD4 + FOXP3 + , and CD68 +  cells at the tumor center (A) and periphery (B) of ccRCC tissues. FAP-expressing CAFs (FAP + CAFs), T-helper, T-cyt, and T-reg lymphocytes were significantly more abundant at the tumor periphery than at the center

Fig. 3

Quantitative expression of stromal cells in ccRCCs with synchronous distant metastasis. Multiplex immunofluorescence (mIF) analysis revealed that tumors debuting with distant metastases had higher concentrations of FAP + CAFs, T-cyt and T-reg lymphocytes, and macrophages. The figure shows the distribution of FAP+, CD8+, and CD68+ cells at the center (A) and the periphery (B) in the primary tumor of a metastasized ccRCC. FAP + CAFs (white arrows) show a spindle morphology when surrounding tumor nests near CD8 (red arrows) and CD68 (yellow arrows) cells. The spindle morphology is accentuated at the tumor center because of the associated sclerosis in the stroma

Fig. 4

Kaplan–Meier curves illustrating the association between cancer-specific survival (CSS) of patients with ccRCC and quantitative expression of FAP + CAFs, T-cyt and T-reg lymphocytes, and macrophages. High percentages (above the 75th percentile (P75)) of macrophages at the center (A) and the periphery of tumors (B) were associated with poor CSS. High levels (above P75) of CD4 + FOXP3 +  cells (T-reg) were also associated with worse CSS (C). The expression of FAP + CAFs and T-cyt, T-regs, and macrophages at the tumor center (all above the P75 threshold) was associated with worse CSS (D). (Rest = tumors with all other combinations, i.e., all cases in which the four biomarkers did not coincide above the P75 threshold)