FCER1G Positively Relates To Macrophage In�ltration In Clear Cell Renal Cell Carcinoma And Contributes To Unfavorable Prognosis By Regulating Tumor Immunity

Background: Tumor-associated macrophages (TAMs) are closely related to unfavorable prognosis of patients with clear cell renal cell carcinoma (ccRCC). However, the important molecules in the interaction between ccRCC and TAMs are unclear. Methods: TCGA-KIRC gene expression data of tumor tissues and normal tissues adjacent to tumor were compared to identify differentially expressed genes in ccRCC. TAMs related genes were discovered by analyzing the correlation between these differentially expressed genes and common macrophage biomarkers. Gene set enrichment analysis was performed to predict functions of TAMs related gene. The �ndings were further validated using RNA sequencing data obtained from the CheckMate 025 study and immunohistochemical analysis of samples from 350 patients with ccRCC. Kaplan–Meier survival curve, Cox regression analysis and Harrell’s concordance index analysis were used to determine the prognostic signi�cance. Results: In this study, we applied bioinformatic analysis to explore TAMs related differentially expressed genes in ccRCC and identi�ed 5 genes strongly correlated with all selected macrophage biomarkers: STAC3, LGALS9, TREM2, FCER1G, and PILRA. Among them, FCER1G was abundantly expressed in tumor tissues and showed prognostic importance in patients with ccRCC who received treatment with Nivolumab; however, it did not exhibit prognostic value in those treated with Everolimus. We also discovered that high expression levels of FCER1G are related to T cell suppression. Moreover, combination of FCER1G and macrophage biomarker CD68 can improve the prognostic strati�cation of patients with ccRCC from TCGA-KIRC. Based on the immunohistochemical analysis of samples from patients with ccRCC, we further validated that FCER1G and CD68 are both highly expressed in tumor tissue and correlate with each other. Higher expression of CD68 or FCER1G in ccRCC tissue indicates shorter overall survival and progression-free survival; patients with high expression of both CD68 and FCER1G have the worst outcome. Combining CD68 and FCER1G facilitates the screening of patients assay conducted Brie�y, para�n-embedded sections of ccRCC TMA were preprocessed with the antigen retrieval procedure. For the antibody against CD68, citric acid was used at a pH of 6.0. For the antibody against FCER1G, ethylene diamine tetraacetic acid was used at a pH of 9.0. Goat serum (10%) was used to block nonspeci�c binding. The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in a moisture chamber. After incubation with horseradish peroxidase-conjugated secondary antibodies (ab205718, goat anti-rabbit IgG polyclonal, 1:2000; Abcam) for 1 hour at room temperature the following day, slides were developed using 3,3'-diaminobenzidine staining for 2 min, and the cell nucleus was stained with hematoxylin. Human tonsil and spleen tissues were used as positive controls for CD68 and FCER1G, respectively (Data not shown). The ccRCC slides incubated only with secondary antibodies were used as negative control (Data not shown). All slides were scanned with Hamamatsu Nanozoomer S60 (Hamamatsu City, and evaluated on the same displayer using the NDP.view software (Version 2.9.20). Given that both CD68 and FCER1G are membrane surface markers that label single cells within tumors, the staining intensity was de�ned as the percentage of area covered by positive cells (0–100) density of positive cells in given (0–3). FCER1G and CD68 expression into TNM staging assessing patients with different prognoses following surgery for ccRCC. Immunohistochemistry an in with CD68 FCER1G. ccRCC,