Sex differences in renal cell carcinoma: a single-cell analysis reveals exhausted CD8+ T-cells highly infiltrated in males

Abstract

Background: Although sex bias has been reported in the development and progression of renal cell carcinoma (RCC), the underlying mechanisms remain enigmatic. Here, we investigated the sex differences in the tumor microenvironment (TME) of RCC and explored a promising combination drug regimen to enhance the efficacy of immunotherapy.

Methods: Single-cell RNA sequencing (scRNA-seq) data from four published datasets were analyzed to investigate the sex differences in RCC patients, and tumor tissues were collected to validate the sex differences using multiplex immunofluorescence (MxIF) and flow cytometry (FCM). The function of the androgen-androgen receptor axis in sex differences was explored in vivo and in vitro experiments.

Results: Our analysis of scRNA-seq data from 220,156 cells, as well as MxIF and FCM assays, revealed that CD8⁺ T-cells infiltrated highly in the TME of male RCC, but were mostly in an exhausted and dysfunctional state. In vitro and in vivo experiments indicated that the dysfunction and exhaustion of CD8⁺ T-cells in male TME were induced by androgen. Clinically, higher serum androgen was significantly associated with a worse prognosis in male RCC patients receiving immunotherapy. Androgen receptor inhibitors could activate tumor-infiltrating CD8⁺ T-cells and enhance the efficacy of immunotherapy of RCC in vivo.

Conclusions: Our study delineated the difference in TME between male and female patients with RCC, and demonstrated that the androgen-androgen receptor axis plays an important role in immunosuppression in male RCC. Our findings suggest that androgen receptor inhibitors in combination with immunotherapy may be a promising treatment option for male RCC patients.

Keywords: Androgen; Exhaustion; Immunotherapy; Renal cell carcinoma; Sex bias; Single-cell transcriptomics; T-cell.

Fig. 1

scRNA-seq profiling of TME of male and female RCC patients. A Sex difference of incidence and clinical stages of 1102 RCC patients who underwent nephrectomy from 1999 to 2020 at SYSUCC. B Meta-analysis of sex-specific pooled hazard ratios in various immunotherapy RCTs. C A schematic representation of scRNA-seq profiling of tumors and adjacent normal kidneys in male and female patients with RCC. D The UMAP plot shows the annotation and color codes for different cell types in tumors and adjacent normal kidney ecosystems. E Heatmap showing the expression of marker genes in the indicated cell types. F, G Histograms and boxplots illustrating the percentage of cell types in different groups. HR: hazard ratio; OS: overall survival; PFS: progression-free survival; RCC: renal cell carcinoma; RCTs: randomized controlled trials; scRNA-seq: single-cell RNA sequencing; SYSUCC: Sun Yat-sen University Cancer Center; UMAP: uniform manifold approximation and projection

Fig. 2

Identification and characterization of epithelial cells and malignant cells in males and females. A UMAP plot representing the subtypes of epithelial cells and malignant cells from male and female samples. B Heatmap showing the expression of marker genes in the epithelial cells and malignant cells. C Histogram showing the percentage of epithelial cells and malignant cells in samples and groups. D GSVA analysis showing the enrichment of specific pathways in malignant cells based on the HALLMARK gene set. E The volcano plot showing DEGs between male (blue dots) and female (red dots) malignant cells. F GSEA of hallmark interferon-γ response and gene ontology antigen presentation and processing via MHC class I signatures in malignant cells between male and female. G The correlation analysis between androgen response score and EMT, angiogenesis and TGF-β score in male malignant cells. DEGs: differential genes expression; EMT: epithelial to mesenchymal transition; GSEA: gene set enrichment analysis; GSVA: gene set variation analysis; TGF-β: transforming growth factor-β; UMAP: uniform manifold approximation and projection

Fig. 3

Characteristics of infiltrating T/NK cells in males and females. A The UMAP plot showing different T/NK cell subtypes, colored and labeled by cell type. B The volcano plot showing DEGs between males (blue dots) and females (red dots) in different T-cells subtypes. C Boxplots illustrating the percentage of infiltrating CD4⁺ and CD8⁺ T-cells in the tumor and adjacent normal kidneys of males and females. D Boxplots illustrating the percentage of infiltrating CD8⁺ T-cell subtypes in tumor and adjacent normal kidneys of males and females. E Differentially enriched pathways were scored per cell by GSVA in tumor-infiltrating CD8⁺ T-cells between males and females. F MxIF images of male and female tumors demonstrating tumor-infiltrating CD3⁺CD8⁺PD1⁺ T-cells. G The pie charts showing the percentage of CD8⁺ T-cell infiltration types. H Violin plots demonstrating CD3⁺, CD3⁺ CD8⁺, CD3⁺ CD8⁺ PD1⁺ infiltration levels in MxIF of tumors from males (n = 45) and females (n = 15). DEGs: differential gene expression; GSVA: gene set variation analysis; MxIF: multiplex immunofluorescence; UMAP: uniform manifold approximation and projection

Fig. 4

Analysis of infiltrating CD8⁺ T-cell transition states in male and female samples. A Pseudotime-ordered analysis and density-distribution map of infiltrating CD8⁺ T-cells in tumors and adjacent normal kidneys of males and females. B Two-dimensional plots showing the change of expression scores for genes related to T-cell exhaustion and dysfunction along with the pseudotime. C Two-dimensional plots showing the dynamic expression of exhaustion and cytotoxicity genes during the CD8⁺ T-cell transitions along the pseudotime in male (blue) and female (red) samples. D The exhausted status and cytotoxic function of tumor-infiltrating CD8⁺ T-cells assessed by flow cytometry in male (n = 10) and female tumor (n = 10) samples

Fig. 5

Androgen contributes to the dysfunction and exhaustion of CD8⁺ T-cells. A Correlation analysis between T-cells status score and androgen response score. B The FCM of human CD8⁺ T-cells in androgen culture. C CD8⁺ T-cells toxicity of OT-I mice in androgen culture. D IHC and ELISA analysis of 44 RCC patients receiving immunotherapy in SYSUCC (males: 32; females: 12). E Prognostic analysis of male RCC patients in different serum androgen after immunotherapy in SYSUCC. ELISA: enzyme-linked immunosorbent assay; FCM: flow cytometry; IHC: immunohistochemistry; HR: hazard ratio. RCC: renal cell carcinoma; SYSUCC: Sun Yat-sen University Cancer Center

Fig. 6

Androgen receptor inhibitors can enhance immunotherapy efficacy. A Flowchart of animal experiment in this study. B, C The tumor growth curves and tumor weight showing the effect of androgen and ENZ on tumor growth. D, E The expression of cytotoxicity and exhaustion markers of CD8⁺ T-cells were evaluated in mouse tumors by IHC. F Schematic representation showing the mechanism of androgen and androgen receptor inhibitors in RCC. ENZ: enzalutamide; IHC: immunohistochemistry; RCC: renal cell carcinoma