PD-1-Positive Tumor-Associated Macrophages Define Poor Clinical Outcomes in Patients With Muscle Invasive Bladder Cancer Through Potential CD68/PD-1 Complex Interactions

Abstract

Tumor-associated macrophages (TAMs) regulate tumor immunity. Previous studies have shown that the programmed cell death protein 1 (PD-1)-positive TAMs have an M2 macrophage phenotype. CD68 is a biomarker of TAMs and is considered to be a poor prognostic marker of several malignancies. Our results show that PD-1-positive TAMs can be a negative survival indicator in patients with muscle-invasive bladder cancer (MIBC), and that the mechanistic effects could result due to a combination of PD-1 and CD68 activity. We analyzed 22 immune cell types using data from 402 patients with MIBC from the TCGA database, and found that a high immune score and M2 TAMs were strongly associated with poor clinical outcomes in patients with MIBC. Further, we analyzed resected samples from 120 patients with MIBC and found that individuals with PD-1-positive TAMs showed a reduction in 5-year overall survival and disease-free survival. Additionally, PD-1-positive TAMs showed a significant association with higher programmed death-ligand 1 (PD-L1) expression, the Ki67 index, the pT stage and fewer CD8-positive T cells. Through the co-immunoprecipitation (co-IP) assay of THP-1 derived macrophages, we found that CD68 can bind to PD-1. The binding of CD68 and PD-1 can induce M2 polarization of THP-1 derived macrophages and promote cancer growth. The anti-CD68 treatment combined with peripheral blood mononuclear cells (PBMC) showed obvious synergy effects on inhibiting the proliferation of T24 cells. Together, these results indicate for the first time that CD68/PD-1 may be a novel target for the prognosis of patients with MIBC.

Keywords: CD68; PD-1; muscle-invasive bladder cancer; prognosis; tumor-associated macrophages.

Figure 1

(A) Illustration of LASSO coefficient profiles of 22 immune cell types. (B) Cross-validation for the LASSO regression model. (C) Kaplan-Meier analysis of OS in high and low immune score patients with MIBC in the TCGA cohort. (D) Kaplan-Meier analysis of DFS in patients with high and low immune scores with MIBC in the TCGA cohort. (E) Heatmap of the related immune cells with high and low immune scores.

Figure 2

(A) Kaplan–Meier analysis of OS in patients with MIBC with M2 TAMs in the TCGA cohort. (B) Kaplan–Meier analysis of DFS in patients with MIBC with M2 TAMs in the TCGA cohort. (C) Kaplan–Meier analysis of OS in patients with MIBC with M2 TAMs in the Shanghai General Hospital cohort. (D) Kaplan–Meier analysis of DFS in patients with MIBC with M2 TAMs in the Shanghai General Hospital cohort. (E) Forest plot showing the hazard ratios with a 95% confidence interval for OS in the TCGA cohort. (F) Forest plot showing the hazard ratios with a 95% confidence interval for DFS in the TCGA cohort. (G) Forest plot showing the hazard ratios with a 95% confidence interval for OS in the Shanghai General Hospital cohort. (H) Forest plot showing the hazard ratios with a 95% confidence interval for DFS in the Shanghai General Hospital cohort.

Figure 3

(A) Immunohistochemical staining of PD-1-positive TAMs (red arrow), PD-1-negative TAMs (purple arrow), strong PD-L1 and weak PD-L1 expression. (B) Kaplan-Meier analysis of OS in patients with MIBC with PD-1-positive TAMs in the Shanghai General Hospital cohort. (C) Kaplan-Meier analysis of DFS in patients with MIBC with PD-1-positive TAMs in the Shanghai General Hospital cohort. (D) PD-1-positive TAMs showed less CD8-postive T cells nearby. ***p < 0.001 (Student’s t-test). (E) The number of PD-1-positive TAMs showed a correlation with PD-L1 expression in the Shanghai General Hospital cohort.

Figure 4

(A) Immunofluorescence staining confirmation for the appearance of PD-1-positive TAMs. (B) mRNA expression of CD68 showed a correlation with PD-1 expression in the TCGA cohort. (C) mRNA expression of CD68 showed a correlation with PD-L1 expression in the TCGA cohort.

Figure 5

(A) Both THP-1 cells and THP-1 derived macrophages expressed PD-1 and CD68. T24 cells expressed PD-L1. (B) A co-IP assay showing the binding between CD68 and PD-1, hinting at the possibility of a CD68 and PD-1 interaction. (C) The binding of CD68 and PD-1 promoted THP-1 derived macrophages to M2 polarization whereas the blockage can reverse the process. (D) Molecular docking showing the interactions of CD68 and PD-1 through possible LAMP-like and IgV domains. SP indicates signal peptide and TM indicates transmembrane domain. (E) The cell viability assays of T24, THP-1 derived macrophages and PBMC co-culture experiments. T24 cell group and THP-1 derived macrophage group were the control groups. ***p < 0.001 (Student’s t-test).