The combination therapy of Everolimus and anti-PD-1 improves the antitumor effect by regulating CD8+ T cells in bladder cancer

Abstract

This study aimed to investigate the efficacy of Everolimus (EVE) in combination with immune checkpoint inhibitors (ICIs) in bladder cancer treatment and the underlying mechanisms. In vitro, MB49 cells were exposed to gradient concentrations (0 nM-100 nM) of EVE for 48 h, to investigate the cell viability and cell proliferative potential. In vivo, we applied a subcutaneous tumor mouse model of bladder cancer and the mice were treated with EVE monotherapy (different doses) or in combination with anti-programmed cell death protein 1 (PD-1) agents to study the impacts on tumor growth and explore the immune mechanism. The influences of treatments on peripheral immune profiles and tumor immune microenvironment were also discussed. EVE could inhibit the growth of MB49 cells in vitro. Though high-dose EVE monotherapy could induce tumor regression in vivo, it also contributed to immunosuppression. High-dose EVE inhibited the expression of PD-L1 by inhibiting Th1 cytokine secretion, while combined therapy with PD-1 inhibitors showed no extra profit. Low-dose EVE in combination with PD-1 inhibitors could effectively suppress tumor growth by increasing periphery CD8⁺ T cell frequency and GZMB⁺ CD8⁺ T cell frequency in the tumor microenvironment. High-dose EVE monotherapy induced tumor regression, but with immunosuppression to some content. Combination therapy with low-dose EVE and PD-1 inhibitor could effectively inhibit the growth of bladder tumors by enhancing the antitumor immunity of CD8⁺ T cells in both periphery and tumor microenvironment.

Keywords: Bladder cancer; Everolimus (EVE); Immune checkpoint inhibitors (ICIs); Mammalian target of rapamycin (mTOR); Programmed cell death ligand 1 (PD-L1).