Clinical implications of T-Cells CX3CR1+, Toll-like Receptor 4 signaling pathway, and immune checkpoints in Non-Muscle Invasive Bladder Cancer

Abstract

Background: This study characterized and compared the molecular profiles of CX3C chemokine receptor 1 (CX3CR1, a marker of T-cell differentiation), Toll-like receptor 4 (TLR4)-mediated interferon signaling pathway, and immune checkpoints in the different histological stages of non-muscle invasive bladder cancer (NMIBC), aiming the investigation of these biomarkers as a criterion of clinical response to immunotherapy. Methods: Seventy-five formalin-fixed paraffin-embedded samples of bladder were obtained from 34 to 96-year-old patients (mean 65 years) with NMIBC diagnosis in University of Campinas (UNICAMP) and Paulinia Municipal Hospital/ Brazil. Subsequently, the samples were divided into 3 groups (n= 25 samples per group): pTis group, high-grade pTa group, and pT1 group; and submitted to immunohistochemistry analysis: TLR4-mediated IFN-γ production signaling pathway (TRIF, TBK1, IRF-3, IFN-γ), CX3CR1+CD8+ T-cells, immune checkpoints (PD-1/PD-L1 and CTLA-4), and regulatory T (Treg) cells (FOXP3). The retrospective anonymous study was approved by the local ethics committee (Clinical Trial: RBR-6swqd2). Results: pTis group showed the lowest activation of TLR4-mediated IFN-γ signaling pathway when compared (p<0.01) to high-grade pTa and pT1 groups. Both the immunoreaction intensity and positive cells percentage were lower (p<0.01) for TLR4, TRIF, IRF-3, and IFN-γ in the pTis group with respect to other groups. No statistical difference was found between high-grade pTa and pT1 groups for these biomarkers. Likewise, CX3CR1 immunoreactivities were remarkably lower (p<0.01) in the pTis group in comparison with high-grade pTa and pT1 groups, which did not show statistical differences between them. Furthermore, immune checkpoints (PD-1/PD-L1 and CTLA4) and FOXP3+ Treg cells immunoreactivities were significantly higher (p<0.01) in the high-grade pTa and pT1 compared to the pTis group. Conclusions: Our data demonstrated that pTis stage was characterized by an immunosuppressive microenvironment in comparison with pTa and pT1 stages, showing decreased TLR4-mediated interferon signaling pathway and low activation of CX3CR1+CD8+ T-cells; which implies in low sensitivity to immunotherapy. The larger number of FOXP3+ Treg cells in pTa and pT1 was correlated with intensified immune checkpoints immunoreactivities, indicating higher sensitivity to immunotherapy. Finally, these biomarkers may be useful in the clinical management of patients with NMIBC.