Tumor Immune Microenvironment in Response to Radiotherapy vs BCG in a Murine Model of Bladder Cancer

Abstract

The role of radiotherapy combined with immunotherapy in patients with non-muscle invasive bladder cancer (NMIBC) is currently being investigated. Radiation induced DNA damage triggering immunogenic tumor cell death and consequent immune responses in bladder tumor microenvironment is not fully understood. We compared local immune response following radiotherapy with BCG-induced immune response using a BBN murine model of urothelial carcinoma (UC). Female C57BL/6 control(no tumor, n=15) or BBN exposed mice(tumor, n=13) received radiotherapy(single external beam dose,15 Gy,n=10), intravesical BCG instillation (3 × 100μL of 1 × 10 8 CFU,n=9) or saline treatment (n=9). Tumor involvement was assessed by pathologic grading of bladder tissue and immune responses quantified using Nanostring Myeloid Innate Immunity Panel. Infiltrating Tcells were evaluated by CD4, CD8 and FOXP3 immunohistochemistry (IHC) and tissue macrophages quantified as M1 or M2 phenotype based on CD86 and CD206 IHC. No tumors were seen in BBN+BCG bladder tissues compared to BBN+saline or BBN+radiotherapy. Intravesical BCG instillation in BBN mice elevated cytosolic DNA sensing pathway as seen by increased mRNA levels of tmem173 (STING) that was consistent with elevated levels of downstream cytokines tnfa, infg, il6 and chemokine cxcl10 . mRNA expression of 3’exonuclease trex1 , that degrades 3′ ends of nicked DNA during immunogenic cell death, was elevated in BBN+BCG as well as BBN+radiation samples. BBN+BCG samples had increased CD8/FOXP3 and CD4/FOXP3 ratios assessed by IHC and increased M1 macrophage shift validated by mRNA expression and IHC. BCG instillation caused a robust proinflammatory immune response compared to radiation in murine UC possibly via STING-dependent induction of proinflammatory cytokines and chemokines concomitant with shift toward M1 macrophage polarization and increased Teff/Treg ratios. Elevated trex1 may degrade DNA damage as a direct response to radiation thereby inhibiting STING activation and cytokine response in radiated samples. These findings highlight the need to uncover mechanisms of radiation induced immune response in NMIBC to optimize utilization of immunotherapy with radiotherapy.