Abstract No. 5 ▪ FEATURED ABSTRACT Adjuvant Macrophage Repolarization to M1 Phenotype Augments Post-Ablation Local Tumor Control and Improves Overall Survival in a Murine Model of Bladder Tumors

Abstract

To investigate the impact of adjuvant macrophage repolarization to inflammatory M1 phenotype on local tumor control and overall survival following irreversible electroporation (IRE) of murine bladder tumors. Mouse bladder cancer cell line (MB49) was used to induce subcutaneous tumors in C57BL/6 (n = 8/group) or NOD-SCID (n = 10/group) mice. Animals were randomized to receive (i) IRE, (ii) supramolecular nanotherapeutic for M1 repolarization (SNT), (iii) IRE+SNT or (iv) sham control. Mice assigned to IRE treatment received electric pulses (875 or 1500V/cm for C57BL/6 mice and 875V/cm for NOD-SCID mice, 90 pulses, 100 us duration) with tweezer electrodes. SNT treatment was administered by intravenous injection (40 mg/kg, 1,3- and 5-days post treatment). Mice were sacrificed at Day 1 or 6 for flow cytometry following IRE or were kept under observation in a survival study. Flow cytometry for macrophage markers (M1: CD80, M2: CD206) and T Cell markers (CD8, CD4 and FoxP3) from tumor samples was performed. Tumor samples underwent immunohistochemistry for cell death, macrophages (F4/80) or T cell markers. Local tumor volume and overall survival were evaluated and compared between cohorts and mouse strains to understand cross-interactions with adaptive immunity. Ablation of subcutaneous tumor with IRE stimulated robust macrophage infiltration that uniformly exhibited an immunosuppressive M2 phenotype at Day 3 and 5. SNT effectively repolarized macrophages at the site of ablation to M1 phenotype, with corresponding change in gene expression of NOS2 and IL-6. There was significantly higher population of CD80+ cells and reduced CD206+ cells at Day 1. Adjuvant SNT did not alter CD4+ and CD8+ cells but greatly reduced FoxP3+ population in vivo. IRE+SNT treated mice had greater reduction in local tumor growth, reduced lung metastases and high overall survival. These findings were reduced in NOD-SCID mice, indicating contribution of adaptive immunity under conditions of M1 repolarization. Repolarization of macrophages attracted to the site of ablation to a M1 phenotype promotes local tumor control, reducing the development of metastatic disease and prolonging survival in a mouse model of bladder cancer.