Abstract 2058: Effect of route of Bacillus-Calmette Guerin administration on systemic immune responses in a murine model of non-muscle invasive bladder cancer
Authors: Grossman Arielle , Atallah Aline , Cotechini Tiziana , Pare Jean-Francois , Siemens Robert , Graham Charles H. 
Published: June 2022

Abstract
Background: Bladder cancer is the 5th most common cancer worldwide, with the most common type being non-muscle invasive bladder cancer (NMIBC). Treatment for high grade NMIBC includes resection of the bladder tumor followed by repeated intravesical administration of Bacillus-Calmette Guérin (BCG), the live-attenuated form of Mycobacterium bovis. Unfortunately, 60-70% of patients will experience disease recurrence. Our current understanding is that BCG acts within the local bladder tumor microenvironment and draining lymph nodes, leading to the activation of a systemic immune response. Using a murine model of NMIBC, here we investigated whether different routes of BCG administration alter the systemic immune response to BCG and affect tumor size.
Methods: To establish syngeneic orthotopic bladder tumors in a mouse model, female C57Bl/6 mice were catheterized and 2.5 x 105 mouse bladder cancer cells (MB49) were instilled into the bladder. Beginning on day 7, mice received either intravesical, intravenous (IV) or intraperitoneal (IP) BCG (8 mg/ml) or saline once weekly for three weeks and bladders were imaged through ultrasound. At endpoint (day 23), tumors, tumor draining lymph nodes (tdLNs), spleen, and bone marrow were harvested and stored. Immune composition of the various lymphoid organs was evaluated using polychromatic flow cytometry. Ex vivo responsiveness to lipopolysaccharide (LPS) was assessed in bone marrow cells by measuring cytokine levels in culture supernatants.
Results: Mice treated with IV BCG had significantly smaller tumor volumes when compared to intravesical BCG treatment. Ex vivo LPS stimulation of bone marrow from BCG-treated mice, regardless of administration route, led to increased release of pro-inflammatory cytokines compared with saline controls. IP and IV BCG treatment skewed immune microenvironments of secondary lymphoid organs towards a cytotoxic phenotype compared with intravesical BCG treatment. Within the lymphoid compartment of tdLNs, the proportion of CD8+ T cells was significantly increased while the proportion of CD4+ T cells was significantly decreased in mice treated with IV and IP BCG compared with intravesical BCG. Conversely, the proportion of CD11b+ dendritic cells (DCs) was significantly increased in tdLNs from mice receiving intravesical BCG compared with BCG administered IV or IP. Within the spleen, the proportion of CD8+CD11b- DCs were significantly increased in the DC compartment following IP BCG treatment compared to other treatment routes.
Conclusion: This provides evidence that route of BCG administration affects the immune composition of secondary lymphoid organs that may be associated with anti-tumor responses. A better understanding of the mechanism of BCG immunotherapy will lead to novel approaches to optimize anti-tumor immune responses and, consequently, improved patient outcomes.
Citation Format: Arielle Grossman, Aline Atallah, Tiziana Cotechini, Jean-Francois Pare, Robert Siemens, Charles H. Graham. Effect of route of Bacillus-Calmette Guerin administration on systemic immune responses in a murine model of non-muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2058.