Identification of Immune Cell Infiltration in Murine Pheochromocytoma during Combined Mannan-BAM, TLR Ligand, and Anti-CD40 Antibody-Based Immunotherapy

SIMPLE SUMMARY: Multiple types of primary tumors and metastases that present with very little if any immune cell infiltration (so-called immunologically “cold” tumors) do not respond to current immunotherapies. In this study, we show that recently developed intratumoral application-based immunotherapy using mannan-BAM, TLR ligands, and anti-CD40 antibody (MBTA therapy) efficiently suppresses tumor growth in a murine bilateral pheochromocytoma model. Moreover, MBTA therapy increases the recruitment of innate immune cells followed by adaptive immune cells not only to primary (injected) tumors but also distal (non-injected) tumors. We also demonstrated that after successful MBTA therapy of subcutaneous pheochromocytoma, long-term immunological memory is driven by CD4(+) T cells. Taken together, this study helps to better understand the systemic effect of MBTA therapy and its use for tumor and metastasis reduction or even elimination. ABSTRACT: Immunotherapy has become an essential component in cancer treatment. However, the majority of solid metastatic cancers, such as pheochromocytoma, are resistant to this approach. Therefore, understanding immune cell composition in primary and distant metastatic tumors is important for therapeutic intervention and diagnostics. Combined mannan-BAM, TLR ligand, and anti-CD40 antibody-based intratumoral immunotherapy (MBTA therapy) previously resulted in the complete eradication of murine subcutaneous pheochromocytoma and demonstrated a systemic antitumor immune response in a metastatic model. Here, we further evaluated this systemic effect using a bilateral pheochromocytoma model, performing MBTA therapy through injection into the primary tumor and using distant (non-injected) tumors to monitor size changes and detailed immune cell infiltration. MBTA therapy suppressed the growth of not only injected but also distal tumors and prolonged MBTA-treated mice survival. Our flow cytometry analysis showed that MBTA therapy led to increased recruitment of innate and adaptive immune cells in both tumors and the spleen. Moreover, adoptive CD4(+) T cell transfer from successfully MBTA-treated mice (i.e., subcutaneous pheochromocytoma) demonstrates the importance of these cells in long-term immunological memory. In summary, this study unravels further details on the systemic effect of MBTA therapy and its use for tumor and metastasis reduction or even elimination.