Abstract Tumor-associated macrophages (TAMs) are a dominant immunosuppressive component of the tumor microenvironment (TME) in many solid cancers, including Lewis lung carcinoma (LLC). While immune checkpoint inhibitors (ICIs) primarily target adaptive immunity, the potential for small molecule checkpoint blockade to modulate the myeloid compartment remains poorly defined. Here, we evaluated the effects of a single-agent small molecule ICI on TAM phenotype and function in the LLC model, integrating molecular imaging with immune profiling. C57BL/6 mice bearing subcutaneous LLC tumors were treated with SRI-42475, a PD-1 targeting small molecule. To noninvasively monitor TAM polarization, longitudinal imaging was performed using a radiolabeled CD206-targeted probe before and after treatment. Imaging allowed for quantification of reduction of CD206 signal in treated tumors, consistent with a loss of M2-like macrophages. Flow cytometry at termination of the study provided an orthogonal assessment of therapy-induced shifts, including decreased expression of CD206 and increased expression of MHC-II and CD86, which indicates a reprogramming toward a pro-inflammatory phenotype. Macrophage remodeling was accompanied by enhanced infiltration and activation of CD8+ T cells within the TME. These findings demonstrate that small molecule checkpoint inhibition can reprogram the myeloid landscape in the absence of additional myeloid-targeting agents. Moreover, CD206-targeted imaging provides a valuable noninvasive biomarker of TAM dynamics and therapeutic response. Together, these results support further investigation of innate immune modulation by small molecule ICIs and highlight the translational utility of macrophage-directed imaging in immunotherapy. Citation Format: Rebecca J. Boohaker, Suzanne E. Lapi, Anna G. Sorace, Chloe Demellier, Volken Tekin, John Tillotson. Macrophage Reprogramming and Immune Checkpoint Blockade Efficacy in the Lewis Lung Carcinoma Model: Utility of longitudinal CD206-Targeted Molecular Imaging [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C048.
