Tumor-associated macrophages (TAMs) represent more than 50% of the immune cells in breast tumors, even in immune dysregulated, ‘cold’ tumors. TAMs that express an M2-like phenotype promote tumor growth and create an immunosuppressive tumor microenvironment (TME). High levels of M2-like TAMs correlate with a poor prognosis. However, macrophages display phenotypic plasticity and can be repolarized from one phenotype to another through signaling molecules, such as cytokines. Macrophages that express an M1-like phenotype are inflammatory and can induce anti-tumor immunity. Administration of pro-inflammatory cytokines reprogram M2 TAMs toward an M1-like phenotype that can reduce tumor growth and stimulate an inflammatory response within the TME. However, systemic delivery of these inflammatory cytokines generates toxic, dose-limiting off target effects. This work seeks to develop a localized delivery approach that enables TAM polarization toward M1 programs while abrogating systemic toxicities. We have developed an injectable alginate cryogel (hydrogel fabricated at -20oC) to act as localized delivery depot for inflammatory cytokines and macrophage-specific chemokines to repolarize TAMs. To delay the release of our chemoattractant relative to our repolarizing cytokines, we are developing a second generation cryogel, loaded with free chemokines and poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulated with cytokines to delay their release. With our first generation cryogel (without nanoparticles), we used qRT-PCR, flow cytometry, Luminex panels, and single cell RNAseq to characterize the impact of treatment. For in vitro studies, bone marrow derived macrophages were extracted from FVB mice, while TAMs and tumor explants were extracted from the spontaneous mammary tumors of PyMT mice. It was found that our cryogel significantly increased M1 markers and decreased M2 markers in all three in vitro models.Peritumoral injection of the cryogel system into FVB female mice with orthotopic mammary tumors resulted in significantly suppressed tumor growth, an increase in T cell infiltration, and an increase in the M1:M2 ratio of TAMs. This activated immune response and elevated presence of T cells suggests that localized chemokine/cytokine treatment ‘primes’ the TME, potentially making it more susceptible to immunotherapies that rely on T cells, such as immune checkpoint blockade.We have concluded that our first-generation gel demonstrates the potential of priming the TME to slow tumor growth. Characterization of our second generation, nano-in-cryogel system is currently underway. Based on mathematical modeling, we hypothesize that the nano-in-cryogel will induce a more pronounced repolarization effect by allowing the chemoattractant to induce M2 TAM migration prior to the release of the inflammatory, reprogramming cytokines from the PLGA nanoparticles. Citation Format: Sydney Ros Henriques, Ori Z. Chalom, Evan B. Glass, Sohini Roy, Abigail E. Manning, Benjamin C. Hacker, Marjan Rafat, Laura C. Kennedy, Fiona E. Yull, Young J. Kim, Todd D. Giorgio. Tumor-associated macrophage reprogramming by novel nano-in-cryogel biomaterials for breast cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2719.