<p>Tumor-associated macrophages (TAMs) play a critical role in the progression of various cancers, including chondrosarcoma, where they can constitute up to 50% of the tumor mass. In chondrosarcoma, TAMs are predominantly of the M2-like phenotype and are linked to more invasive and higher-grade disease. Despite the challenge of chondrosarcoma’s chemo- and radio-resistance due to its slow cell division and unique extracellular matrix, targeting TAMs has emerged as a promising strategy. Therapeutic approaches include inhibiting TAMs recruitment, reprogramming TAMs to a tumoricidal M1 phenotype, and depleting TAMs. Current clinical studies are exploring combinations of TAMs-targeting agents with chemotherapy in many cancers, and some agent like mifamurtide, a TLR4 agonist, is already used with chemotherapy in cancer treatment, for pediatric non-metastatic osteosarcoma. Recent preclinical studies have shown that targeting TAMs in chondrosarcoma can slow tumor growth but no preclinical data shown the relevance of immunomodulator and chemotherapy co-treatment. Our research further evaluates the potential mifamurtide and chemotherapy co-treatment using a 3D co-culture model and a murine xenograft model of chondrosarcoma. Our findings suggest that mifamurtide enhances the chemosensitivity of CH2879/THP-1 spheroids. In the CH2879 xenograft model, its combination with doxorubicin led to improved antitumoral efficacy and a decrease in intermediate-stage tumor-associated macrophages. These results highlight the importance of TAMs in modulating treatment response. This study provides a preclinical proof of concept for the efficacy of combining mifamurtide with doxorubicin in managing chondrosarcoma, highlighting the potential of immunomodulator and chemotherapy co-treatment in improving treatment outcomes.</p>