High-throughput heterospheroid-based screening identifies drugs that reprogram tumor-associated macrophages

The tumor microenvironment (TME) provides a niche for immune evasion and immunotherapy resistance, in part, by recruiting pro-tumor M2-like macrophages. In the present study, using heterospheroids consisting of cancer cells and macrophages, we identified TAM activators, which are compounds that reprogram M2-like tumor-associated macrophages (TAMs) toward the antitumor M1-like phenotype. THP-1- or human peripheral monocyte-derived macrophages were co-cultured with liver cancer cells in an ultra-low attachment dish to generate heterospheroids. Cell surface marker expression and macrophage infiltration into the heterospheroids were assessed by flow cytometry and fluorescence microscopy, respectively. Lipopolysaccharide (LPS) and interferon-{gamma} (IFN{gamma})-induced M1 marker expression was observed on the macrophages in the homospheroids; however, this induction was suppressed in heterospheroids. Microscopic imaging revealed that macrophage infiltration into the heterospheroids was decreased in the presence of LPS and IFN{gamma}, which prompted us to develop a high-content imaging screen. We identified two compounds [alprostadil (prostaglandin E1) and HX531] with TAM-activating activity. RNA-seq analysis revealed that HX531 modulated the immune and IFN response in cancer cells and cell division in macrophages. Moreover, alprostadil promoted the M1-like polarization of TAMs, increased tumor-infiltrating CD8+ T cells, and enhanced anti-PD-1 antibody therapeutic efficacy in a syngeneic mouse xenograft model. In conclusion, the heterospheroid culture recapitulates the immunosuppressive TME, which prevents the M1 polarization of TAMs. It provides a new platform for screening TAM activators and will enable the development of novel cancer immunotherapeutics when combined with high-content imaging analysis.