Single-cell RNA-seq analysis of a soft-tissue sarcoma model reveals the critical role of tumor expressed MIF in shaping macrophage heterogeneity. [CITE-Seq 2]

The standard of care is unsuccessful to treat recurrent and aggressive soft-tissue sarcomas. Interventions aimed at targeting components of the tumor microenvironment have shown promise for many solid tumors, yet have been only marginally tested for sarcoma, partly because knowledge of the sarcoma microenvironment composition is limited. We employed single-cell RNA-sequencing to characterize the immune composition of an undifferentiated pleiomorphic sarcoma mouse model, showing that macrophages in the sarcoma mass exhibit distinct activation states. Sarcoma cells used the pleiotropic cytokine Macrophage Migration Inhibitory Factor (MIF) to interact with macrophages expressing the CD74 receptor to switch macrophages’ activation state and pro-tumorigenic potential. Blocking the expression of MIF in sarcoma cells favored the accumulation of macrophages with inflammatory and antigen-presenting profiles, hence reducing tumor growth. These data may pave the way for testing new therapies aimed at re-shaping the sarcoma microenvironment, in combination with the standard of care. We performed scRNA sequencing to profile the major cell types present in the microenvironment of a murine sarcoma model. We transplanted p53-/- Ccne1+ mouse sarcoma cells subcutaneously on polyurethane scaffolds into 6 recipient mice, and allowed to grow until reaching ~1cm3 in size. The tumors were extracted, digested to single-cell suspensions, and partially enriched for CD45+ immune cells. The samples were individually hashtagged using 6 unique sequenceable oligonucleotides, pooled together in equal proportions, and were captured on the 10X Genomics Chromium platform for single-cell 3’ RNA sequencing, using 2 reactions to target ~10,000 cells total.