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]

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, with or without the silencing of tumor-expressed MIF. To see how tumor Mif expression affected the composition of the immune microenvironment, p53-/- Ccne1+ mouse sarcoma cells were first transduced with Cas9 and either a control sgRNA (sgCTR), or a guide targeting Mif (sgMif). The MIF-KO and MIF-WT sarcoma cells were transplanted subcutaneously on polyurethane scaffolds into 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 hashtagged using sequenceable oligonucleotides in order to later identify cells as coming from either MIF-KO tumors or MIF-WT tumors, pooled together, and captured on the 10X Genomics Chromium platform for single-cell 3’ RNA sequencing, using two reactions to target ~20,000 cells total.