Single-cell RNA-seq analysis of a soft-tissue sarcoma model reveals the critical role of tumor expressed MIF in shaping macrophage heterogeneity. [bulk RNA-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 bulk RNA-seq of sarcoma cells sorted from whole mouse tumors. To see how tumor Mif expression affected cell-autonomous pathways, p53-/- Ccne1+ mouse sarcoma cells were first transduced with (i) Cas9 and either a control sgRNA (sgSCR) or a guide targeting Mif (sgMif) in the case of the knockout experiment, or with (i) Cas9 and either a control shRNA (shSCR) or one targeting Mif (shMif), in the case of the knockdown experiment. The MIF-KO/KD and MIF-WT sarcoma cells were transplanted subcutaneously on polyurethane scaffolds into recipient mice, and allowed to grow until reaching ~1cm3 in size. Tumors were extracted, digested into single-cell suspensions, FACS-purified on the basis of a fluorescent marker, and submitted to RNA-seq.