Fig 2: Macrophages promote sarcoma growth(A) Flow cytometry of the main immune populations infiltrating STS, gated in CD45+ (n = 3 independent human sarcomas). DDLPS, dedifferentiated liposarcoma; GIST, gastrointestinal stromal tumor; LMS, leiomyosarcoma.(B) Schematic representation of the UPS mouse models used (left) and analysis of immune cells that populate the murine sarcoma mass (right).(C) Schematic representation of the treatment schedule administered to test the effect of anti-CSF1R on sarcoma growth.(D) Relative proportion of macrophages (CD45+ F4/80+) infiltrating tumors treated with IgG control or anti-CSF1R.(E) Relative tumor weight after treatment with IgG control or anti-CSF1R (left) and relative % of dsRED+ tumor cells in flow cytometry (right).(F) CD8+ and CD4+ T cells in proportion to all CD45+ cells infiltrating tumors treated with IgG control or anti-CSF1R.(G) Schematic representation of the in vivo experiments to test the role of macrophages in promoting sarcoma growth. Bone-marrow-derived macrophages (BMDMs) were generated by macrophage colony stimulating factor (M-CSF) stimulation in vitro and then co-injected with tumor cells. The relative tumor weights (tumor cells alone versus co-injected with BMDMs) are shown on the right.Unless otherwise indicated, results are presented as mean ± SEM, and results are compared by Student’s t test. *p < 0.05 and **p < 0.01. ns, not significant. See also Figure S1.

Fig 3: RNA and antibody-based epitope expression in brain infiltrating leukocytes(A) Example of gating leukocyte populations using CITE antibodies. Briefly, CNS native myeloid cells (microglia and BAM) were gated based on both populations lowly expressing CD45 and then differentiated from each other based on higher I-A-I-E and CD38 expression in BAM. Peripheral leukocytes were gated based on high CD45 expression. Within the peripheral leukocyte population: B cells were defined as having CD19 and B220 expression. T cells were defined based on high CD3 expression and low NK1.1 expression, then subpopulations further distinguished on the basis of CD4 and CD8 expression. Myeloid cells were distinguished from adaptive peripheral immune cells by high CD11b expression, and then further subdivided on the basis of Ly6C expression levels and Ly6G expression.(B) Example of gating leukocyte populations using CyTOF antibodies.(C) Biaxial plots of CITE epitope expression on the x axis and its corresponding mRNA expression on the y axis. Discordant expression of mRNA and protein can be observed.(D) UMAPs derived from CITE-seq data clustered by RNA transcription cluster color coded by transcriptional cluster (left) or canonical cell ID determined by CITE-antibody gating (right). While canonically identified cells generally group within the same cluster, there is appreciable dispersion of canonical cell types among clusters, indicating the utility in using CITE antibody tags rather than mRNA alone to identify canonical cell types.

Fig 4: MIF silencing in the tumor cells re-shapes the myeloid microenvironment(A) Relative proportions in flow cytometry of immune cells (CD45+) infiltrating MIF-WT (sgCTR) and MIF-KO (sgMIFif) sarcomas.(B) Relative proportions in flow cytometry of monocytes (CD45+LyC6+F4/80-) and macrophages (CD45+LyC6-F4/80+) infiltrating MIF-WT (sgSCR) and MIF-KO (sgMif) tumors.(C) Relative proportions in flow cytometry of CD4+ and CD8+ T lymphocytes infiltrating MIF-WT (sgCTR) and MIF-KO (sgMif) tumors.(D) Scoring the expression of four macrophage gene signatures (of the previously defined tumor-infiltrating macrophage subtypes; Figure 2) in macrophages coming from MIF-WT (sgCTR) or MIF-KO (sgMif) tumors, using Seurat’s Module Score method.(E) Pathway analysis of macrophages infiltrating tumors generated by MIF-WT (sgCTR) or MIF-KO (sgMif) tumor cells.(F) Differential expression of interferon-related genes in FACS-sorted myeloid cells (CD11b+) from the MIF-WT (sgCTR) and MIF-KO (sgMif) tumors.(G) Differential expression of genes related to activation and exhaustion of T cells in FACS-sorted CD8+ T cells from the MIF-WT (sgCTR) and MIF-KO (sgMif) tumors.Unless otherwise indicated, results are presented as mean ± SEM and results are compared by Student’s t test. *p < 0.05 and ***p < 0.001. See also Figure S4.