Fig 2: MIF silencing in the tumor cells reduces sarcoma growth(A) Survival analysis of human soft tissue sarcoma in the TCGA, stratified by MIF mRNA expression level.(B) Western blot verification of MIF protein expression in mouse and human sarcoma cells.(C) qRT-PCR analysis of Mif mRNA expression levels in normoxic (20% O2) or hypoxic (3% O2) culture conditions.(D) Schematic representation of the generation of MIF-KO and KD sarcoma models and assessment of MIF KO or KD by western blot.(E) Relative growth in vivo of MIF-WT (sgCTR) and MIF-KO (sgMif) sarcoma cells, represented as tumor mass at day 21 (left panel) and relative proportion of total digested tumor that was sarcoma cells (right panel).(F) Relative growth in vivo of MIF-WT (shSCR) and MIF-KD (shMif) sarcoma cells, represented as tumor mass at day 21 (left panel) and relative proportion of total digested tumor that was sarcoma cells (right panel).(G) Relative expression of Cd74, Cxcr2, Cd44, and Cxcr4 in dsRed+ tumor cells, CD74+ myeloid cells, and CD3+ T cells. All cells were FACS sorted from whole mouse tumors.(H) Relative proliferation in vitro of MIF-WT (shSCR) and MIF-KD (shMif) sarcoma cells (left) and of MIF-WT (sgCTR) and MIF-KO (sgMif) sarcoma cells (right).(I) Relative capacity of spheroid formation for the MIF-WT (sgCTR) and MIF-KO (sgMif) sarcoma cells in vitro.Unless otherwise indicated, results are presented as mean ± SEM and results are compared by Student’s t test. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S3.

Fig 3: 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.

Fig 4: Single-cell transcriptomic profiling of the sarcoma-associated myeloid cells(A) Schematic representation of the experimental design for scRNA-seq analysis (top panel). t-distributed stochastic neighbor embedding (t-SNE) depicting the main immune cell types of the sarcoma mass of six murine p53KOCcne1+ UPS tumors (left bottom panel). Markers of major immune clusters are overlaid on the t-SNE (right bottom panel).(B) t-SNE derived from sub-clustering of mononuclear phagocytes only (left panel). CL1–4 refer to clusters 1–4 as described in the text. Key myeloid genes broadly or differentially expressed by these cell subtypes are overlaid on the t-SNE (right panel). See also Table S1.(C) Expression of top marker genes differentially expressed by the myeloid cell clusters.(D) Circos plot of intercellular MIF signaling between all cell types in murine UPS tumors, with directionality of signaling indicated by arrowheads. The primary sources of Mif signaling are sarcoma cells (red arrows), stromal cells, and Cd36/Spp1 MΦ, while the primary targets of Mif signaling are cells expressing Cd74 along with Cxcr4 and/or Cd44: inflammatory mono/MΦ, MHC class II high MΦ, Mrc1/Cx3cr1 MΦ, and monocytic dendritic cells (moDCs).(E) Expression of Mif and its receptor and co-receptor genes Cd44, Cd74, Cxcr2, and Cxcr4 by tumor or myeloid cells (right panel).See also Figure S2 and Table S1.

Fig 5: rMIF mitigated ferroptosis during H/R by increasing GSH content independent of GPX4.A Cell viability in different groups, with rMIF added at different time points (n = 6). B Cell viability in different groups after upregulation and downregulation of MIF (n = 6). C Free iron ions content of HK-2 cells in different groups after upregulation and downregulation of MIF (n = 6). D MDA content of HK-2 cells in different groups after upregulation and downregulation of MIF (n = 6). E Representative micrographs of C11 BODIPY immunofluorescence in HK-2 cells after upregulation and downregulation of MIF (n = 3). The scale bar represents 20 μm. F Representative image taken by TEM of mitochondria in HK-2 cells after upregulation and downregulation of MIF (n = 3). The scale bar represents 2 μm. G GSH content of HK-2 cells in different groups after upregulation and downregulation of MIF (n = 6). H Western blot results of P-AMPK and GPX4. (n = 3). Data are presented as means ± SD. Statistical analysis: one-way ANOVA with Tukey’s test for cell viability, Fe2+, MDA, GSH, and Western blots (n = 3–6 per group). * indicates p < 0.05, ** indicates p < 0.001.