Extended Data Fig. 5: β-glucosylceramide, rather than cholesterol, is released by tumor cells to mediate the protumorigenic polarization in macrophages.
a, b, Quantification results of Cholesterol (a) (n = 6 per group) and fatty acids (b) (n = 4 per group) on YUMM1.7 CM prior (Yumm1.7) and after treatment with lipid removal reagent (Y1.7 w.o. Lipids). Data are pooled from two independent experiments. c, Immunoblot of YUMM1.7 cells stably transduced with lentivirus expressing short hairpin RNA against scramble or HMGCR sequence. d, qPCR analysis of indicated genes in BMDMs exposed to CM isolated from YUMM1.7 shCTRL or from YUMM1.7 shHMGCR (n = 3). Data are representative of three independent experiments. e, qPCR analysis of mRNA expression of indicated genes in BMDMs treated with CM in the absence or presence of 1μg/ml α-CD36 antibody (n = 3 per group). Data are representative results of two independent experiments. f, Quantification of BODIPY staining in BMDM cultured with regular culture medium (Ctrl) or with YUMM1.7 CM in the absence or presence of 5μg/ml α-Mincle antibody; Ctrl (n = 9), CM (n = 9), CMα-Mincle (n = 8). Data are pooled from three independent experiments. g, h, Immunoblot and quantification of the indicated proteins (g) (n = 4 per group) and qPCR analysis of mRNA expression of ARG1 and MRC1 (h) of WT or Mincle-KO BMDMs exposed to regular culture medium (Ctrl) or or with YUMM1.7 CM for 18 h (n = 9 per group). Data are pooled from three independent experiments. i, Proliferation of CFSE-labeled T cells activated with anti-CD3 and anti-CD28 alone or co-cultured with WT or Mincle-KO BMDMs previously treated with CM in a ratio 2:1 for 72 h; T cells (n = 6), WT (n = 8), ΚΟ (n = 9). Data are pooled from three independent experiments. j, Quantification result of indicated β-glucosylceramide levels from CM derived from YUMM1.7 shCTRL and YUMM1.7 shUGCG cells (n = 3 per group). k, qPCR analysis of the indicated genes in BMDMs treated with Ctrl or CM derived from YUMM1.7 shCTRL and YUMM1.7 shUGCG cells alone or in presence of αMincle antibody (5μg/ml) (n = 3). Data are representative of two independent experiments. l–m, Quantification result of indicated β-glucosylceramide levels in serum and tumor interstitital fluid (TIF) isolated from YUMM1.7 melanoma-bearing mice (l) or Braf/Pten melanoma-bearing mice (m) (n = 5 per group). All data are mean ± s.e.m and were analyzed by two-tailed, unpaired Student’s t-test (a–f), paired Student’s t-test (g, j, l–m), one-way ANOVA with Sidak’s multiple comparison test (i), one-way ANOVA with Tukey’s multiple comparison test (k).
