Fig. 4: GLUT1 inhibition targets glycolytic CAF and promotes intratumoral T cell infiltration.

a Glucose uptake of glyCAF quantified by flow cytometry of 2-NBDG (FITC) MFI relative to non-glyCAF (n = 5 mice). b Multiplex immunofluorescence staining of CAF marker CD90 (red) glyCAF marker CD73 (green), and CD3 (white) at the tumor margin. Images are representative of two independent experiments with n = 3 mice. c Quantifications of the number of CD73⁺ CD90⁺ cells (glyCAF) in ~1 mm² ROIs encompassing the tumor margin (Ctrl: n = 17 ROI, GLUT1i: n = 14 ROI from n = 3 mice). d Proportion of CD73⁺ glyCAF (dsRED⁻, CD45⁻, CD31⁻, CD90⁺, CD73⁺) by flow cytometry (n = 10 mice). e Proportion of CD90⁺ CAF (dsRED⁻, CD45⁻, CD31⁻, CD90⁺) by flow cytometry (n = 10 mice). f Averaged expression of mCAF, iCAF, glyCAF, and apCAF signature genes (top 10 DEG per cluster) in CAFs from mouse tumors treated with GLUT1i or control (NT) (n = 4 mice). g Immunofluorescence staining of CD8⁺ cells (green) in Ctrl and GLUT1i treated Ccne1⁺ tumors (Scale bars 500 μm (top) and 100 μm (bottom)). Images are representative of two independent experiments with n = 3 mice. h Quantifications of CD8⁺ cells/field in ROIs encompassing the tumor margin or the tumor parenchyma in Ctrl and GLUT1i treated Ccne1⁺ tumors (n = 3 mice) and i relative proportions of tumor infiltrating CD8⁺ T cells (CD45⁺CD8⁺) cells determined by flow cytometry (n = 10 mice). j Quantifications of CD8⁺ cells/field in ROIs encompassing the tumor margin or the tumor parenchyma in WT and Glut1-KD tumors (Ctrl, Parenchyma: n = 6 ROI, Margin: n = 7 ROI; GLUT1i, Parenchyma: n = 8, Margin n = 8 ROIs from n = 3 mice) and k relative proportions of tumor infiltrating CD8⁺ T cells (CD45⁺CD8⁺) cells determined by flow cytometry (n = 8 mice). l Multiplex immunofluorescence staining of CAF marker CD90 (red), glyCAF marker CD73 (green), and CD8 (white) at the tumor margin. White arrows illustrate association between glyCAF and CD8⁺ T cells. Scale bars 100 μm. Images representative of three independent experiments with n = 3 mice. m Quantifications of the distance to the nearest CD8⁺ T cell using multiplex immunohistochemistry images. Dots represent individual cell-cell interactions (n = 24 CD73⁺ CD90⁺, n = 17 CD73⁻ CD90⁺, n = 24 CSF1R⁺) acquired from n = 3 mice each group. P-values determined by one-way ANOVA with Tukey’s multiple comparisons test. Unless otherwise indicated, results are presented as mean ± SEM and p-values are derived by a two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.