Figure 5. HFD induces distinct metabolic adaptations in MC38 tumor cells and CD8⁺ TILs.

(A-C, E-J) Analysis of RNA-sequencing data performed on cells sorted from day 12 MC38 tumors from CD-fed and HFD-fed animals.

(A) Principal component analysis of the top 400 genes with the largest variance from CD8⁺ TILs versus CD8⁺ T cells from the dLN in animals fed HFD or CD.

(B) Volcano plot comparing gene expression levels in CD8⁺ TILs from CD and HFD tumors. Genes with FDR-corrected p-value < 0.05 are highlighted. Dotted lines indicate 1.5-fold change.

(C) Volcano plot depicting differentially expressed metabolic genes in MC38-GFP tumor cells. Metabolic genes were defined as the union of the following GO gene subsets: GO:0006520 Cellular Amino Acid Metabolic Process, GO:0005975 Carbohydrate Metabolic Process, and GO0006629 Lipid Metabolic Process, or GO:0006099 Tricarboxylic acid cycle, excluding transcription factors. Dotted lines indicate 1.5-fold change.

(D) Phd3 expression in day 23 MC38 tumors measured by qPCR.

(E-F) Heatmaps showing relative expression in CD8⁺ TILs (E) and MC38 tumor cells (F) of genes that are significantly differently expressed between CD and HFD tumor cells (Phd3) or CD8⁺ TILs (Cherp, Dgat1, Elovl6, Ldlrap1).

(G-H) Average expression for genes involved in FAO from tumor cells (G) and CD8⁺ TILs (H).

(I-J) Heatmaps depicting glycolytic genes in CD8⁺ TILs (I) versus tumor cells (J).

(K-L) Ex vivo LipidTox neutral lipid staining in CD8⁺ TILs (K) and GFP⁺ MC38 cells (L) in day 10-14 tumors.

(M) Quantification of C16-BODIPY uptake ex vivo in MC38-RFP tumor cells.

(N-P) Quantification of C16-BODIPY uptake in ex vivo CD8⁺ T cells (N). Representative histograms for ex vivo C16-BODIPY uptake in CD8⁺ T cells isolated from dLN (O) or tumor (P) from day 10-14 MC38 tumors.

(Q-S) Quantification of C16-BODIPY uptake ex vivo from dissociated tumors: B16-OVA-RFP tumor cells (Q) or CD8⁺ TILs isolated from B16-OVA-RFP (R) and E0771 tumors (S) or dLN.

(T-U) Expansion index (T) and representative flow plots (U) measuring proliferation of CTV-labeled CD- and HFD-derived naïve CD8⁺ T cells after 48 and 72 hours on 1, 2, or 4 μg/mL each of plate-bound anti-CD3 and anti-CD28, with or without supplementation of BSA-conjugated free fatty acids (FFAs).

Data represent ≥ two independent experiments with ≥ 6 mice per group. Graphs display mean +/− SD (K-N, Q-T). (ns p>0.05, *p≤0.05, **≤<0.01).

See also Figure S5.