Fig 2: APOE regulates Cxcl1 expression in tumor cells and fibroblasts. A, Dot plot of Ldlr, Vldlr, Lrp1, and Lrp8 in orthotopic KPC samples. Color represents average expression, while size of the dot represents expression frequency. B, Dot plot of LDLR, VLDLR, LRP1, and LRP8 in human PDAC. Color represents average expression, while size of the dot represents expression frequency. C, Violin plot of normalized LDLR expression in human PDAC. D, Heat map of differentially expressed genes in in vitro 7940b KPC cells treated with vehicle (n = 3) compared with 7940b KPC cells treated with 0.3 μg/mL murine recombinant APOE (n = 3) for 48 hours. Red, high expression; blue, low expression. E, qRT-PCR analysis of Cxcl1 and Cxcl5 mRNA levels relative to Cyclophilin A in four KPC cell lines (7940b, mT3, mT4, mT5). Dotted line represents fold induction compared with vehicle-treated cells normalized to 1. Statistical significance was determined using one-way ANOVA with Tukey test for multiple correction. F, Survival analysis of PDAC patients stratified by plasma CXCL1 levels. CXCL1 low, n = 38; CXCL1 high, n = 38. Statistical significance was determined using log-rank (Mantel–Cox) test. G, qRT-PCR analysis for Cxcl1 mRNA levels relative to Cyclophilin A in WT fibroblasts (BLK6318) and CAFs (FB1) treated with vehicle (n = 2–3) or 0.3 μg/mL recombinant ApoE (n = 2–3) for 48 hours. Statistical significance was determined by two-tailed t tests. H, qRT-PCR analysis of Cxcl1 and Cxcl5 mRNA levels relative to Cyclophilin A in WT (n = 6) and ApoE–/− (n = 5) tumors. Statistical significance was determined using two-tailed t test. n.s., not significant. I, Coimmunofluorescence staining of CXCL1 (green), CK19 (red), αSMA (white), and DAPI (blue) in WT and ApoE–/− orthotopic KPC tumors. J, Experimental design schematic. K, qRT-PCR analysis of Cxcl1 mRNA levels relative to Cyclophilin A in 7940b tumor cells alone control (n = 6), 7940b cells cultured with WT macrophage CM (n = 6), 7940b cells cultured with ApoE–/− macrophage CM (n = 6), and 7940b cells cultured with ApoE–/− macrophage CM with 0.3 μg/mL recombinant ApoE (n = 3). Statistical significance was determined by two-tailed t tests between groups.

Fig 3: Human APOE levels are elevated in PDAC and correlate to patient survival. A, UMAP analysis of the 13 identified cell populations in human adjacent/normal pancreas (n = 3) and PDAC tumors (n = 16). B, Dot plot of APOE in all identified cell populations in human single-cell data set. Color represents average expression. Size of the dot represents expression frequency. C, UMAP visualization of four identified myeloid cell subpopulations in the human PDAC tissue. D, Feature plot of APOE expression in all identified myeloid cell populations in human PDAC. Gray, low expression; blue, high expression. Black outline denotes APOE-positive macrophages. E, Violin plot of normalized gene expression of APOE in PDAC and adjacent normal pancreas macrophages in human PDAC. Statistical significance was determined using nonparametric Wilcoxon rank sum test. F, UMAP visualization of human PDAC fibroblast subpopulations. G, Violin plot of normalized gene expression of APOE in human myCAF and iCAF populations. H, Violin plot of normalized gene expression of APOE in PDAC and adjacent normal pancreas iCAFs in human PDAC. Statistical significance was determined using nonparametric Wilcoxon rank sum test. I, Violin plot of normalized expression of APOE in human monocytes. Statistical significance was determined using nonparametric Wilcoxon rank sum test. J, Human APOE concentration (μg/mL) in plasma from healthy donors (n = 15), chronic pancreatitis patients (n = 17), and PDAC patients (n = 155). Statistical significance was determined using one-way ANOVA with Tukey test for multiple comparisons. n.s., not significant. K, Survival analysis of PDAC patients stratified by plasma APOE levels. APOE low, n = 32; APOE high, n = 32. Statistical significance was determined using log-rank (Mantel–Cox) test.

Fig 4: Loss of APOE results in reduced tumor burden and fibrosis. A, Experimental scheme for orthotopic transplantation of 7940b, KPC tumor cells. B, Final tumor weight (g) in WT (n = 10) and ApoE–/− (n = 13) mice. Statistical significance was determined using two-tailed t test, with a P < 0.05 considered statistically significant. C, Representative IHC for APOE in WT and ApoE–/− mice. Scale bar, 100 μm. D, Representative IHC staining for Ki-67, cleaved caspase-3 (CC3), F4/80, and CD3 in WT and ApoE–/− mice. Scale bars, 100 μm. E, Quantitation of IHC stain as a percentage area per 20× field in WT (n = 4–8) and ApoE–/− mice (n = 5–8). Statistical significance was determined by two-tailed t tests. n.s., not significant.

Fig 5: Antitumor phenotype in ApoE–/− mice is rescued upon T-cell depletion. A, Experimental design schematic for T-cell depletion in WT and ApoE–/− mice. B, Final tumor weight (g) from WT (n = 6), WT anti-CD4/CD8 (n = 3), ApoE–/− (n = 6), and ApoE–/− anti-CD4/CD8 (n = 6). Statistical significance was determined with a nonparametric Mann–Whitney test. C, Representative SPADE analysis of cellular infiltrate in WT tumor. Identified populations include nonimmune cells, CD8 T cells, CD4 T cells, B cells, immature myeloid cells, macrophages, and CD11c+ myeloid cells. The SPADE plot is colored to indicate CD45 expression. Red, high expression; blue, low expression. D, Manual gating quantitation of cell populations in WT (n = 4), WT anti-CD4/CD8 (n = 2), ApoE–/− (n = 4), and ApoE–/− anti-CD4/CD8 (n = 5) tumors. Populations include CD4 T cells (CD3+ CD4+) and CD8 T cells (CD3+ CD8+) E, total myeloid cells (CD45+ CD11b+), macrophages (CD11b+ F4/80+), CD11c+ myeloid cells (CD11b+ CD11c+), and immature myeloid cells (Ly-6C+ Ly-6G+). Statistical significance was determined by two-tailed t tests between groups. F, Representative SPADE analysis colored by Ly-6G expression in WT, WT anti-CD4/CD8, ApoE−/−, and ApoE–/− anti-CD4/CD8 tumors. Red, high expression; blue, low expression.