Fig 2: Phospho-tau aggregates and ApoE were visible in the fenestrated capillaries of choroid plexus and sTREM2 in the macrophages. Brain sections containing choroid plexus (CP) in the lateral ventricles and third ventricles from control, AD, and DS subjects were stained with anti-TREM2 and anti-Tau (t-Tau or p-Tau), or anti-TREM2 and anti-ApoE antibodies and analyzed by confocal microscopy. DAPI was used for nuclear staining. In control brain, both TREM2 and anti-t-Tau were present in CP cuboidal epithelial cells (CPE) surrounding a core of fenestrated capillaries and connective tissues (a). Some soluble t-Tau protein was visible inside fenestrated capillaries and TREM2 was visible in stromal capillary (vesicles) and in the stromal macrophages (a and b). In DS and AD brain dense fibrillary p-Tau present in psammoma bodies (calcified intracellular inclusion structures), was visible in the stroma and TREM2 co-localized in the vesicles and in stromal macrophages (c and d). In control CP, both proteins TREM2 (red) and ApoE (green) were visible in CPE cells and in stromal macrophages that appeared normal with healthy structure (e). In contrast, in DS brains ApoE was visible in the “amyloid biondi” bodies (complex filamentous ring-like structures associated with lipid droplets, showing with an arrow) and lipofuscin (yellow or brown intracellular structures composed of lipid molecules (f). Scale bar in a = 50 µm, b–f = 25 µm.

Fig 3: ApoE-/- mice have fewer monocytic-MDSCs and increased CD8+ T-cell infiltration. A, Experimental scheme for orthotopic transplantation of 7940b, KPC tumor cells. B, tSNE visualization of the 6 cell populations identified using CyTOF in WT and ApoE–/- tumors. Populations identified include macrophages (blue), immature myeloid cells (orange), CD8 T cells (green), CD4 T cells (red), B cells (purple), and nonimmune (brown). C, Manual gating quantitation of cell populations in WT (n = 5–6) and ApoE–/- (n = 7) tumors. Populations include total immune (CD45+), B cells (CD45+ CD19+), total myeloid (CD45+ CD11b+), macrophages (CD11b+ F4/80+), TAMs (F4/80+ CD206+; F4/80+ PD-L1+), granulocytic-MDSCs (Ly-6C+ Ly6G+), monocytic-MDSCs (Ly-6C+ Ly-6G-), total T cells (CD45+ CD3+), CD4 T cells (CD3+ CD4+), Tregs (CD4+ CD25+), and CD8 T cells (CD3+ CD8+). D, Representative immunofluorescence staining of CD8 (green) and DAPI (blue) in WT and ApoE-/- tumors. Scale bars, 100 µm. Right, quantitation of percent CD8-positive area in a 20× field in WT (n = 4) and ApoE–/- mice (n = 5). Statistical significance was determined by two-tailed t test. E, Representative coimmunofluorescence staining of CD8 (green), GZMB (red), and DAPI (blue) in WT and ApoE–/- tumors. Scale bars, 50 µm. Right, quantitation of the number of Gzmb+ CD8+ double-positive cells in at least three, 40× fields in WT (n = 4) and ApoE–/- mice (n = 4). Statistical significance was determined by two-tailed t test.

Fig 4: 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.

Fig 5: 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), aSMA (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.