Fig 2: SSDE-induced alterations in inflammatory mediators and lung histopathology in vivo. (A–D) Cytokine release in BALF for (A) TNF-a; (B) IL-6; and (C) CXCL1 following single and repetitive exposure to SSDE. (D–G) Representative histology of lungs following single and repetitive exposure to saline and SSDE. (D) Single exposure, saline; (E) single exposure, SSDE; (F) 7-day exposure, saline; and (G) 7-day exposure, SSDE. Green arrow indicates perivascular inflammation. Yellow arrows indicate representative infiltrating granulocytes. Scale bar in D is 120 µm. Images taken at 20× with 150% optical zoom. (H–J) Representative images of lung immunofluorescence of LYVE-1 are shown. (H) Seven-day exposure, saline; (I) 7-day exposure, SSDE; (J) quantification of fluorescent intensity of LYVE-1 in saline- and SSDE-exposed mice. Quantification data are mean ± SEM, four lung sections were imaged per mouse, with five mice each in saline and control groups (total of 20 images per group). Error bars are standard error of mean (*p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001).

Fig 3: Quantitation of the staining for the various markers in the tumor and stromal components separately in the 4 models (OCICx 1, 3, 8, 16) over passage number. The percent pixel positivity is shown for each marker (A) CA-9, EF5, CD31; (B) Ki67 and LYVE1. Values plotted are mean ± SD. Passage 0 indicates biopsy piece.

Fig 4: Cells undergoing endothelial-mesenchymal transition produce collagen in the lung tissue of UUO rats. Three-color confocal laser scanning microscopy of cells co-expressing LYVE-1 (green), a-SMA (red) and collagen I (blue). Lymphatic vessels co-expressing LYVE-1 and a-SMA are surrounded by collagen I. Magnification, x400. LYVE-1, lymphatic vessel endothelial receptor-1; a-SMA, a-smooth muscle actin; UUO, unilateral ureteral obstruction; EPL, eplerenone.

Fig 5: Generation of an Immunotherapy-Responsive Pancreas Cancer Animal Model(A) Representative immunofluorescent (IF) staining of wild-type mouse pancreas (top row), primary tumors isolated from a genetically engineered KPC mouse (middle row), and a polyclonal KPC TECs following orthotopic implantation into B6 pancreas at 3 weeks post-implantation (bottom row). Images depict cytokeratin+ (CK) tumor cells, CD8+ T cells, F4/80+ macrophages, Ly6G+ neutrophils, PD-L1, aSMA cancer-associated fibroblast (CAFs), CD31+ endothelial cells, and Lyve-1+ lymphatic venules. Scale bar, 25 µM.(B) Histogram overlay of CB-eGFP+ KPC clones generated from three independent KPC mice. Shown are representative clones (n = 3) from the three independent polyclonal KPC tumor epithelial cell lines (KPC1–3).(C) Timeline for orthotopic tumor cell injection, aPD-L1 treatments (blue arrows), and IVIS imaging.(D) Representative IVIS imaging of tumor bioluminescence at 7 days (prior to treatment) and 14 days following the initial aPD-L1 injection (day 21 post-tumor).(E) Radiance of tumor bioluminescence following orthotopic injection of a CB+KPC clone (KPC3b) and following aPD-L1 treatment. Data are mean ± SEM. *p < 0.05 (unpaired two-tailed Student’s t test). n = 5 mice per group.(F) Radiance of tumor bioluminescence following orthotopic injection of a CB+KPC clone (KPC2a) and following treatment with either aPD-1 or aPD-L1. Data are mean ± SEM. *p < 0.05 and **p < 0.005 (unpaired two-tailed Student’s t test). n = 5 mice per group.(G) Representative IF of CD8+ T cells, cytokeratin (CK+) tumor cells, and PD-L1 in wild-type mouse pancreas, primary tumor isolated from a genetically engineered KPC mouse, and KPC2a (CB+) orthotopic tumors isolated from B6 mice ± aPD-L1. Scale bars, 50 µM (left and middle columns) and 10 µM (right column, inset).(H) CD8+ T cell number per all nucleated cells in IF sections. Data are mean ± SEM. n = 3–5 mice per group.(I) CD8+ T cell number per field of view in IF sections. Data are mean ± SEM. n = 3–5 mice per group.(J) PD-L1 intensity was quantified through pixel intensity measurement by Fiji 2.0.Data in (H)–(J) are mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.005 (one-way ANOVA, with a post hoc test to correct for multiple comparisons). Data are representative of n = 3–6 mice per group. See also Figure S1.