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sc-12-0923_sm_SupplFigure1.tiff2702KSupplementary Figure S1: NPRA as a biomarker for melanoma, colon and pancreatic cancer. Human melanoma, colon and pancreatic cancer tissue microarray (TMA), prepared in the histology laboratory of the Moffitt Cancer Center Tissue Core Facility was used to test for expression of NPRA. The TMA slide was stained using a Ventana Discovery XT automated system (Ventana Medical Systems, Tucson, AZ), according to the manufacturer's protocol, as described 2. The TMA slide was scored for intensity and cellularity by an expert pathologist, as described 2. The product of the intensity and percentage scores was used as the final score. The final score was classified as: 0, negative; 1-3, weak; 4-6, moderate; and 7-9, strong. A median analysis of NPRA scores and the frequency in each disease group of having a score at or below the median was performed. (A) Scores for NPRA in different stages of melanoma are shown. These include, normal skin (N) (n=21), NEVI (compound Nevi, junctional nevi, intradermal nevi) (n=21), Clark's atypical dysplastic nevi (DN) (n=37). (B) Scores for each sample of different stages of Colon cancer are shown. These include, normal (N) (n = 24); adenoma (ADE) (n = 24) and adenocarcinoma (ADC) (n = 78). (C) Scores for each sample of different stages of pancreatic cancer are shown. These include, normal pancreatic duct (N) (n = 54); Intraductal carcinoma IDC (n = 68) and neuroendocrine carcinoma (NC) (n = 158). The bar represents the mean sample score for each category.
sc-12-0923_sm_SupplFigure2.tif812KSupplementary Figure S2: Control antibody staining. A) Specificity of antibodies was optimized by immunohistochemical staining of WT LLC-1 tumor sections using corresponding host- and isotype-matched control antibodies. Control and test sections were represented in the same row. Rb IgG: rabbit IgG. B) LLC-1 cells and GFP expressing MSC were stained with antibodies for CEA (upper panel) and GFP (lower panel). Scale bar in all the images is 20 μm.
sc-12-0923_sm_SupplFigure3.tiff2702KSupplementary Figure S3: Lung vasculature in NPRA-KO mice. Lungs isolated from WT and NPRA-KO mice were sectioned and stained for vWF. Cells expressing vWF were counted and compared between WT and KO sections. n = 4 mice/group, 1 section/mouse. Fluorescent cells were counted, expressed as means ± SEM, and compared with unpaired student t-test. *p < 0.05.
sc-12-0923_sm_SupplFigure4.tiff2702KSupplementary Figure S4: LLC-1 cells were injected s.c. into the flanks of WT and NPRA-KO mice. GFP-BM MSCs were injected i.v. on day 10 and tumors were extracted on day 21. Tumor dimensions (A) and weights (B) were measured and statistically compared with Student's t-test. (C-D) Tumor sections were stained for carcinoembryonic antigen (CEA) antigen and examined with a fluorescence microscope (400× magnification; n = 4 mice/group; 1 section per mouse). Fluorescent cells were counted, expressed as means ± SEM, and compared with unpaired student t-test. **P < 0.01. Scale bar in the images is 20 μm.
sc-12-0923_sm_SupplFigure5A.tiff2702KSupplementary Figure S5: After LLC-1 cells were injected s.c. into the flanks of WT and NPRA-KO mice, GFP-BM MSCs were injected i.v. on day 10 followed by tumor extraction on day 21. (A) Tumor sections were stained for CAFs (S100A4, Vimentin) and endothelial cells (vWF). Cells from WT and KO tumor sections were quantified (n = 3 mice; one field/ section/mice) and compared with student's t-test. (B) Tumor sections were dual stained for GFP and characteristic markers such as S100A4, Vimentin, and vWF. The dual stained cells were counted (n = 3 mice; one field/ section/mouse) and the WT and KO sections were compared with student's t-test. **p < 0.01, ***p < 0.001, ****p < 0.0001
sc-12-0923_sm_SupplFigure5B.tiff2702KSupplementary Figure S5: After LLC-1 cells were injected s.c. into the flanks of WT and NPRA-KO mice, GFP-BM MSCs were injected i.v. on day 10 followed by tumor extraction on day 21. (A) Tumor sections were stained for CAFs (S100A4, Vimentin) and endothelial cells (vWF). Cells from WT and KO tumor sections were quantified (n = 3 mice; one field/ section/mice) and compared with student's t-test. (B) Tumor sections were dual stained for GFP and characteristic markers such as S100A4, Vimentin, and vWF. The dual stained cells were counted (n = 3 mice; one field/ section/mouse) and the WT and KO sections were compared with student's t-test. **p < 0.01, ***p < 0.001, ****p < 0.0001
sc-12-0923_sm_SupplFigure6.tiff2702KSupplementary Figure S6: LLC-1 tumors were generated by s.c. injection of LLC-1 cells followed after 10 days by i.v. injection of GFP-MSC. The tumors were excized on day 21. The tumor sections (n = 3 mice; one field/ section/mouse) were dual stained for NPRA expression and for specific cells, (a) endothelial cells (CD31), (b) CAFs (SMA), and (c) MSCs (GFP). Manders coefficient was calculated in imageJ to assess the dual stain expression. Scale bar in the images is 20 μm.
sc-12-0923_sm_SupplFigure7.tiff2702KSupplementary Figure S7: LLC-1 cells with or without MSCs were injected s.c. into the flank region of NPRA-KO mice. The tumors were measured at the end of 3 weeks. Tumor A) volumes and B) weights were represented as means ± SEM and compared with student t-test in Graphpad prism. L: LLC-1 cells; M: MSCs; **P ≤ 0.01.

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