Fig 2: Switching culture conditions from Wnt3a-conditioned medium to Wnt3a free medium induces most efficient differentiation of mouse colonoids. Mouse colonoids derived from C57Bl/6 mice crypts were cultured for two days in WNR CM, Wnt3a CM + rENR or rWENR medium before medium change to rENR or maintenance in original media. After four additional days of culture, colonoids were assayed for markers of epithelial cell types by ISH/IHC and qPCR. (A, B) Colonoids were stained with in situ hybridization for LGR5 for the quantification of stem cells (brown dots), immunohistochemistry for EpCam (epithelial cells) and hematoxylin (nuclei); (C) Quantification of Ki67 histochemistry for measurement of proliferation; (D) immunohistochemistry for Chromogranin A (enteroendocrine cells) and Alcian Blue counterstain (mucus and Goblet cells); (E) quantification of goblet cells; (F) quantification of Mucin 2 mRNA levels; (G) immunohistochemistry of Ki67 as a proliferation marker, with an Alcian Blue counterstain (mucus and Goblet cells); (H) quantification of chromogranin A immunohistochemistry; (I) quantification of enterocytes using mRNA transcript levels of alkaline phosphatase (ALPI). qPCR analysis was performed on biological triplicates for each treatment group at each timepoint. Graphs in (B, C) are the average expression by IHC/ISH of the indicated markers in the stem cell conditions from three independent studies. Graphs in (E, F, H, I) are the average fold increase in markers by IHC (E) or qPCR (F, H, I) after differentiation compared to levels in the stem cell conditions from three independent experiments. Scale bars in A indicate 20 microns and in C and E indicate 50 microns. IHC/ISH quantification was performed on an average of 30 colonoids per slide. Graphs and images are representative of the average of biological duplicates from three independent experiments. Statistical significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.005.

Fig 3: iNOS Downregulation promoted EMT and metastasis in vivo.A, SW480 cells and CTCs were cultured on slides for 36 hours and followed Immunofluorescence staining. The green and red signal represents the staining of iNOS and EpCAM, respectively (Scale bars, 25 µm). B, the percentages of iNOS gene expression among the CTC samples, n = 15. C, cells treated as in A, the representative images are shown for immunofluorescence staining with the indicated antibodies. EpCAM and CD45 are the epithelial and immune cell marker, respectively. The corresponding color signal represents the staining as indicated (Scale bars, 25 µm). D, the RNA-seq data were analyzed by using IPA, heatmap shows the transcriptomic profiling of colorectal cancer metastatic signaling induced by iNOS knockdown. E, representative H&E images of section from SCID mouse lymph node and stomach bearing metastatic colon cancer of SW480-shiNOS (n = 10) and SW480-shControl cells (n = 10). (Scale bars, 50 µm). F, the statistics of the metastatic tumors between the two groups of experimental mice, P < 0.0055 by Fisher exact test. G, RNA-seq shows the selected metastasis suppressors were downregulated by iNOS knockdown.