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. Author manuscript; available in PMC: 2023 Feb 3.
Published in final edited form as: Sci Transl Med. 2022 Aug 3;14(656):eabn7571. doi: 10.1126/scitranslmed.abn7571

Figure 3: Vimentin represses colony formation ex vivo and tail vein experimental metastasis in vivo.

Figure 3:

A) Schema of colony formation assay using shRNA. After isolation, organoids are infected with either NT-shRNA or either of two shRNA sequences against vimentin (shVim #1 and shVim #2). After puromycin selection to eliminate non-infected cells, organoids are dissociated into clusters and embedded in Matrigel.

B) Representative DIC images of colonies infected with NT-shRNA, shVim #1, or shVim #2 after 5 days (GEMM) or 10 days (PDX) in Matrigel. Scale bars are 100 μm.

C) Number of colonies per well. Each dot represents the number of colonies in one well, bars show the medians. NS = not significant, **P=0.008, ***P<0.001 ****P<0.0001 (one-way ANOVA). n=89, r=3.

D) Tail vein assay schema to study the late steps of the metastatic cascade. Fluorescent organoids are infected with NT-shRNA, shVim #1, or shVim #2. After selection, organoids are dissociated into clusters and injected into the tail vein of NSG mice. After 4 weeks, the number of metastases in the lungs is assessed.

E) Representative epifluorescence images of whole lungs. Macrometastases indicated by arrows are detected by tdT signal.

F) Number of macrometastases per lung. Each dot corresponds to a lung. Bars show medians. **P=0.0036, ***P=0.0004 (Kruskal-Wallis). n=24 mice, r=2 biological replicates.