Figure 3.
Lgr5− Cancer Cells Are the Disseminating Cells in CRC
(A) Experimental setup: mice bearing metastatic CRC were used to sample blood from the portal vein. Blood was analyzed using FACS for the presence of circulating tumor cells.
(B) Cumulative FACS profile of circulating Lgr5+ CSCs and Lgr5− cancer cells (n = 4). The color coding links individual circulating tumor cells to the corresponding blood sample in Figure S3B.
(C) Heatmap of differentially expressed genes in Lgr5+ and Lgr5− cancer cells isolated from primary tumors, CTCs, and liver metastasis. Genes marked with green are known to be upregulated in intestinal stem cells; genes marked in red indicate known intestinal differentiated cell markers.
(D) Representative confocal images of spontaneous liver metastases grouped per diameter range. Dashed lines highlight the metastasis edges. Scale bar, 100 μm.
(E) Metastases are subdivided in lesions composed of only Lgr5− cancer cells (red) or lesions containing Lgr5+ CSCs (green) and grouped per diameter range (the analysis includes 132 metastatic lesions, n = 6).
(F) Intravital multi-day imaging of liver metastases hatching from Lgr5− cancer cells. Dashed lines highlight metastasis edges. Yellow boxed areas are enlarged next to the corresponding panels. Scale bars, 50 μm.
(G) Representative images of a liver metastasis followed overtime by intravital imaging demonstrating growth and regression upon depletion of Lgr5+ CSCs via diphtheria toxin (DT) administration. Dashed lines highlight metastasis border. Scale bar, 100 μm.
(H) Size of DT-treated metastatic lesions followed by intravital imaging (n = 5), normalized to the first time point. The red line represents the example shown in (G).
(I) Representative histochemistry images of livers of mice subjected mesenteric vein injection of FACS-sorted Lgr5− cancer cells and treated with either vehicle or DT treatment for 4 weeks. ROI, region of interest. Asterisks indicate metastatic foci; dashed lines highlight metastasis edges. Boxed areas are enlarged next to the corresponding overview images. Scale bars, 500 μm.
(J) Representative examples of livers of mice of mice subjected mesenteric vein injection of CRC Lgr5eGFP and treated with either vehicle or DT treatment for 4 weeks.
(K) Number of metastatic foci derived from mesenteric vein injection of Lgr5+ CSCs or Lgr5− cancer cells.
(L) Metastatic load upon injection of Lgr5+ CSCs or Lgr5− cancer cells. Similar shapes represent paired experiments (n = 4). Data are presented as median with interquartile range; p values were calculated using the paired t test (∗p < 0.05).
(M–O) Representative examples of metastases generated from Lgr5+ CSCs (M) and Lgr5− cancer cells (N), boxed area enlarged in (O). Scale bars, 100 μm.