Figure 3: The metastatic ability of lung TICs is dependent on miR-1246 and miR-1290.
(a) ISH for miR-1246 and miR-1290 in paired primary lung adenocarcinoma and lymph node metastases. Scale bars, 600 μm (50 μm in the inset). (b) Association between miR-1246 or miR-1290 expression in primary tumours by ISH and status of lymph node (N) and distant metastases (M). (c,d) Transwell migration assay and quantification of tumoursphere cells treated with either zip1246 or zip1290. The migrated cells were stained with Giemsa (c) and counted 6 h after plating with 100,000 cells (d); n=3. Scale bars, 50 μm. (e) Fluorescence and bright-field images of mouse lung for assessing metastasis. A total of 1 × 106 tumoursphere cells treated with either zip1246 or zip1290 were injected through the tail-vein. Lung metastatic nodules on day 13, 27 and 33 after transplantation are shown. Scale bars, 1 cm. (f–h) H&E (f) and E-cadherin immunohistochemistry staining (g) for mouse lung sections as shown in e (day 27). Quantitative analysis of the number of metastatic pulmonary nodules per lung is shown (h). Scale bar, 200 μm (f) and 50 μm (g); n=4. (i,j) H&E staining for mouse liver sections after injecting 1 × 106 tumoursphere cells on day 27 via tail vein (i). Quantitative analysis of the number of metastatic nodules in the liver per mouse is shown (j). Nodules (see inset) are shown in higher magnification. Scale bar, 700 μm (inset, 200 μm); n=4. (k,l) Fluorescence and bright-field images (top panel) of the mouse lung for assessing metastasis. A total of 1 × 106 tumoursphere cells treated with either zip1246 or zip1290 were injected subcutaneously. Whole-lung tissues were collected on day 60 (k) and until the subcutaneous tumours reached a similar size (12 mm in diameter) (l) after transplantation. The metastatic nodules are shown in higher magnification (see inset). Quantitative analysis of the number of metastatic lung nodules is shown (lower panel). n=4. Scale bars, 1 cm. All error bars represent±s.e.m. and statistical significance was calculated using Student’s t-test; *P<0.05.
