Figure 3. The lineage TFs GATA6 and HOPX limit metastatic colonization.

(A) H2030 cells stably expressing two separate doxycycline (DOX) inducible shRNAs, (a) or (b), against GATA6 were cultured in the absence or presence of DOX. GATA6 level was measured by qRT-PCR.

(B) H2030 cells from (A) were injected at indicated cell numbers into the arterial circulation of mice fed with regular (-DOX) or DOX (+DOX) diet. Metastatic burden was measured by bioluminescent imaging. The bar chart shows the whole body bioluminescent units (BLU) collected at Day 36 (shGATA6(a)) or Day 40 (shGATA6(b)) and normalized to Day 0. Right: representative images of mice injected with H2030-shGATA6(a) cells on Day 36. p-values calculated by Mann-Whitney test.

(C) 5 × 10⁴ H2030-BrM3 cells overexpressing GATA6, HOPX, or both were injected as in (B). The scatter plot shows the metastatic burden at Day 42. p-values calculated by Mann-Whitney test.

(D) qRT-PCR of GATA6 and HOPX in PC9 cells expressing the indicated shRNAs. shCon: a control shRNA.

(E) 5 × 10⁴ PC9 cells expressing the indicated shRNAs were injected intracardially into DOX treated mice. The scatter plot shows the metastatic burden at Day 48. Bottom: representative images of mice with metastases. p-values calculated by Mann-Whitney test.

(F) Kaplan-Meier curve showing the metastasis free survival of mice inoculated with 2.5 × 10⁴ PC9-shGATA6/shHOPX(b) cells as in (B) or injected with PC9-shGATA6/shHOPX(b) cells that co-express GATA6 and HOPX cDNAs (+Rescue). p-values calculated by log-rank test.

(G) Hematoxylin and eosin staining of metastases in the (a) brain, (b) bone and (c) lung of mice harvested from (E). Scale bars, 100 μm.

(H) Volumes of subcutaneous tumors formed by PC9-shGATA6/shHOPX(a) cells. Mean ± SEM. p-values calculated by Student's t-test. ns= not significant.

All bar charts represent the mean ± SEM. See also Figure S3.