Extended Data Fig. 8: A loss-of-function screen in vivo identifies SFRP2 as survival regulator in lung disseminated indolent breast cancer cells.
From: Crosstalk with lung epithelial cells regulates Sfrp2-mediated latency in breast cancer dissemination
a, Volcano plot of RNAseq expression data of D2.0R cells in vivo compared to the other groups (as in Fig. 3a). In blue, candidate genes selected for step 2 validation. b, Step 2 validation of candidate genes. Subpopulations of D2.0R-EGFP cells bearing a single shRNA for the indicated gene were individually generated (3 shRNA sequences/gene). Cells with shRNA for the same gene were mixed together in equal amount, injected in tail vein of BALB/c nude mice (n = 6 mice for Control and n = 3 for the other groups) and processed as in Fig. 4c. Unpaired t-test with Welch’s correction. c, Subpopulations of D2.0R-EGFP-shSfrp2 cells were mixed and injected in the tail vein with an equal amount of D2.0R-mCherry-shControl. After 3 days to allow seeding and extravasation in the lung parenchyma, lungs were collected and GFP + and mCherry + simultaneously quantified to rule out pre-dissemination role of SFRP2 (n = 4 mice). Scale bar, 1 mm. Unpaired t-test with Welch’s correction. d, In vitro growth curves of D2.0R-EGFP cells bearing the indicated shRNAs for Sfrp2. Confluency values at indicated time points were log10-transformed and linear regression was calculated (n = 2 independent experiments). Line was forced to go through the origin. Solid line, mean of best-fit line; dashed lines, 95% confidence bands. e, Relative expression levels of Sfrp2 in D2.0R.EGFP cells on plastic, isolated from mammary fat pad or lung-disseminated (n = 5 wells for Control group, n = 3 mice for the other groups). Unpaired t-test. f, Histogram showing the induction of SFRP family members by AT1-like conditioned media in both D2.0R and 4TO7 cells. Mean normalized pooled samples (n = 9) from independent experiments (n = 3–4). Mann-Whitney test.
