Extended Data Fig. 1: Characterization of the metastatic variants of SUM159 breast cancer cell line identifies DACT1 as a highly expressed gene in the bone metastatic M1a subline.
From: TGF-β-induced DACT1 biomolecular condensates repress Wnt signalling to promote bone metastasis
a, Schematic summary of the establishment of a series of isogenic sublines with different primary tumor and metastasis potential from the parental SUM159 triple negative breast cancer cell line. Parental SUM159 cells were stably labeled with a retroviral triple reporter (TR) expressing GFP, thymidine kinase, and firefly luciferase (F-luc) and injected into the mammary fat pad of nude mice. A primary tumor was isolated, cultured, and re-injected by either tail-vein or mammary fat pad injection. Successful outgrowths were then isolated and cultured. b, Isolated cell lines were injected orthotopically into NSG mice and monitored for primary tumor growth. n = 5 mice/group. Student’s t-test. Representative of 2 independent experiments. c, d, e, Ex vivo imaging of bones and lungs was conducted once primary tumors reached mean diameter >1 cm. Values for lung (c) and bone (d) metastasis were thresholded to 0 at values below 104 and 105 photon/sec, respectively, to remove background noise. Individual hindlimbs were treated as independent data points. Rates of successful outgrowth were enumerated per group (e). f, Representative ex vivo bioluminescence images of spontaneous metastasis to lung and bone from each derived subline from (c-e). g, The development of bone metastasis after intracardiac injection of each derivative was monitored by bioluminescent imaging and was compared to SUM159-TR. Mann-Whitney U test. n = 6 mice/group. h,i, Representative bioluminescent (h) and X-ray images (i) at day 0 and day 25 of mice from (g). j, Gene expression values from microarray analysis were used to generate a list of genes up-regulated >4-fold in M1a compared to either SUM159PT-TR or M1L1. k, Heatmap representation of the expression levels of the 11 differentially expressed genes from (j). l, RT–qPCR analysis of DACT1 mRNA levels normalized to Gapdh in the indicated SUM159 sublines. n = 4 technical repeats, representative data from 2 independent experiments. m, n) Flow cytometry measurement of 7x-TCF-GFP Wnt reporter activity in BM2 (m) or HPL1 (n) cells with Wnt3a and Wnt inhibitor ICG-001 (25 μM) treatment. n = 3 biological replicates. Student’s t-test. Experiment independently repeated 3 times. Data represents mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005 in g with exact p values in Source Data. Numerical source data for b-e, g, l-n are provided.
