TGF-β regulates Sca-1 expression and plasticity of pre-neoplastic mammary epithelial stem cells

The epithelial-mesenchymal plasticity, in tight association with stemness, contributes to the mammary gland homeostasis, evolution of early neoplastic lesions and cancer dissemination. Focused on cell surfaceome, we used mouse models of pre-neoplastic mammary epithelial and cancer stem cells to reveal the connection between cell surface markers and distinct cell phenotypes. We mechanistically dissected the TGF-β family-driven regulation of Sca-1, one of the most commonly used adult stem cell markers. We further provided evidence that TGF-β disrupts the lineage commitment and promotes the accumulation of tumor-initiating cells in pre-neoplastic cells.

(A) The plot shows expression pattern of surface markers in MMC and ANV2 cells, acquired with highthroughput surface profiling. Data are shown as log fold change (MFI = median fluorescence index).
(B) The plot shows independent validations of selected, differentially expressed surface markers in MMC and ANV2 cells, identified with high-throughput surface profiling and analyzed with flow cytometry. Results are from at least three independent experiments and are presented as mean ± SD, paired t test (MFI = median fluorescence index). (A) The scheme shows an establishment of experimental models MMC and ANV2, as described in Knutson et al., The JI 2006;117(3). (B) Plot shows the percentage of breast cancer stem-like cells, as characterized by CD24-CD44+ immunophenotype, in the culture of MMC, ANV2 and ANV5 cells. Three biological replicates per cell line were analysed with flow cytometry and are shown as mean ± SD (t test).

Supplementary Figure 2
(C) Plot shows capacity of MMC, ANV2 and ANV5 cells to retain JC-1 as a proxy of ABC transporter activity in standard cell culture conditions. Results are from four measurements per subline and are presented as mean ± SD (t test).
(E) Representative images and scatter plot show spheroid size of MMC, ANV2 and ANV5 cell lines in standard cell culture conditions, as determined with spheroid formation assay. Results are from two independent experiments (Mann-Whitney u test, scale = 500 µm).
(F) Plot shows in vivo behaviour of MMC (n = 15; 2.5x10 5 cells injected per mouse) and ANV2 cells (n = 14; 5x10 4 cells injected per mouse) implanted into mammary fat pad. The growth curves were derived from plots shown in Figure 2F (MMC, EV clones) and Supplementary Figure 6E        (A) Scheme shows experimental workflow, used for determination of gene expression changes upon exposure to experimental treatment for 96 h. Sca-1 +/fractions were sorted, washed and seeded into cell culture media containing vehicle (PBS) or TGF-β1 (1 ng/mL). Viable, single cells (pool) were sorted as controls and processed in the same way.
(B-C) Plot shows changes in gene expression of TGF-β target genes Id1 and Id3 in sorted subpopulations of Comma-Dβ cells, exposed to vehicle (PBS) or TGF-β1 (1 ng/mL) for 96 h directly after sorting. Results are presented as mean ± SD (paired t test, *P < 0.05).  (A-C) Plot shows relative surface Sca-1 expression after exposure of ANV2, ANV5 and MMC cells to vehicle (PBS with 0.1% BSA) or 1 µM of selected Type II receptor chimeras for 72 h. The results are presented as mean ± SD from two independent experiments (*P < 0.05) and were analyzed with flow cytometry (MFI = median fluorescence index).
(D) Plots show % of Sca-1 Comma-Dβ cells in validation of recombinant ligand quenching by receptor chimeras. Cells were exposed to vehicle (PBS with 0.1% BSA), ligands and/or chimeras for 72 h. The results are presented as mean ± SD from two technical replicates and were analyzed with flow cytometry. (E) Plot shows concentration of active and total TGF-β1, as determined by ELISA in serum-free cell culture medium, condition by MMC, ANV2 and ANV5 cells for 24 h. Results are from four biological replicates.