Fig. 7

Inhibition of the TGF-β pathway during cellular transformation reduces metaplastic phenotypes in basal epithelial-derived tumors. A Activation of the TGF-β pathway and EMT collaborate to drive cellular dysfunction and growth arrest. Adapted variants that emerged from stasis cultures exhibit notable changes in gene expression associated with growth and differentiation and are stabilized in a hybrid EMT state. Inhibition of the TGF-β pathway eliminates these selective pressures during the expansion of basal epithelial cells. B Basal epithelial cell populations: (1) P3, cultured in MEGM + DMSO for three passages, (2) variant, cultured in MEGM + DMSO for eight passages, and (3) A83-01, cultured in MEGM + 500 nM A83-01 for three passages, were oncogenically transformed through sequential transduction with TERT and SV40, followed by selection (puromycin and hygromycin) and lentiviral transduction of oncogenic KRAS^G12V followed by a second round of selection (blasticidin and IRES-RFP). Western blot analysis was performed on lysates from these oncogenically transformed basal epithelial cells using antibodies against RAS and Actin (as a loading control). C At each passage, Oncogenically transformed basal epithelial cells were counted. A growth curve of the cumulative population doubling over time was plotted. D Transformed basal epithelial populations were suspended in soft agar in each well of a 6-well plate at a concentration of 20,000 cells/well and cultured in DMEM/F12 + 20% FBS. After 4 weeks, the resulting colonies were stained with crystal violet, imaged, and counted using the ImageJ software. E P-cadherin, N-cadherin, Slug, Snail, Twist, and Zeb were analyzed qPCR using cDNA produced from oncogenically transformed basal epithelial cell populations. F Variant and A83-01-treated oncogenically transformed basal epithelial cells were immunostained for P-cadherin and N-cadherin, then visualized by flow cytometry. G Variants and A83-01-treated oncogenically transformed basal epithelial cells (4 × 10⁶) were injected subcutaneously with 50% Matrigel into 8-to 12-week-old female NSG mice. Tumors formed over a period of 3–6 months. FFPE sections from five tumors were subjected to multiplexed immunohistochemical analysis for p63, cytokeratin 13 (CK13), Slug + Snail, and cytokeratin 5 + vimentin. Human-specific lamin A/C was used to identify the tumor cells