Figure 7. The TGF-β-induced CD44+/CD24− cell state increases the adaptability of cell populations.
(A and B) A549 cells exposed to TGF-β acquire phenotypic and molecular changes characteristic of a CD44+/CD24− cell state. Upon TGF-β withdrawal, the cells return to their original cell state, as indicated by the FACS analysis in (A) and the RT-PCR analysis in (B). See Figure 7—figure supplement 1 for quantification of FACS analysis. (C) The chart indicates the number of DNA joint points in TGF-β-naïve and TGF-β-treated A549 cells. Each dot represents the analysis of a single cell. The breakpoint matrix (utilized to calculate DNA joint points) is generated using Ginkgo (http://qb.cshl.edu/ginkgo). A variable bin size of 175 kb is used. p-value *<0.05, unpaired t-test. (D) The table depicts the IC50 values of A549, A549-3W△ and A549-6W△ cells in the context of treatment with the indicated drugs. (E and F) TGF-β treatment increased the adaptability of cells. Cells that were transiently exposed to TGF-β for 3 or 6 weeks (A549-3W△, A549-6W△) were then treated with the indicated drugs upon TGF-β withdrawl. (E) The number of colonies (mean ± SD) that have survived epitaxol (1.6 μM), tunicamycin (3.2 μM) and cisplatin (1 mM) treatment. Notably, the concentration of drugs used in this experiment corresponds to approximately >100X the IC50. Two independent experiments, each with three replicates, were carried out and approximately 50 fields were counted for each sample. (p-value *<0.05, **<0.005, paired t-test.) (F) A549 tunicamycin-resistant clones were grown in regular/drug-free medium for a week and then retested for sensitivity to tunicamycin (3.2 μM) or epitaxol (1.6 μM). The plot represents mean ± SD number of colonies surviving 5 days after treatment compared with untreated cells, from two independent experiments each with three replicates (p-value **<0.005, paired t-test). (G) Heat map of normalized read counts across segment breakpoints (using Euclidian distance and the ward-clustering method) of the indicated cells. Each horizontal line across the y-axis represents an individual cell, whereas the x-axis annotates the CNA across chromosomes from chr1 on the left to the sex chromosomes on the right. A heat map of cisplatin-naïve cells is shown on the left and of cisplatin-resistant cells on the right. (H) Cluster dendogram of normalized read counts across segment breakpoints (using Euclidian distance and the ward-clustering method) of cisplatin-resistant-A549, cisplatin-resistant-A549-3W△ and cisplatin-resistant-A549-6w△. The cluster dendogram and heat-map of normalized read counts were generated using Ginkgo. (I) Schematic of proposed model. When cells transit into a CD44+/CD24− state, they acquire mesenchymal-like features and autocrine secretion of TGF-β that leads to the downregulation of HDR genes. This process results in a hyper-mutable phenotype that spurs genetic diversity and intra-tumor clonal heterogeneity. Consequently, following a Darwinian model of cancer evolution, the transition of cancer cells into a CD44+/CD24− state or exposure to TGF-β leads to an increased adaptability to any given perturbation.
Figure 7—figure supplement 1. A549 cells undergo changes in the surface expression of CD44 and CD24 before, during and after exposure to TGF-β.
