Immune-induced epithelial to mesenchymal transition in vivo generates breast cancer stem cells

Abstract

The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell-dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24(-/lo)CD44(+) phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse.

Figure 1. Induction of EMT in vivo requires CD8 T cells

Panel A shows tumor growth in control, CD4-depleted, and CD8-depleted mice. Each point is the mean (s.e.m.) of measurements from 3 tumors. A repeat experiment yielded identical results. Panel B, tumor growth (mean and s.e.m., n=3) in CD8-depleted mice that had been injected with parental E cells on day 0 and rechallenged at day 47 with antigen-loss variant tumor cells (M3). Panel C, shown are the proliferation rates (counts per minutes incorporation of thymidine) of antigen-negative tumor cells incubated with RPMI-1640 + 10% FBS (control medium), medium with 20% FBS, conditioned medium from resting CD8 T cells (resting CD8 CM), conditioned medium from anti-CD3/anti-CD28 stimulated T cells, or conditioned medium from ConA-stimulated T cells cocultured with irradiated splenocytes. Each bar is the mean (s.e.m., n=3). Panel D, shown are the percentages of tumor cells which lost neu and had reduced CD24 (epithelial marker) expression following exposure of epithelial (E) tumor cells to control (regular) medium, tumor-primed CD8 T cells (PCD8), or irradiated tumor-primed CD8 T cells (irPCD8). Each bar is the mean (s.e.m.) of 3 separate samples.

Figure 2. Stable cell lines from tumor cells that underwent EMT were established

Panel A shows expression levels of E-cadherin and claudin 3 in E and M cell lines. The E-cadherin experiments were repeated independently 3 times with similar results. Claudin 3 was measured using flow cytometry and each bar is the mean (s.e.m.) of 2 separate experiments. Panel B shows expression of N-cadherin, and Snail, and matrix metalloproteinase (MMP) activity for all cell lines. The bars represent the signal intensity derived using RT-PCR. RT-PCR results are representative of 2 experiments that gave nearly identical results. For MMP activity each bar is the mean (s.e.m.) of 3 samples. Panel C shows light microscope pictures (40X) of the cell lines. Bar=100 μm.

Figure 3. Mesenchymal cells are CD24^−/loCD44⁺ and highly tumorigenic

Panel A shows a representative histogram demonstrating that mesenchymal (M) tumor cells are CD24^−/lo CD44⁺. Dark and light tracings represent specific and isotype fluorescence, respectively. Panel A also shows graphs that summarize CD24 and CD44 expression in the E and M cells. Shown are the average (± s.e.m.) relative mean fluorescent intensities (rMFI) calculated from 3 experiments. Panel B shows tumor growth curves of the E (left) and M3 (right) cell lines injected into neu-tg mice. Each data point is the mean (s.e.m.) tumor size calculated from three mice. Numbers indicate the dose of cells injected. On the right, images of the tumors (20X) and spheroids (20X) corresponding to E and M3 cell lines. The far right graph shows the mean (± s.e.m, n=5 independent experiments) spheroid forming efficiency of each line. Panel C (left) is a summary of dot plots from CD24, CD44, and neu expression in tumors derived from sorted CD24⁻CD44⁺ M3 mesenchymal cells. This experiment is representative of 4 similar experiments done with each of the mesenchymal cells lines. Quadrants were established using M3 cell line cells (not shown). Inset numbers indicate percentage of gated cells in upper right quadrant. Panel D shows histograms of neu expression in cells from a mesenchymal cell line (Line), flank tumors (Flank), and mammary fat pad tumors (Fat pad) are shown. Inset numbers represent the relative mean fluorescent intensity. Nearly identical results were seen with 4 different samples of each cell type which is shown averaged in accompanying graph.

Figure 4. Mesenchymal cell lines have elevated expression of drug pumps, DNA repair enzymes and are resistant to environmental insults

Panel A (left) shows western blot analysis of BCRP and PGP drug pumps in all cell lines. β actin was used as a loading control. Western blots are representative of three experiments for each protein. Panel A (middle) shows inhibition of growth (i.e. compared to control treated cells) of the tumor cells lines after in vitro treatment with mitoxantrone. Panel A (right) shows the apoptotic response to etoposide as assessed using flow cytometry. Each bar is expressed as the % apoptotic cells following treatment. A representative experiment is shown for MTT and apoptotic assays, both performed twice. Panel B (left) depicts the expression levels of MGMT in the tumor cell lines by real time PCR. Results are expressed as fold expression over E and represent one experiment. Panel B (right) shows the results of a clonogenic assay of the tumor cells following exposure to escalating doses of BCNU. Each data point is the mean (s.e.m.) of 3 replicates. Panel C (left) shows the results of real-time PCR analysis of the double stranded DNA repair genes. Results are expressed as fold expression over E. Panel C (right) shows the results of clonogenic assay after escalating doses of gamma irradiation. Each data point is the mean (± s.e.m.) of 3 replicate plates.

Figure 5. Gene expression of luminal or basal epithelial markers in mesenchymal cells suggests that EMT is incomplete

Panel A shows the RT-PCR results depicting expression of CK8 (left) and CK14 (right) in the tumor cell lines. Results are representative of 3 independent experiments. Panels B and C show RNA microarray analyses of common luminal epithelial and basal markers in the cell lines, respectively. Shown are the mean (s.e.m.) signal intensities of 2 microarray analyses for each cell line. Each bar represents the signal intensity for a unique probe.