Triple negative breast cancer initiating cell subsets differ in functional and molecular characteristics and in γ-secretase inhibitor drug responses

Abstract

Increasing evidence suggests that stem-like cells mediate cancer therapy resistance and metastasis. Breast tumour-initiating stem cells (T-ISC) are known to be enriched in CD44(+) CD24(neg/low) cells. Here, we identify two T-ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44(+) CD24(low+) subpopulation generates CD44(+) CD24(neg) progeny with reduced sphere formation and tumourigenicity. CD44(+) CD24(low+) populations contain subsets of ALDH1(+) and ESA(+) cells, yield more frequent spheres and/or T-ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA-MB-231 model, metastatic potential. CD44(+) CD24(low+) but not CD44(+) CD24(neg) express activated Notch1 intracellular domain (N1-ICD) and Notch target genes. We show N1-ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44(+) CD24(low+) cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44(+) CD24(low+) cells, but CD44(+) CD24(neg) were resistant. While GSI hold promise for targeting T-ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T-ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti-cancer drug responsiveness.

Keywords: GSI; Notch1; Sox2; breast cancer stem cells; metastasis.

Figure 1. CD44⁺CD24^low+ and CD44⁺CD24^neg population characteristics

1. A. CD44 and CD24 in MDA-MB-231, DT-22 and MCF7. Unstained controls are shown.

2. B. Surface expression of CD44 and CD24 in DT-22 at passage four (P4) was similar to that at passage 11 (P11).

3. C,D. Mean ± SEM serial mammospheres formed/10⁴ cells seeded from sorted CD44⁺CD24^low+ and CD44⁺CD24^neg from MDA-MB-231 (*p = 0.0003) (C) and DT-22 (*p = 0.0001 Student's t-test) (D).

4. E. Mean ± SEM soft agar colonies from MDA-MB-231 (*p = 0.00024) and DT-22 (*p = 0.0016).

5. F,G. ESA⁺ and ALDH1⁺ are detected in a minority of CD44⁺CD24^low+ but not in CD44⁺CD24^neg populations. CD24 and CD44 were assayed together with either ESA or Aldefluor assays as described. Cells gated CD44⁺CD24^neg and CD44⁺CD24^low+ from MDA-MB-231 (F) and DT-22 (G) were assayed for percentage of surface ESA (left) and percentage of ALDH1⁺ cells (right).

Figure 2. CD44⁺CD24^low+ give rise to both CD44⁺CD24^low+ and CD44⁺CD24^neg progeny while CD44⁺CD24^neg yield only CD44⁺CD24^neg

CD44⁺CD24^low+ or CD44⁺CD24^neg MDA-MB-231 were sorted and 100,000 cells cultured over 21 days.

1. CD44 and CD24 analysis before and 12 days after sorting.

2. Population growth from sorted CD44⁺CD24^low+ and CD44⁺CD24^neg.

3. Proportions of CD44⁺CD24^low+ or CD44⁺CD24^neg cells arising from CD44⁺CD24^low+ cells.

4. Growth curves of progeny arising from CD44⁺CD24^low+ over time. Findings validated in DT-22 and DT-25, see in Supporting Information Fig S3.

5. Model depicting largely asymmetric division of CD44⁺CD24^low+ cells generating mostly CD44⁺CD24^neg progeny with a modest increase in CD44⁺CD24^low+ over time.

6. CD44 and CD24 analysis of dissociated spheres initiated by single isolated CD44⁺CD24^low+ or CD44⁺CD24^neg cells. Scale bar = 75 µm.

Figure 3. CD44⁺CD24^low+ generate orthotopic tumours with higher frequency and shorter latency

Sorted CD44⁺CD24^neg or CD44⁺CD24^low+ from MDA-MB-231 and DT-22 cells were injected orthotopically as described.

1. Tumour formation from indicated numbers of sorted CD44⁺CD24^neg (red) or CD44⁺CD24^low+ (blue) DT22 cells is graphed over time.

2. Calculated T-ISC frequency from limiting dilution assays with DT22 CD44⁺CD24^neg and CD44⁺CD24^low+.

3. Tumours from 10,000, 1000 and 100 sorted CD44⁺CD24^neg (red) or CD44⁺CD24^low+ (blue) MDA-MB-231 cells.

4. Mean MDA-MB-231 xenograft volume from 10,000 injected CD44⁺CD24^neg (red) or CD44⁺CD24 (blue) cells (means graphed ± SEM, *p = 0.019, comparative analysis of growth curves).

5. Primary tumours (1 cm diameter) were excised and metastasis monitored by IVIS. Representative IVIS images are shown.

6. Bioluminescence (photons × 10⁸/s) of metastatic tumour burden from CD44⁺CD24^low+ tumours and CD44⁺CD24^neg is graphed (mean graphed ± SEM, *p = 0.001, comparative analysis of growth curves).

7. Gene set analysis (GSA) shows preferential expression of lung and brain metastasis signatures in CD44⁺CD24^low+ versus CD44⁺CD24^neg. Shown are ordered gene scores for each gene in the line plot and the average fold change in the heatmap (orange = high expression in CD44⁺CD24^low+ and blue = low); enrichment score and p-value shown in upper left (see also bone mets signature and similar GSA for sorted DT-22 in Supporting Information Fig S4).

Figure 4. Notch1-ICD, Notch2-ICD, Notch4-ICD and embryonic transcription factors are preferentially expressed in CD44⁺CD24^low+

CD44⁺CD24^neg (CD24^neg) and CD44⁺CD24^low+ (CD24^low+) subpopulations of MDA-MB-231 and DT-22 cells were flow sorted.

1. Western blots.

2. Relative abundance of Notch 1, 2, 3 and 4 (top) expression by qPCR and mean fold increase in CD24^low+ versus CD24^neg ± SEM (bottom) are shown for MDA-MB-231.

3. Notch target gene expression is enriched in MDA-MB-231 CD44⁺CD24^low+ cells.

4. GSA shows CD44⁺CD24^low+ preferentially express Notch target genes defined by GSI washout.

5. GSA shows CD44⁺CD24^low+ preferentially express NOS-targets and NOS-TFs. The ordered scores for each gene in the line plot and the average fold change in the heatmap are shown (orange indicates high in CD44⁺CD24^low+ and blue, low) with enrichment scores and p values (see similar GSA for ES-TFs for both lines and NOS-targets, and NOS-TFs in sorted DT-22 in Supporting Information Fig S5).

Figure 5. Notch1-mediated Sox2 activation governs stem cell-like phenotype of CD44⁺CD24^low+ cells

1. A. SOX2 qPCR in HC-11 cells ± N1-ICD, N2-ICD, N3-ICD or N4-ICD overexpression, graphed as fold change versus parental controls, normalized to HPRT. *p = 0.018.

2. B. ChIP shows N1-ICD and control histone H3 binding to indicated CSL binding sites in murine SOX2 promoter in HC-11 (top). Schematic of murine SOX2 promoter (bottom).

3. C–E. Controls or N1-ICD overexpressing MDA-MB-231 cells were assayed for N1-ICD and Sox2 by Western (C) SOX2 expression by qPCR (*p = 0.006) (D) and relative binding of N1-ICD to SOX2 promoter (E) and means graphed ± SEM.

4. F–H. Effects of siSOX2 were assayed in MDA-MB-231 ± N1-ICD overexpression on spheres formed/10,000 plated cells (F), % of CD44⁺CD24^low+ (G) and % ALDH1⁺ cells (H). Graphs show mean ± SEM; For (F), ***p = 0.019, **p = 0.032 and *p = 0.026; for (G), ***p = 0.0049, **p = 0.0048 and *p = 0.0007 and for (H), ***p = 0.04, **p = 0.03 and *p = 0.01 (Student's t-test compared to control, C).

5. I. Cell cycle profiles of siSOX2 in MDA-MB-231 ± N1-ICD overexpression.

6. J. Proliferation curves are unchanged by siSOX2 in MDA-MB-231 cells ± N1-ICD overexpression.

Figure 6. Gamma-secretase inhibitors target the CD44⁺CD24^low+ but not the CD44⁺CD24^neg population

CD44⁺CD24^low+ (CD24^low+) and CD44⁺CD24^neg (CD24^neg) cells sorted from MDA-MB-231 (A and B) and DT22 (C and D) ± RO4929097.

1. A,C. Cleaved Notch1 and Sox2 levels before (C) and after 24 h of 10 µM RO4929097 in CD24^low+ cells.

2. B,D. Serial mammospheres from indicated sorted cell populations ±10 µM RO4929097 (mean ± SEM, Student's t-test). The p values in panel B: *p = 0.00002; **p = 0.0018; ***p = 0.004; panel D: *p = 0.00004; **p = 0.003; ***p = 0.0015. The GSI, DAPT, showed similar effects on sorted MDA-MB-231, DT-22 and DT-25 see Supporting Information Fig S6A–F.

3. E. Migration and matrigel invasion of sorted populations from MDA-MB-231 ± 10 µM RO4929097 (RO) generated by xCELLigence real time cell analysis, graphed as mean ± SD. CD24^low+ cells show significantly greater migration and invasion compared to CD24^neg cells (p = 0.0003 at T = 48 h). RO significantly inhibited migration (p = 0.0002 at T = 48hrs) and invasion (p = 0.0002 at T = 48 h) of CD24^low+ cells. CD24^neg cells were unaffected (see also data for DT-22 in Supporting Information Fig S6G).

4. F. Cell cycle profiles of sorted DT-22 populations after 48 h ±10 µM RO were unchanged. Cell cycle profiles and proliferation curves of sorted populations were similar with and without RO treatment in MDA-MB-231 and DT-22 (see Supporting Information Fig S6H–J).

5. G. Volume of DT-22 xenografts arising from CD24^low+ or CD24^neg ±14-day RO4929097 treatment as described, graphed as mean ± SEM, Student's t-test. *p = 0.005 (Day 40); *p = 0.008 (Day 48); *p = 0.004 (Day 55).

6. H. Tumours arising from sorted CD24^low+ or CD24^neg DT-22 cells (n = 18 each group) were excised at mean 1.2 cm, dissociated into single cell suspensions and stained for surface CD44 and CD24 expression. All cells were CD44⁺. Mean CD24 expression in each tumour group is shown as a mean % of total ± SEM (*p = 0.0003, Student's t-test).

Figure 7. Heterogeneity within CD44⁺CD24^low+ subpopulations with regard to GSI sensitivity

1. MDA-MB-231 cells were flow sorted into either CD44⁺CD24^neg, CD44⁺ CD24^low+ ESA− and CD44⁺CD24^low+ ESA⁺ subpopulations and analysed for frequency of sphere forming cells by limiting dilution assay ±10 µM RO4929097 (RO). Mean of spheres/cells plated are plotted from triplicate data from each of two independent experiments. The p values for 10,000 cells: ***p = 0.01; **p = 0.03; *p = 0.01 and 0.03; for 5000 cells: *p = 0.001 and 0.007; for 2500 cells: *p = 0.001 and 0.007; for 1250 cells: *p = 0.003 and 0.006; for 600 cells: *p = 0.001 and 0.007; for 300 cells: *p = 0.02 and 0.007 (Student's t-test).

2. MDA-MB-231 cells were flow sorted into either CD44⁺CD24^neg, CD44⁺CD24^low+ALDH1⁻ and CD44⁺CD24^low+ALDH1⁺ subpopulations and analysed for frequency of sphere forming cells by limiting dilution assay ±10 µM RO. Mean spheres/cells plated are plotted from triplicate data from each of two independent experiments. The p values for 10,000 cells: ***p = 0.01; **p = 0.01; *p = 0.001 and 0.01; for 5000 cells: ***p = 0.01; **p = 0.05; *p = 0.008 and 0.001; for 2500 cells: ***p = 0.01; **p = 0.02; *p = 0.001 and 0.003; for 1250 cells: *p = 0.047 and 0.00045; for 600 cells: *p = 0.005 and 0.00045; for 300 cells: *p = 0.007 and 0.0002 (Student's t-test). Cells used in (A) and (B) above were sorted simultaneously.

Figure 8. CD44⁺CD24^neg cells convert to CD44⁺CD24^low+ upon Notch1 activation

1. A–D. N1-ICD was expressed MDA-MB-231 and N1-ICD-CD44⁺CD24^neg (A,B,D) and N1-ICD-CD44⁺CD24^low+ (C,D) populations were sorted and compared with their counterparts in vector control MDA-MB-231 cells. Progeny of each were counted, and the proportion of cells expressing CD24 analysed by flow cytometry over a period of 15 days is graphed as mean ± SEM. Note that cell number increase over time was similar with and without N1-ICD expression (Fig 5J). NI-ICD expression is shown by western blot in controls and in N1-ICD-CD44⁺CD24^neg and N1-ICD-CD44⁺CD24^low+ (D).