Markers of tumor-initiating cells predict chemoresistance in breast cancer

Abstract

Purpose: Evidence is lacking whether the number of breast tumor-initiating cells (BT-ICs) directly correlates with the sensitivity of breast tumors to chemotherapy. Here, we evaluated the association between proportion of BT-ICs and chemoresistance of the tumors.

Methods: Immunohistochemical staining(IHC) was used to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and proliferating cell nuclear antigen, and TUNEL was used to detect the apoptosis index. The significance of various variables in patient survival was analyzed using a Cox proportional hazards model. The percentage of BT-ICs in breast cancer cell lines and primary breast tumors was determined by ALDH1 enzymatic assay, CD44(+)/CD24(-) phenotype and mammosphere formation assay.

Results: ALDH1 expression determined by IHC in primary breast cancers was associated with poor clinical response to neoadjuvant chemotherapy and reduced survival in breast cancer patients. Breast tumors that contained higher proportion of BT-ICs with CD44(+)/CD24(-) phenotype, ALDH1 enzymatic activity and sphere forming capacity were more resistant to neoadjuvant chemotherapy. Chemoresistant cell lines AdrR/MCF-7 and SK-3rd, had increased number of cells with sphere forming capacity, CD44(+)/CD24(-) phenotype and side-population. Regardless the proportion of T-ICs, FACS-sorted CD44(+)/CD24(-) cells that derived from primary tumors or breast cancer lines were about 10-60 fold more resistant to chemotherapy relative to the non- CD44(+)/CD24(-) cells and their parental cells. Furthermore, our data demonstrated that MDR1 (multidrug resistance 1) and ABCG2 (ATP-binding cassette sub-family G member 2) were upregulated in CD44(+)/CD24(-) cells. Treatment with lapatinib or salinomycin reduced the proportion of BT-ICs by nearly 50 fold, and thus enhanced the sensitivity of breast cancer cells to chemotherapy by around 30 fold.

Conclusions: These data suggest that the proportion of BT-ICs is associated with chemotherapeutic resistance of breast cancer. It highlights the importance of targeting T-ICs, rather than eliminating the bulk of rapidly dividing and terminally differentiated cells, in novel anti-cancer strategies.

Figure 1. Aldehyde dehydrogenase (ALDH1) expression correlates with clinical outcome of breast cancer patients.

(A) Immunohistochemical staining shows tumors with poor clinical response (progressive or stable disease, PD/SD) to neo-adjuvant chemotherapy express high ALDH1 (>20% positive cancer cells) in pre-chemotherapy samples, and tumors with partal response (PR) express low ALDH1 (≤20% positive cancer cells). High proliferating cell nuclear antigen (PCNA)(>25% positive cancer cells) and poor apoptosis are observed in tumors with PD/SD after neo-adjuvant chemotherapy. Representative images of ALDH1 (×200), PCNA (×200) and Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP labeling (TUNEL,×400). (B) Surgically removed tumor samples obtained after neoadjuvant chemotherapy from patients with low ALDH1 have higher percentage of TUNEL-staining cells (r = −0.892, p<0.001), but lower percentage of PCNA-staining cells (r = 0.804, p<0.001). (C–D)Kaplan-Meier curves with log rank tests show statistical difference in overall survival (Log Rank = 7.987, p = 0.005) (C) and disease-free survival (Log Rank = 19.347, P<0.001) (D) between patients with high ALDH1 expression and low ALDH1 expression.

Figure 2. Breast tumor initiating cell (BT-IC) proportion correlates with poor clinical response to neo-adjuvant chemotherapy.

(A) The example of ALDH1⁺ primary cancer cells in the progressive disease (PD) samples (symbol “•”, n = 13) is 13.2% whereas 0.4% in partial response (PR) samples (symbol “⧫”, n = 13) (p = 0.007). All these 26 cancer tissue samples were obtained before chemotherapy. (B) Breast tumors with progressive or stable disease(PD/SD) (7 cases) have more cancer cells CD44⁺/CD24⁻ phenotype than those with PR (7 cases) (35.0%±9.13% vs. 1.56%±0.33%, p<0.001). (C) Mammosphere formation of primary cancer cells in breast tumors with PD/SD is 10–20 fold higher than those with PR (15.2% vs. 0.83%, p<0.001).

Figure 3. BT-ICs are intrinsic chemoresistance independent of individual tumors and cell lines.

(A) The percentage of adriamycin resistant MCF-7 (AdrR/MCF-7) cells and epirubicin resistant SKBR3 (sk-3rd) cells with the phenotype of CD44⁺CD24⁻ is significantly higher than parental MCF-7 line and SKBR3 line tested by FACS (42.38±4.80% vs. 1.82%±0.64%, 64.34±2.62% vs. 0.68%±0.16%, p<0.001). (B) FACS-sorted CD44⁺CD24⁻ MCF-7 and CD44⁺CD24⁻ AdrR/MCF-7 was about 10–60 fold higher resistant to adriamycin relative to the parental cells and non-CD44⁺CD24⁻ cells. CD44⁺CD24⁻ SK-3rd and primary cells were about 25 fold higher resistant to epirubicin relative to parental cells and non-CD44⁺CD24⁻ cells by using cell viability assay. (C)Compared with non-BT-ICs, chemotherapy (adriamycin or epriubicin 0.5µg/ml for 24 hours) increased Annexin V positive cells of non-CD44⁺/CD24⁻cells, which derived from tumors with high or low proportion T-ICs, MCF-7, AdrR/MCF-7 and SK-3rd breast cancer cell lines containing different number of T-ICs (p<0.001). (D) MDR1 (multidrug resistance 1) and ABCG2(ATP-binding cassette sub-family G member 2) protein expression is both up-regulated in CD44⁺/CD24⁻ cells from different cell lines and primary cells. All the experiments were repeated 3 times independently.

Figure 4. Reducing the proportion of BT-ICs sensitizes breast cancer cells to chemotherapy.

(A)Treatment with salinomycin or lapatinib significantly reduced mammosphere formation in CD44⁺/CD24⁻ MCF-7 cells and primary cells by approximately 10 fold relative to DMSO control (p<0.001). (B) Treatment with salinomycin or lapatinib reduced the percentage of ALDH1⁺ in CD44⁺/CD24⁻ MCF-7 cells by 5–10 fold and primary cells by 50 fold. (C) Addition of lapatinib or salinomycin reduced the percentage of cloning efficiency of CD44⁺CD24⁻ MCF7 cells by approximately 10–30 fold (p<0.001). (D) The sensitivity of CD44⁺/CD24⁻ MCF-7 cells to adriamycin increased in the presence of lapatinib or salinomycin, as the dead cells increased from 27.8%±0.85% and 32.8%±2.6% to 75.1%±4.1%% and 77.9%±4.8% respectively determined by FACS analysis of Annexin V and PI staining(*p<0.01, **p<0.001, mean±SD). All the experiments were repeated 3 times independently.