Mammosphere Formation Efficiency Research Articles

Abstract 2319: Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumors

Abstract Sulforadex (SFX) is a novel therapeutic comprising synthetic sulforaphane (SFN) stabilised within α-cyclodextrin. SFN has been studied as an anti-cancer compound for many years however its development has hitherto been hampered due to its inherent instability. Breast cancer stem-like cells (CSCs) have been identified in all molecular subtypes and are believed to be the drivers of breast cancer metastasis. We investigated SFX effects on breast CSC activity using mammosphere formation and aldehyde dehydrogenase (ALDH) activity in patient samples and patient-derived xenograft (PDX) tumours. Mammosphere formation efficiency (MFE) assessed in cells from primary (n = 12) and metastatic (n = 15) samples treated with SFX (5 μM) or vehicle control (water) showed a significant reduction in breast CSC activity following SFX treatment. A reduction greater than 50% in the MFE was observed in both primary (0.52%±0.06 vs 0.19±0.02, p<0.001) and metastatic (0.93%±0.07 vs 0.43±0.04, p<0.001) cells treated with SFX. Importantly, SFX also inhibits breast CSC activity of early (HBCx34) and metastatic (BB3RC31) patient-derived xenograft (PDX) tumours. Using a 14 day in vivo ‘window’ treatment, we observe that SFX (300mg/Kg/day) decreases ALDH+ cells using ALDEFLUOR assay (HBCx34 6.3%±0.4 vs 3%±0.6, p<0.01; BB3RC31 3%±0.6 vs 1±0.2, p<0.05) and MFE (HBCx34 0.64%±0.09 vs 0.35%±0.03, p<0.01; BB3RC31 0.89%±0.06 vs 0.78±0.04). The majority of breast cancers express the estrogen receptor (ER) but these hormone-responsive breast cancers frequently develop resistance to hormonal therapies (e.g. tamoxifen). We have established that CSCs in ER+ BC lack ER expression, representing a potential mechanism of therapeutic resistance. However, SFX targets such surviving CSCs identified by ALDH positivity and mammosphere-initiating capacity following tamoxifen treatment in vivo in ER+ PDX tumours. Combinatorial treatment of tamoxifen (10 mg/kg/day) and SFX (300mg/Kg/day) in a 14 day ‘window’ treatment reduced ALDH+ cells (HBCx34 10%±0.4 vs 4.2%±0.4, p<0.01; BB3RC31 11.3%±1.1 vs 6.9±0.6, p<0.05) and MFE (HBCx34 0.81%±0.07 vs 0.34%±0.02, p<0.01; BB3RC31 0.47%±0.06 vs 0.27±0.04, p<0.05) compared to tamoxifen treatment alone. Mechanistically SFX potently inhibited the canonical Wnt pathway in MCF-7 cells and their endocrine-resistant derivatives and future studies will explore SFX activity on other CSC regulatory pathways. Our data demonstrate that SFX might produce clinically meaningful improvements to endocrine therapy in ER+ BC by reversing CSC mediated resistance. Clinical trials of endocrine therapy in combination with SFX are currently being planned. Citation Format: Bruno M. Simões, Denis Alferez, Rachel Eyre, Kath Spence, Angélica Santiago-Gomez, Iris Tanaka, Bertram Kohler, David Howat, Sacha J. Howell, Robert B. Clarke. Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2319. doi:10.1158/1538-7445.AM2015-2319

Abstract 2312: Identifying novel cancer stem cell target for triple-negative breast cancer

Abstract Triple negative breast cancer is characterized by its aggressive clinical behavior with high incidence of metastasis to the lungs and brain. These patients do not respond to hormonal therapy and show intrinsic resistance to conventional chemotherapy. Robust evidence indicates that treatment-resistance and metastases may arise from a subpopulation of cells with tumor-initiating capacity called breast cancer stem cells (BCSC). We are one of the first groups to demonstrate that residual tumors after exposure to chemotherapy are enriched for BCSC. We have previously described a treatment-resistant gene signature of 493 genes derived from patient biopsies.This finding narrowed down the possible cancer stem cell growth related genes from genome wide to only 493 genes and provided candidate genes for screening potential targets that affect BCSC self-renewal. Re-evaluation of the candidate genes determined a previously unidentified cancer gene, Hematological and Neurological Expressed 1-Like (HN1L) as the target of cancer stem cell self-renewal. The objective of this study was to investigate the role of HN1L in regulating BCSC and metastasis in TNBC, and to determine the mechanism of action of HN1L in BCSC. Knocking down HN1L by shRNA in SUM159 and MDAMB231 cell lines significantly decreased mammosphere forming efficiency (MSFE) and CD44+/CD24low/- population. To assess the contribution of HN1L to BCSC and tumor growth, a patient derived human-cancer-in-mouse xenograft model and two cancer cell line xenograft models were employed. To ensure targeted delivery, siRNA was packaged into DOPC liposomes and delivered into mice via intraperitoneal injection. Results showed that silencing HN1L alone or in combination with chemotherapy in xenografts reduced tumor volume and BCSC population as measured by MSFE, CD44+/CD24low/- markers, ALDF+ cell population and limiting dilution assay. Liposomal HN1L siRNA treated mice also showed lower level of lung metastasis. A gene signature obtained from microarray analysis on HN1L siRNA treated tumor tissues highly correlated with better prognosis in TNBC patients. Overexpression of HN1L in TNBC cell lines activated STAT3 signaling, induced CD44+/CD24low/- population and cancer cell migration. Moreover, chemoresistance were also observed with HN1L overexpression. HN1L colocalized with CNTFR and STAT3 in cytoplasm and nucleus by immunofluorescence staining. Changes in HN1L expression level resulted in corresponding changes in phospho-STAT3. Furthermore, CNTFR knockdown compromised the effect of HN1L overexpression in activating STAT3.These results suggest that the HN1L gene plays an important role in BCSC through regulating CNTFR-STAT3 signaling pathway, and is associated with poor prognosis. Taken together, the results from our study demonstrate for the first time that HN1L is a novel target for TNBC. Targeting HN1L and/or CNTFR-STAT3 pathway might provide an alternative therapeutic strategy for TNBC. Citation Format: Yi Liu, Dong Soon Choi, Sergio Grandos-Principal, Wei Qian, Lacey Burey, Helen Wong, Crisitian Rodriguez-Aguayo, Anil Sood, Zheng Li, Stephen Wong, Heidi Weiss, Bhuvanesh Dave, Melissa Landis, Jenny C. Chang. Identifying novel cancer stem cell target for triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2312. doi:10.1158/1538-7445.AM2015-2312

Abstract 349: Autophagy, cancer stem cells, and trastuzumab resistance in three dimensional cultures of HER2+ breast cancer cells

Abstract HER2 is overexpressed in 20% invasive breast tumors and correlates with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used to treat HER2+ tumors; however more than half of the patients are resistant or acquire resistance during treatment. Multicellular tumor spheroids (MS) are a 3D growth model that mimics the structure of in vivo avascular tumors. Autophagy has been proposed as a tumoral escape mechanism. The cancer stem cell (CSC) hypothesis postulates that tumor growth is maintained by a small fraction of cells with unlimited proliferative potential that are also able to differentiate into cell populations that become the bulk of the tumor. CSCs are thought to be more resistant to chemotherapy and radiotherapy and related with tumor recurrence. We have previously demonstrated that MS present different subpopulations showing basal autophagy and apoptosis that increase towards the center of the spheroid, and these characteristics make them more resistant to Tz than monolayers. We also observed that Tz inhibited basal apoptosis in MS and was capable of inducing autophagy. Our aim was to analyze alternative mechanisms of resistance of HER2+ breast cancer cells against Tz. For this purpose, we used BT474 (HER2+) human breast cancer cell line, from which we developed a resistant subline (BT474-MR). MS were cultured one per well in agar covered plates. We observed that cells from BT474 MS treated with Tz for 15 days upregulated the autophagy marker LC3 and showed 100% Tz resistance when compared with cells from MS treated with non-related human IgG as control. BT474 cells were treated with the autophagy inhibitor 3-metyiladenine that was dose dependently cytotoxic both in 2D and 3D cultures. Interestingly, the higher concentration used was critical for spheroids survival but not for monolayers. BT474-MR cells were more sensitive to autophagy inhibition than parental cells (75.7 vs 100% cell viability, p<0.05). In order to investigate if the increase in autophagy after Tz treatment could favor a CSCs propagation, we used a mammosphere assay, which enriches for mammary progenitor cells and mammary CSCs. BT474 cells were cultured in low-attachment plates without serum and supplemented with B27; formed mammospheres were treated with Tz for 7 days, after which they were disaggregated and cultured again as secondary mammospheres. We found that Tz, rather than favor CSC growth, was able to effectively target this cell population, significantly decreasing secondary mammosphere-forming efficiency compare to IgG control (227 vs 164 respectively, p<0.05). In conclusion, Tz exerted a differential effect in cells cultured as MS, inducing autophagy related to resistance. Since BT474 CSCs were a specifically aim of this immunotherapy, it is valid to propose that autophagy addiction of resistant cells could be associated with an increase in CSCs that are no longer eliminated by Tz. Citation Format: Cristina E. Rodríguez, Sara Reidel, Maria Adela Jasnis, Elisa Bal de Kier joffe, Gabriel L. Fiszman. Autophagy, cancer stem cells, and trastuzumab resistance in three dimensional cultures of HER2+ breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 349. doi:10.1158/1538-7445.AM2015-349

Abstract 2314: d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells

Abstract The transmembrane tyrosine kinase receptor HER2, overexpressed in ∼20% of human breast cancers (BCs), identifies an aggressive tumor subtype and is reportedly an important regulator of breast cancer-initiating cell activity (BCIC). We found that ∼90% of HER2+ BC patients constitutively express a splice isoform of wild-type HER2 (WTHER2) gene characterized by the lack of exon 16 (d16HER2), a deletion that promotes the generation of a particularly aggressive HER2 isoform and that forms stable and constitutively activated d16HER2 homodimers. Our comparison of the tumorigenic potential of the human d16HER2 and WTHER2 genes in the corresponding transgenic mouse models revealed a significantly shorter tumor latency period (p< 0.001) and a higher tumor incidence in the d16HER2 mice (p<0.001), suggesting a role for this variant in HER2-driven activation of BCICs. In this context, our preliminary analyses of HER2-positive primary mammary tumor cell lines MI6 and MI7 derived from spontaneous transgenic d16HER2 mice showed a significantly higher mammosphere-forming efficiency and higher levels of stem cell marker transcripts, including CD44, Wnt, Notch and Bmi1, compared to transgenic mouse WTHER2 tumor cells (WTHER2_1 and WTHER2_2). Mammospheres generated from human HER2-overexpressing breast cancer cell lines BT474 and MDA-MB-361, which also express the d16HER2 variant, exhibited an increase in the relative abundance of d16HER2 mRNA compared with that in the same parental cells cultured in adhesion conditions, as indicated by qPCR analyses. Experiments in mice injected into the mammary fat pad with the d16HER2- and WTHER2-positive cell lines at different serial dilutions indicate a consistently higher “stemness potential” of MI6 and MI7 cells compared to WTHER2_1 and WTHER2_2 cells, strongly suggesting that the d16HER2 variant plays a greater role than WTHER2 in regulating BCICs of HER2-driven mammary tumors. Supported by Minister of Health and AIRC Citation Format: Lorenzo Castagnoli, Ada Koschorke, Gaia C. Ghedini, Lorenzo Galvani, Valentina Ciravolo, Cristina Ghirelli, Arianna Palladini, Alessia Lamolinara, Manuela Iezzi, Pier Luigi Lollini, Tiziana Triulzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2015-2314

Mitochondrial biogenesis is required for the anchorage-independent survival and propagation of stem-like cancer cells.

Here, we show that new mitochondrial biogenesis is required for the anchorage independent survival and propagation of cancer stem-like cells (CSCs). More specifically, we used the drug XCT790 as an investigational tool, as it functions as a specific inhibitor of the ERRα-PGC1 signaling pathway, which governs mitochondrial biogenesis. Interestingly, our results directly demonstrate that XCT790 efficiently blocks both the survival and propagation of tumor initiating stem-like cells (TICs), using the MCF7 cell line as a model system. Mechanistically, we show that XCT790 suppresses the activity of several independent signaling pathways that are normally required for the survival of CSCs, such as Sonic hedgehog, TGFβ-SMAD, STAT3, and Wnt signaling. We also show that XCT790 markedly reduces oxidative mitochondrial metabolism (OXPHOS) and that XCT790-mediated inhibition of CSC propagation can be prevented or reversed by Acetyl-L-Carnitine (ALCAR), a mitochondrial fuel. Consistent with our findings, over-expression of ERRα significantly enhances the efficiency of mammosphere formation, which can be blocked by treatment with mitochondrial inhibitors. Similarly, mammosphere formation augmented by FOXM1, a downstream target of Wnt/β-catenin signaling, can also be blocked by treatment with three different classes of mitochondrial inhibitors (XCT790, oligomycin A, or doxycycline). In this context, our unbiased proteomics analysis reveals that FOXM1 drives the expression of >90 protein targets associated with mitochondrial biogenesis, glycolysis, the EMT and protein synthesis in MCF7 cells, processes which are characteristic of an anabolic CSC phenotype. Finally, doxycycline is an FDA-approved antibiotic, which is very well-tolerated in patients. As such, doxycycline could be re-purposed clinically as a 'safe' mitochondrial inhibitor, to target FOXM1 and mitochondrial biogenesis in CSCs, to prevent tumor recurrence and distant metastasis, thereby avoiding patient relapse.

Abstract P6-12-08: The effects of the BKM120 in combination with herceptin on HER2+ BC cells and BCSCs

Abstract Introduction: Breast cancer stem cells (BCSCs) are suspected to be responsible for tumour recurrence, metastasis as well as chemo-resistance. Dysregulated PI3K/Akt signaling is implicated in the pathogenesis of a number of breast cancers, including the HER2+ breast cancer. This study evaluated the efficacy of BKM120 monotherapy and BKM120 plus Herceptin in treatment of normal cells and the BCSCs of the HER2+ breast cancer . Methods:The BCSCs of SK-BR-3 cells were isolated by Serum free cells suspension culture. The impacts of BKM120 monotherapy and BKM120 combined with Herceptin on normal breast cancer cells and BCSCs were assayed by MTT assay, wound-healing assay and plate clone formation assay. The mammosphere-forming efficiency (MFE) of breast cancer cells treated with BKM120 and BKM120 plus Herceptin were also calculated. The nature of the drug interaction of BKM120 and Herceptin was evaluated by using the combination index (CI) according to the method of Chou and Talalay. The impacts of BKM120 monotherapy and BKM120 combined with Herceptin in PI3K/AKT pathway on the normal cancer cells and BCSCs of SK-BR-3 were assayed by Western bloting. Results: BKM120 showed significant antiproliferative activity in the BCSCs, as well as in normal SK-BR-3 cells. After treated with BKM120, invasion abilities of SK-BR-3 cells was significantly weakened(P<0.01).The colony forming efficiency and the MFE was also decreased compared with the control (P<0.01). Additionally, the pAKT and pS6 expression levels were inhibited. The effects of BKM120 in combination with Herceptin were investigated, which was called Combination Index, showed synergism between the two agents in BCSCs as well as in the normal cells. Furthermore, BKM120 plus Herceptin showed more significantly suppression on following aspects in BCSCs and normal cancer cells : proliferation, invasion ability, colony forming as well as MFE. In addition, the levels of pAKT,pS6 in the BKM120 plus Herceptin group were decreased more obviously than those in control and monotherapy group respectively(p<0.05). Conclusions:BKM120 can inhibit the growth of normal cancer cells and BCSCs of SK-BR-3, by inhibiting cell proliferation, impairing the invasion abilities and the MFE, and reducing the expression of pAKT(Thr308,S473)and pS6(Thr389).Moreover, these results suggest that the combination of PI3K Inhibitor BKM120 with Herception may provide a novel therapy strategy for HER2+ BC. Citation Format: Jin Zhang, Feng Yu, Jingjing Liu, Xiaobei Zhang, Yunhui Hu. The effects of the BKM120 in combination with herceptin on HER2+ BC cells and BCSCs [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-12-08.

Abstract P2-06-04: Proteosome inhibitor bortezomib inhibits NFκB and effectively overcomes cancer stem cell escape triggered by Wnt inhibitor therapy in FOXC1+ basal-Like/claudin-low breast cancer

Abstract Cancer stem cells (CSCs) are considered to be an important contributing factor towards treatment failure, cancer recurrence, and mortality. CSCs are known to be more enriched in the basal-like and claudin-low subtypes of breast cancer. The Wnt/β catenin signaling pathway is well known as a regulator of embryonic development and stem cell biology, and is prominently active in basal-like and claudin-low breast cancers. We have previously reported that transcription factor FOXC1 plays a critical role in mediating aggressive cell traits in basal-like/claudin-low breast cancer. In this study, we sought to investigate the link between Wnt signaling and FOXC1 and its potential in regulating CSC biology in basal-Like/claudin-low breast cancer. We observed that exposure of the MDA-MB-231 basal-like/claudin-low cell line (low constitutive FOXC1 expressor) to Wnt3a (a canonical Wnt signaling ligand), resulted in increased expression of FOXC1. Reciprocally, overexpression of FOXC1 in MCF10A human mammary epithelial cells led to a pronounced increase in Wnt signaling activity, strongly suggestive of a direct or indirect positive feedback loop between Wnt signaling and FOXC1. More importantly, BT549 and HS578t basal-like/claudin-low cells (high constitutive FOXC1 expressors) proved to be more sensitive to treatment with the Wnt inhibitor ICRT3 as evidenced by decreased cell viability when compared to MCF7 (luminal) or SKBR3 (HER2) breast cancer cell lines. Furthermore the decrease in cell viability appeared to be proportionate to the level of FOXC1 expression. Upon pharmacological inhibition with ICRT3 and biological inhibition with siRNA knockdown of LRP6, (a canonical Wnt signaling cell surface receptor) a decrease in FOXC1 expression level was observed in a dose and time dependent manner. This effect was particularly pronounced in mammosphere cultures enriched for BT549 cancer stem-like cells. Inhibition of Wnt signaling reduced mammosphere formation efficiency of BT549 cells, suggesting that Wnt inhibition targets cancer stem cells (CSCs) in the basal-like/claudin-low breast cancer subtype. More importantly, however, after an initial 4 day incubation period, some cells are observed to persist and later display renewed enhancement of mammosphere formation ability. Profiling of such cells interestingly revealed depletion of FOXC1+ve cells but persistence of cells displaying pronounced up regulation of stereotypical embryonic stem cell Transcription Factors OCT4, SOX2 and NANOG, strongly suggestive of a potential primitive stem cell/quiescent cell state escape mechanism. qRT-PCR based pathway activation analysis revealed marked activation of NFκB signaling in the residual cells that withstood Wnt inhibition. Simultaneous pharmacologic inhibition with Wnt inhibitor ICRT3 and the proteasome inhibitor Bortezomib (known to inhibit NFκB signaling) effectively targeted BT549 cancer stem cells in mammosphere culture and prevented the persistence/emergence of any residual cells. Taken together, our findings suggest that combination therapy approaches are likely required to effectively target breast cancer stem cells. Citation Format: Partha S Ray, Connie Y Tsai, Tania Ray, Bomy Kim, Tor W Jensen. Proteosome inhibitor bortezomib inhibits NFκB and effectively overcomes cancer stem cell escape triggered by Wnt inhibitor therapy in FOXC1+ basal-Like/claudin-low breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-04.

Abstract P1-07-30: FOXC1/FOXA1 transcriptional balance in breast cancer: From acquisition of mesenchymal and stem cell traits to occult lymph node independent breast cancer metastasis

Abstract Background: Distant metastatic spread of cancer cells to other organs from the primary site of origin currently constitutes the most significant contributor to cancer-related morbidity and mortality. Epithelial-to-mesenchymal transition (EMT) is a biologic transformation of cancer cells from a non-migratory phenotype to a migratory one, and is thought to initiate the metastatic cascade in cancer. EMT has also been reported to trigger acquisition of stem cell traits in breast cancer. Transcription factor (TF) Forkhead box C1 (FOXC1), strongly associated with the basal-like and claudin-low breast cancer molecular subtypes, is a powerful EMT inducer and is also a marker of stem/progenitor cells. In contrast, TF forkhead box A1 (FOXA1), strongly associated with luminal subtypes, is an EMT repressor and a luminal differentiation marker, thus seemingly exerting reciprocally opposite transcriptional effects to that of FOXC1. We hypothesized that effective EMT program activation status in breast cancer might be better predicted by examining the expression ratio of an EMT inducer and EMT repressor, such as FOXC1/FOXA1, theoretically being more reflective of net transcriptional effect than either component alone. Methods: Herein we utilize RNA-Seq profiling of the HRAS-transformed MCF10A cell series, a well characterized and widely accepted in vitro model of breast cancer progression and metastasis, to correlate measured FOXC1/FOXA1 ratios to dynamic shifts in EMT marker expression in 3D matrigel cultures and to stem cell traits observed in primary and secondary mammosphere suspension cultures. We further test the ability of the FOXC1/FOXA1 expression ratio to predict lymph node independent breast cancer metastasis and death in independent human breast cancer gene expression datasets. Results: RNA-Seq and qRT-PCR profiling confirmed progressive increase in FOXC1/FOXA1 ratio to correlate with a progressive loss of E-cadherin expression and synchronous gain of EMT markers N-cadherin, Fibronectin, and Vimentin. FOXC1/FOXA1 ratio was found to be directly proportional to mammosphere formation efficiency, a surrogate indicator of stem cell enrichment. In patients without any evidence of nodal metastasis, elevated FOXC1/FOXA1 ratio was associated with significantly decreased 10 year Overall Survival (HR 2.58;95%CI 1.39 to 4.80, p = 0.003, 295 patient Van de Vijver dataset), 10 year Disease-specific Survival (HR 1.74;95%CI 1.16 to 2.61, p = 0.008,1992 patient Curtis dataset) and predicted the development of lung metastasis. Conclusion: Elevated FOXC1/FOXA1 expression ratio indicates EMT program activation in breast cancer, and predicts the associated occurrence of lymph-node-independent distant metastasis and death in human patients. These findings may allow for the early (pre-symptomatic) diagnosis of clinically occult (node negative) metastasis by using the FOXC1/FOXA1 ratio as a biomarker of metastasis and permit institution of appropriate therapy earlier than currently possible. The current study improves our understanding of EMT and highlights the importance of future studies geared towards unraveling mechanisms involved in regulating FOXC1 and FOXA1 expression in breast cancer. Citation Format: Partha S Ray, Tor W Jensen, Tania Ray, Omid Gholamalamdari, Connie Y Tsai, Bomy Kim, Rohit Bhargava, K V Prasanth. FOXC1/FOXA1 transcriptional balance in breast cancer: From acquisition of mesenchymal and stem cell traits to occult lymph node independent breast cancer metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-30.

Abstract P2-06-02: Breast cancer stem-like cell activity correlates with tumour progression to metastasis but not with clinical or tumour characteristics

Abstract Introduction: Breast cancers exhibit cellular heterogeneity, containing both stem-like and more differentiated cells. The activity of cancer stem cells (CSC) is likely to be dependent on the microenvironment or niche. Using 158 patient tumour samples, correlations between niche-independent breast CSC activity and clinical and tumour characteristics were tested. Methods: 104 early breast cancer surgical samples and 54 unrelated metastatic samples from pleural or ascitic fluid were harvested. To test CSC activity, isolated cells were grown in both primary (formation) and secondary (self-renewal) mammosphere (MS) culture. Tumour initiating activity was also tested by transplanting breast cancer fragments or cells into the sub-cutaneous flanks of NSG mice (n=84 early and n=10 metastatic). Results: No correlation was found between MS growth, MS formation (%), MS self-renewal (%) or in vivo tumour initiation and breast cancer sub-type, grade, node status or Nottingham prognostic index. 33% of the samples that formed MS in vitro initiated tumours in vivo while only 9% that failed to form MS initiated tumour growth. Metastatic compared to early BC samples grew MS more frequently (53/54 compared to 81/104), and had a higher primary MS formation efficiency (1% vs 0.6%; P<0.001) although rates of MS self-renewal were similar. Tumour initiation in vivo was also more frequent in metastatic than early breast cancer samples (7/10 versus 25/84; P<0.02). Conclusions: In summary, niche-independent breast CSC activity measured in vitro by MS assay and in vivo by xenograft growth is not directly correlated with standard clinical parameters. However, both in vitro and in vivo CSC activity are increased in metastatic samples. These results suggest that breast CSC activity is independent of other prognostic indicators but may predict for poor outcome tumours. Relapse free survival data are maturing and will be presented with analysis of primary tumour ALDH1 expression. Citation Format: Sacha J Howell, Denis Alferez, Katherine Spence, Rachel Eyre, Fran Shaw, Bruno Simoes, Angelica Santiago-Gomez, Maria Bramley, Mohamed Absar, Zahida Saad, Sumohan Chatterjee, Cliona Kirwan, Ashu Gandhi, Anne C Armstrong, Andrew M Wardley, Gillian Farnie, Robert B Clarke. Breast cancer stem-like cell activity correlates with tumour progression to metastasis but not with clinical or tumour characteristics [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-02.

Leptin as a Mediator of Tumor‐Stromal Interactions Promotes Breast Cancer Stem Cell Activity.

Breast cancer stem‐like cells (BCSC) are responsible for tumor initiation, maintenance, and metastases. BCSC rely for their properties on complex interactions with the tumor microenvironment through networks of cytokines and growth factors. Leptin, a well‐known cytokine involved in breast cancer progression, plays a crucial role in the tumor‐stromal crosstalk. Thus, we investigated how tumor microenvironmental factors and especially leptin may affect BCSC activity using MCF‐7 breast cancer cells and patient‐derived samples.Treatment of mammosphere cultures with conditioned media (CM) from cancer associated fibroblasts and breast adipocytes resulted in a significant increase in mammosphere‐forming efficiency (MFE). Depletion of leptin from stromal cell‐CM completely abrogated this effect. Accordingly, mammosphere cultures exhibited increased leptin receptor expression and leptin exposure enhanced MFE. Microarray analyses revealed a similar expression profile of genes involved in stemness and cell proliferation in mammospheres treated with stromal cell‐CM and leptin. Finally, leptin significantly increased MFE in patient‐derived samples (metastatic ascites and pleural effusion fluid) and leptin mRNA levels in peritumoral adipose tissue of breast cancer patients were positively correlated with MFE (r= 0.449; P 蠄 0.05).Our study highlights leptin as an important paracrine molecule that mediates the microenvironment effects on BCSC activity.

Differential effect of phosphorylation-defective survivin on radiation response in estrogen receptor-positive and -negative breast cancer.

Survivin is a key member of the inhibitor of apoptosis protein family, and is considered a promising therapeutic target due to its universal overexpression in cancers. Survivin is implicated in cellular radiation response through its role in apoptosis, cell division, and DNA damage response. In the present study, analysis of publically available data sets showed that survivin gene expression increased with breast cancer stage (p < 0.00001) and was significantly higher in estrogen receptor-negative cancers as compared to estrogen receptor-positive cancers (p = 9e-46). However, survivin was prognostic in estrogen receptor-positive tumors (p = 0.03) but not in estrogen receptor-negative tumors (p = 0.28). We assessed the effect of a survivin dominant-negative mutant on colony-formation (2D) and mammosphere-formation (3D) efficiency, and radiation response in the estrogen receptor-positive MCF7 and estrogen receptor-negative SUM149 breast cancer cell lines. The colony-formation efficiency was significantly lower in the dominant-negative survivin-transduced cells versus control MCF7 cells (0.42 vs. 0.58, p < 0.01), but it was significantly higher in dominant-negative population versus control-transduced SUM149 cells (0.29 vs. 0.20, p < 0.01). A similar, non-significant, trend in mammosphere-formation efficiency was observed. We compared the radiosensitivity of cells stably expressing dominant-negative survivin with their controls in both cell lines under 2D and 3D culture conditions following exposure to increasing doses of radiation. We found that the dominant-negative populations were radioprotective in MCF7 cells but radiosensitive in SUM149 cells compared to the control-transduced population; further, Taxol was synergistic with the survivin mutant in SUM149 but not MCF7. Our data suggests that survivin modulation influences radiation response differently in estrogen receptor-positive and estrogen receptor-negative breast cancer subtypes, warranting further investigation.

Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer.

IntroductionTriple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors.MethodsiNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models.ResultsHigh endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal.ConclusionsConsidering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0527-x) contains supplementary material, which is available to authorized users.

Vitamin D compounds reduce mammosphere formation and decrease expression of putative stem cell markers in breast cancer

Breast cancer stem cells (BCSCs) are a subset of tumor cells that are believed to be the cells responsible for the establishment and maintenance of tumors. Moreover, BCSCs are suggested to be the main cause of progression to metastasis and recurrence of cancer because of their tumor-initiating abilities and resistance to conventional therapies. Ductal carcinoma in situ (DCIS) is an early precursor in breast carcinogenesis which progresses to invasive ductal carcinoma (IDC). We have previously reported that a vitamin D compound, BXL0124, inhibits the progression of DCIS to IDC. In the present study we sought to determine whether this effect was mediated through an influence on BCSCs. In MCF10DCIS cells treated with vitamin D compounds (1α25(OH)2D3 or BXL0124), the breast cancer stem cell-like population, identified by the CD44+/CD24−/low and CD49f+/CD24−/low subpopulations, was reduced. To determine the effects of vitamin D compounds on cancer stem cell activity, the MCF10DCIS mammosphere cell culture system, which enriches for mammary progenitor cells and putative BCSCs, was utilized. Untreated MCF10DCIS mammospheres showed a disorganized and irregular shape. When MCF10DCIS cells were treated with 1α25(OH)2D3 or BXL0124, the mammospheres that formed exhibited a more organized, symmetrical and circular shape, similar to the appearance of spheres formed by the non-malignant, normal mammary epithelial cell line, MCF10A. The mammosphere forming efficiency (MFE) was significantly decreased upon treatment with 1α25(OH)2D3 or BXL0124, indicating that these compounds have an inhibitory effect on mammosphere development. Treatment with 1α25(OH)2D3 or BXL0124 repressed markers associated with the stem cell-like phenotype, such as CD44, CD49f, c-Notch1, and pNFκB. Furthermore, 1α25(OH)2D3 and BXL0124 reduced the expression of pluripotency markers, OCT4 and KLF-4 in mammospheres. This study suggests that vitamin D compounds repress the breast cancer stem cell-like population, potentially contributing to their inhibition of breast cancer.This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’.

Abstract 1236: A gemini vitamin D analogue, BXL0124, represses mammosphere formation and decreases expression of stem cell markers in MCF10DCIS breast cancer cells

Abstract It has been suggested that cancer stem cells (CSCs), which represent a subset of tumor cells, are responsible for the possible origination and maintenance of tumors. Moreover, CSCs are believed to be the main cause of progression to metastasis and recurrence of cancer because of their tumor-initiating abilities and resistance to conventional therapies. Ductal carcinoma in situ (DCIS) is an early precursor in breast carcinogenesis which progresses to invasive ductal carcinoma (IDC). The involvement of CSCs in the progression of DCIS to IDC has yet to be elucidated. In order to investigate the role of CSCs in DCIS progression to IDC, we utilized the MCF10DCIS.com cell line in the mammosphere cell culture system, which enriches for mammary progenitor cells and mammary CSCs. We have previously reported that a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), inhibits the progression of DCIS to IDC. Therefore, we have determined whether BXL0124 plays a role in regulating the CSC or progenitor cell populations, resulting in reduced tumorigenicity. MCF10DCIS.com cells were plated in ultra-low attachment plates and treated with BXL0124 or 1α25(OH)2D3 over the course of 1, 3 and 5 days. Starting at day 2, MCF10DCIS.com cells began to form mammospheres of 100 µm in size. MCF10DCIS.com mammospheres showed enrichment in the CD44+/CD24- and the CD49f+/CD24- populations compared to monolayer cell culture. The phenotype of MCF10DCIS.com mammospheres showed a disorganized, undefined and irregular shape. However, when the MCF10DCIS.com mammospheres were treated with BXL0124 or 1α25(OH)2D3, mammospheres form a more organized, symmetrical and circular shape, which is similar to the phenotype of the spheres formed by the non-malignant, normal mammary epithelial cell line, MCF10A. In addition, the mammosphere forming efficiency (MFE) was decreased upon BXL0124 and 1α25(OH)2D3 treatment, showing that there is an inhibitory effect on mammosphere development. Treatment with BXL0124 repressed markers associated with the stem cell-like phenotype, such as CD44, Notch1 and JAG1. Furthermore, BXL0124 demonstrated the reduced expression of MMPs, such as MMP2, 9, 14, 15, and 16, in mammospheres, suggesting that BXL0124 reduces the tumorigenicity and invasive potential of MCF10DCIS.com cells by targeting the stem cell-like population. In conclusion, the Gemini vitamin D analog BXL0124 represses the CSC population, potentially contributing to the inhibition of the progression of DCIS to IDC. (This work was supported in part by the National Institutes of Health National Cancer Institute R01-CA127645 and the National Institute of Environmental Health Sciences Grant ES005022). Citation Format: Joseph Wahler, Hubert Maehr, Milan Uskokovic, Nanjoo Suh. A gemini vitamin D analogue, BXL0124, represses mammosphere formation and decreases expression of stem cell markers in MCF10DCIS breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1236. doi:10.1158/1538-7445.AM2014-1236

Abstract 3018: SHIP2 plays an oncogenic role in breast cancer stem cells through JNK/vimentin activation and its phosphatase activity

Abstract Background and Objectives: Over-expression of SH2-containing-5’-inositol phosphatase-2 (SHIP2) which converts phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] into phosphatidylinositol (3,4)-biphosphate [PI(3,4)P2] has been shown to correlate with decreased disease-free survival in breast cancer. However, its role in breast cancer stem cells (BCSCs) remains unclear. Results: We analyzed 60 clinical breast cancer specimens and found that the percentage of SHIP2+ cells was positively correlated with that of CD24-CD44+ cells. Among 20 estrogen receptor (ER)-negative samples, 17 had greater SHIP2 expression in CD24-CD44+ subpopulation than the remaining subpopulation. Examination of mouse xenografts derived from primary human breast cancer specimens revealed that SHIP2 protein and its tyrosine 1135 phosphorylation were significantly higher in BCSCs, identified as CD24-CD44+ or ALDH+, than non-BCSCs. SHIP2 silencing or inhibitor of SHIP2 phosphatase significantly decreased mammosphere-forming efficiency, percentage of ALDH+ subpopulation in vitro and tumorigenicity of BCSCs in vivo. SHIP2 shRNA knockdown and SHIP2 phosphatase inhibitor suppressed p-AktSer473 levels. Addition of PI(3,4)P2 could partially rescue Akt phosphorylation and ALDH+ subpopulation in SHIP2-overexpressing AS-B634 cells treated with SHIP2 phosphatase inhibitor. Whereas, PI(3,4)P2 failed to reverse the effects of SHIP2-silencing on Akt activation and ALDH+ subpopulation. Futhermore, over-expression of SHIP2 enhanced the expression of epithelial-mesenchymal transition markers including vimentin (VIM), which was mainly expressed in ER-negative breast cancer cells with higher level in mammospheres than monolayer culture. Ablation of c-Jun N-terminal kinase 1 (JNK1), JNK2 or VIM hampered the increases of both ALDH+ population and tumorigenicity induced by SHIP2 over-expression. BCSCs displayed greater expression of phospho-JNK than non-BCSCs. Futhermore, silencing of JNK suppressed the upregulation of VIM by SHIP2 in breast cancer. Conclusion: Our study is the first to demonstrate that SHIP2 is crucial for maintaining the properties of BCSCs. Within ER-negative breast cancer, there was a significant correlation of greater expression of SHIP2 in BCSCs than non-BCSCs. We documented a novel functional role of SHIP2 in enhancing mammosphere formation and ALDH+ subpopulation in vitro, promoting tumorigenicity in vivo and inducing the expression of EMT markers including VIM. The upregulation of VIM was mediated by JNK activation. Furthermore, the phosphatase activity of SHIP2 also contributed to its role in BCSCs. In summary, our study has uncovered an oncogenic role of SHIP2 in promoting BCSC features and its underlying molecular mechanisms, providing the impetus for the design of novel BCSC-directed therapeutics by targeting SHIP2. Citation Format: Chiung-Hui Fu, Ruey-Jen Lin, John Yu, Wen-Wei Chang, Guo-Shiou Liao, Wen-Ying Chang, Ling-Ming Tseng, Yi-Fang Tsai, Jyh-Cherng Yu, Alice L. Yu. SHIP2 plays an oncogenic role in breast cancer stem cells through JNK/vimentin activation and its phosphatase activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3018. doi:10.1158/1538-7445.AM2014-3018

STAT3 signaling is activated preferentially in tumor-initiating cells in claudin-low models of human breast cancer.

In breast cancer, a subset of tumor-initiating cells (TIC) or "cancer stem cells" are thought to be responsible for tumor maintenance, treatment resistance, and disease recurrence. While current breast cancer stem cell markers (e.g., CD44(high) /CD24(low/neg) , ALDH positive) have allowed enrichment for such cells, they are not universally expressed and may actually identify distinct TIC subpopulations in the same tumor. Thus, additional markers of functional stem cells are needed. The STAT3 pathway is a critical regulator of the function of normal stem cells, and evidence is accumulating for its important role in breast cancer stem cells. However, due to the lack of a method for separating live cells based on their level of STAT3 activity, it remains unknown whether STAT3 functions in the cancer stem cells themselves, or in surrounding niche cells, or in both. To approach this question, we constructed a series of lentiviral fluorescent (enhanced green fluorescent protein, EGFP) reporters that enabled flow cytometric enrichment of cells differing in STAT3-mediated transcriptional activity, as well as in vivo/in situ localization of STAT3 responsive cells. Using in vivo claudin-low cell line xenograft models of human breast cancer, we found that STAT3 signaling reporter activity (EGFP(+) ) is associated with a subpopulation of cancer cells enriched for mammosphere-forming efficiency, as well as TIC function in limiting dilution transplantation assays compared to negative or unsorted populations. Our results support STAT3 signaling activity as another functional marker for human breast cancer stem cells thus making it an attractive therapeutic target for stem-cell-directed therapy in some breast cancer subtypes.

395P - The Effects of the Bkm120 Combination with Docetaxel on Breast Cancer Cells and Breast Cancer Stem Cells

ABSTRACT Aim: Buparlisib (BKM120) is an oral pan-phosphatidylinositol 3-kinase inhibitor, targeting all four isoforms of class I PI3K (a, b, γ, and d). This study evaluated the effects of the inhibitor on breast cancer normal cell lines (MDA-MB-231, MCF-7, SK-BR-3) and the BCSCs in vitro. Methods: The BCSCs were typically enriched via mammosphere culture. The inhibition of drugs on cancer cells were tested by MTT assay. The tumorigenicities and invasion abilities were detected by wound-healing assay and plate clone formation assay. The mammosphere-forming efficiency of breast cancer cells treated with different concentrations of the BKM120, an operational surrogate of BCSCs, were also performed. The nature of the drug interaction of was evaluated by using the combination index (CI) according to the method of Chou and Talalay. The impact of BKM120 and combined with docetaxel on PI3K/AKT/mTOR pathway was assessed by Western blotting. Results: All three kinds of BCSCs were appeared resistant to BKM120 in vitro, compared with the non-tumorigenic cancer cells. In addition, when three cancer cells treated with the IC50 dose of BKM120, the invasion abilities of cancer cells significantly decreased (P Conclusions: BKM120 can inhibit the growth of normal cancer cells and BCSCs, by inhibiting cell proliferation, descenting the invasion abilities, and reducing the expression of pAKT and pS6 indicating that treatment with BKM120 and docetaxel may be an efficient therapy for normal cancer cells and BCSCs. Disclosure: All authors have declared no conflicts of interest.

A novel oncogenic role of inositol phosphatase SHIP2 in ER-negative breast cancer stem cells: involvement of JNK/vimentin activation.

Overexpression of SH2-containing-5'-inositol phosphatase-2 (SHIP2) correlates with poor survival in breast cancer. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here, we showed that the percentage of SHIP2(+) cells was positively correlated with that of CD24(-) CD44(+) cells in 60 breast cancer specimens. Among 20 estrogen receptor (ER)-negative samples, 17 had greater SHIP2 expression in CD24(-) CD44(+) subpopulation than the remaining subpopulation. Data mining of microarray analysis of 295 breast tumors showed a significant correlation of higher SHIP2 expression with distant metastasis. Examination of patient-derived mouse xenografts revealed that SHIP2 protein and its tyrosine 1135 phosphorylation were significantly higher in BCSCs, identified as CD24(-) CD44(+) or aldehyde dehydrogenase (ALDH(+)), than non-BCSCs. SHIP2 silencing or inhibitor of SHIP2 phosphatase significantly decreased mammosphere-forming efficiency, ALDH(+) subpopulation in vitro and tumorigenicity of BCSCs in vivo. Overexpression of SHIP2 enhanced the expression of epithelial-mesenchymal transition markers including vimentin (VIM), which was mainly expressed in ER-negative breast cancer cells with higher level in mammospheres than monolayer culture. Ablation of c-Jun N-terminal kinase 1 (JNK1), JNK2, or VIM diminished the increased ALDH(+) population and tumorigenicity, induced by SHIP2 overexpression. BCSCs displayed greater expression of phospho-JNK than non-BCSCs and silencing of JNK suppressed SHIP2-mediated upregulation of VIM. Furthermore, SHIP2 overexpression enhanced Akt activation, but Akt inhibition failed to influence SHIP2-induced phospho-JNK/VIM upregulation. In conclusion, SHIP2 plays a key role in BCSCs of ER-negative breast cancers through activation of Akt and JNK with upregulation of VIM and may serve as a target for therapy directed at BCSCs.

Chemical inhibition of acetyl-CoA carboxylase suppresses self-renewal growth of cancer stem cells.

Cancer stem cells (CSC) may take advantage of the Warburg effect-induced siphoning of metabolic intermediates into de novo fatty acid biosynthesis to increase self-renewal growth. We examined the anti-CSC effects of the antifungal polyketide soraphen A, a specific inhibitor of the first committed step of lipid biosynthesis catalyzed by acetyl-CoA carboxylase (ACACA). The mammosphere formation capability of MCF-7 cells was reduced following treatment with soraphen A in a dose-dependent manner. MCF-7 cells engineered to overexpress the oncogene HER2 (MCF-7/HER2 cells) were 5-fold more sensitive than MCF-7 parental cells to soraphen A-induced reductions in mammosphere-forming efficiency. Soraphen A treatment notably decreased aldehyde dehydrogenase (ALDH)-positive CSC-like cells and impeded the HER2's ability to increase the ALDH+-stem cell population. The following results confirmed that soraphen A-induced suppression of CSC populations occurred throughACACA-driven lipogenesis: a.) exogenous supplementation with supraphysiological concentrations of oleic acid fully rescued mammosphere formation in the presence of soraphen A and b.) mammosphere cultures of MCF-7 cells with stably silenced expression of the cytosolic isoform ACACA1, which specifically participates in de novo lipogenesis, were mostly refractory to soraphen A treatment. Our findings reveal for the first time that ACACA may constitute a previously unrecognized target for novel anti-breast CSC therapies.

Comparison of mammosphere formation from breast cancer cell lines and primary breast tumors.

Breast cancer stem cells (BCSCs) can be enriched by culturing of cells in non-adherent non-differentiating conditions. However, culturing mammospheres from primary breast tumors are costly and difficult to control. In order to overcome problems associated with using primary human tissues, continuous breast cancer cell lines have been developed from various sources. In this study, a luminal subtype breast cancer cell line MCF-7 and a basal subtype cell line MDA-MB-231 were chosen. We explored the optimal culturing system for BCSCs from the two cell lines and primary breast tumors. Then, mammosphere formation efficiency (MFE), CD44(+)/CD24(-/low)ESA(+)Lin(-) cell proportion in mammospheres, and tumorigenecity of mammospheres generated from the two breast cancer cell lines and primary breast tumors were compared. Enzymatic digestion of 60 mins and the addition of B27 to the culture medium were optimal for mammosphere culturing. Mammospheres could be formed in all the three cells, in which MCF-7 had the highest MFE. After 3 weeks culture, CD44(+)/CD24(-/low)ESA(+)Lin(-) cell proportion in mammospheres from MCF-7, MDA-MB-231 cells and primary breast tumors was 95.0%±2.5%, 82%±22% and 21.5%±1.0%, respectively. A total of 1,000 cells from MCF-7, MDA-MB-231 mammospheres but not primary mammospheres were tumorigenic. This study validates the use of breast cancer cell lines as models to elucidate the nature of BCSCs.