Mammosphere Formation Efficiency Research Articles

Microfluidics separation reveals the stem-cell–like deformability of tumor-initiating cells

Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.

Histone Deacetylase Inhibitors Stimulate Dedifferentiation of Human Breast Cancer Cells Through WNT/β‐Catenin Signaling

Recent studies have shown that differentiated cancer cells can dedifferentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti-cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self-renewal and expansion of hematopoietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells toward the more resistant stem cell-like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA-231, were sorted based on aldehyde dehydrogenase (ALDH) activity and subsequently ALDH-negative and ALDH-positive cells were treated with one of two known HDAC inhibitors, VA or suberoylanilide hydroxamic acid. In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single-cell-sorted ALDH-negative cells spontaneously generated ALDH-positive cells in vitro. Treatment of ALDH-negative cells with HDAC inhibitors promoted the expansion of ALDH-positive cells and increased mammosphere-forming efficiency. Most importantly, it significantly increased the tumor-initiating capacity of ALDH-negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β-catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC-inhibitor-induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer.

Relationship between expressions of Notch1 and drug resistance of breast carcinoma

Objective To investigate the role of Notchl in the chemotherapy process of breast cancer by studying the the efficiency of mammosphere formation, the ratio of stem cells in MCF-7 and MCF-7/ADR, and the expression status of Notchl in MCF-7, MCF-7/ADR and stem cells respectively. Methods Real time fluorescent quantitative reverse transcriptase polymerase chain reaction （RT-qPCR） and Western blotting were employed to detect the expression status of Notchl in breast cancer cell lines MCF-7, MCF-7/ADR and ALDH1 ＋/CD44 ＋/CD24- breast cancer stem cells （BCSCs）. Serum-free me- dium was available to assess the efficiency of mammosphere formation. Flow cytometry was used to define the ratio of BCSCs and select them in MCF-7 and MCF-7/ADR. Results The expression status of Notchl [ （0. 0748±0. 0043 ） vs （0. 0461±0. 0022 ）, P 〈 0.05 ] and the ability of mammosphere formation were significantly higher in MCF-7/ADR than in MCF-7 （P 〈 0. 05）. There was significant difference in the ra- tio of BCSCs between MCF-7/ADR and MCF-7 （ 11.40% vs 1.89% ）. Furthermore the expression score of Notchl in BCSCs was significantly higher than in non-BCSCs [ （0. 3096±0. 0324 ） vs （0. 0428 ±0. 0061 ）, P 〈 0.05 ]. Conclusion The high level expression of Notchl in breast cancer and BCSCs suggests regulating Notchl may play an important role in reversing chemotherapy resistance of breast cancer. Key words: Breast neoplasm; Stem cell; Notchl

Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells

Basal breast cancer, common among patients presenting with inflammatory breast cancer (IBC), has been shown to be resistant to radiation and enriched in cancer stem cells. The Notch pathway plays an important role in self-renewal of breast cancer stem cells and contributes to inflammatory signaling which promotes the breast cancer stem cell phenotype. Herein, we inhibited Notch signaling using a gamma secretase inhibitor, RO4929097, in an in vitro model that enriches for cancer initiating cells (3D clonogenic assay) and conventional 2D clonogenic assay to compare the effect on radiosensitization of the SUM149 and SUM190 IBC cell lines. RO4929097 downregulated the Notch target genes Hes1, Hey1, and HeyL, and showed a significant reduction in anchorage independent growth in SUM190 and SUM149. However, the putative self-renewal assay mammosphere formation efficiency was increased with the drug. To assess radiosensitization of putative cancer stem cells, cells were exposed to increasing doses of radiation with or without 1 μM RO4929097 in their standard (2D) and self-renewal enriching (3D) culture conditions. In the conventional 2D clonogenic assay, RO4929097 significantly sensitized SUM190 cells to ionizing radiation and has a modest radiosensitization effect in SUM149 cells. In the 3D clonogenic assays, however, a radioprotective effect was seen in both SUM149 and SUM190 cells at higher doses. Both cell lines express IL-6 and IL-8 cytokines known to mediate the efficacy of Notch inhibition and to promote self-renewal of stem cells. We further showed that RO429097 inhibits normal T-cell synthesis of some inflammatory cytokines, including TNF-α, a potential mediator of IL-6 and IL-8 production in the microenvironment. These data suggest that additional targeting agents may be required to selectively target IBC stem cells through Notch inhibition, and that evaluation of microenvironmental influences may shed further light on the potential effects of this inhibitor.

Abstract 5210: Impact of tumor microenvironment on expansion of tumor initiating cells in breast cancer metastasis

Abstract Breast cancer metastasis which ultimately results in breast cancer death, is an event believed to be initiated by the migration of tumor initiating cells (TIC) from the primary tumor to niches for micrometastatic disease. Recent data suggests the tumor microenvironment promotes TIC. The clinical relevance of secreted factors from the microenvironment on TIC surrogate, mammosphere (MS) formation and MS sensitivity to drug therapy was investigated using breast cancer patient fluids inherently conditioned by the tumor microenvironment: post-operative seromas and malignant pleural effusions. Fluids from 20 patients with breast cancer (8 seromas and 13 pleural effusions) and mesenchymal stem cells (MSC) from healthy donors were collected on IRB approved protocols. Cellular components were eliminated from patient-derived fluids using density-gradient centrifugation. MSC conditioned media (MSC-CM) was collected from 3D cultures of primary MSC. Luminex multiplex array platform was used to characterize 79 cytokine and growth factor components of all fluids. In addition, MSC-CM and patient-derived fluids were added to cultures of breast cancer cell lines: MCF-7, an estrogen receptor (ER)-positive cell line; SUM149, a triple-negative inflammatory breast cancer cell line; and SUM159, a triple-negative metaplastic breast cancer cell line and MS forming efficiency was examined. Our results show that pleural effusions and seromas are enriched for factors also secreted by MSC such as MCP-1, GRO, IL-6 and IL-8. We found remarkable similarities regarding the cytokines and growth factors profile in pleural effusions and seromas. Both patient-derived fluids have comparable amount of IL-2, IL-3, IL-4 and IL-10, as well as, VEGF, EGF and FGF-b. IL-1β and IL-16 were significantly different between pleural effusions and seromas. Seroma fluid from bilateral drains in a patient with an invasive cancer and a contralateral benign mastectomy had very similar cytokine concentrations. Moreover, MSC-CM and pleural fluids from ER+ and ER- patients increased the MS formation efficiency of both triple-negative cell lines while seroma fluids from ER+ and ER- patients increased the MS formation efficiency of ER-positive cell line MCF-7. Finally, we evaluated the impact of a panel of drugs on cell cultures grown with MSC-CM and patient-derived fluids. We found that the effect of chemotherapies on MS formation can be attenuated by patient-derived fluids. Seroma and pleural effusion fluids from breast cancer patients have similar cytokine profiles, change MS formation efficiency of standard breast cancer cell models, and mediate sensitivity to therapy. Here we demonstrate that host and microenvironmental factors are critical for determining resistance to therapy. Future studies will investigate the prognostic implications of factors that promote TIC survival in the fluid tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5210. doi:1538-7445.AM2012-5210

Abstract 3492: γ-secretase inhibitor PF-03084014 diminishes the tumor-initiating cells and demonstrates synergy with docetaxel in breast cancer xenograft models

Abstract Notch signaling is known to be a survival pathway for tumor-initiating cells. In this report, we demonstrate that the γ-secretase inhibitor PF-03084014 significantly enhances the antitumor activity of docetaxel in multiple xenograft models of triple-negative breast cancer. Mechanistic evaluation revealed that PF-03084014 perturbs the Notch signaling pathway and suppresses the function of tumor initiating cells (TIC). In MDA-MB-231Luc model, treatment of docetaxel led to a significant increase of CD133+/CD44+ and ALDH+ subpopulations by FACS analysis. In combination with PF-03084014, these two unique cell subpopulations were significantly diminished. Correspondingly, the functional analyses by tumor re-implant and mammosphere-forming efficiency assays revealed that docetaxel-therapy promoted the tumor initiating capability of the remaining cells, in which an increased stem cell property and Notch pathway activation were observed through gene signature changes. In contrast, PF-03084014 co-treatment with docetaxel substantially hampered the self-renewal ability of these cells. Notch target gene analysis demonstrated the biological relevance of PF-03084014-induced activity. To characterize the function of CD133+/CD44+ subpopulation, MDA-MB-231Luc tumors were de-bulked by the treatment with docetaxel. Subsequently, the CD133+/CD44+ and CD133-/CD44- subpopulatons were isolated and re-implanted in SCID-bg mice using a limiting dilution approach. The results showed that CD133+/CD44+ cells gave rise to tumors with a 100 % take rate (10/10), whereas CD133-/CD44- cells were not tumorgenic (0/10). In addition, CD133+/CD44+ cells exhibited much higher tumorigenicity compared with the respective adherent parental cell line. PF-03084014 treatment caused a significant delay of CD133+/CD44+ tumor growth. The ability of PF-03084014 to suppress TICs was also observed in other breast cancer xenografts, including patient derived models. This data suggests that anti-TIC is one of the contributing mechanisms for the synergistic activities of PF-03084014 in combination with docetaxel. Our work provides potential therapeutic opportunities for PF-03084014 to improve conventional cytotoxic therapy by inhibiting Notch signaling in tumor-initiating cells and other bulk tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3492. doi:1538-7445.AM2012-3492

P‐Cadherin Is Coexpressed with CD44 and CD49f and Mediates Stem Cell Properties in Basal‐like Breast Cancer

Although the luminal progenitor cell of the normal mammary gland hierarchy has been proposed as the cell-of-origin for basal-like breast cancers, finding the cancer stem cell (CSC) phenotype for this malignancy has proven a difficult task, mostly due to the lack of specific markers. Recently, basal-like sporadic and familial cases of breast cancer have been linked to BRCA1 gene inactivation, which enables the upregulation of the target-repressed CDH3/P-cadherin gene, an important biomarker of basal-like breast carcinomas. Previously, we demonstrated that P-cadherin overexpression can mediate aggressive behavior in these tumors. Thus, our aim was to test whether P-cadherin mediates stem cell properties in basal-like breast carcinomas. Using a series of breast cancer cell lines and primary tumors, we showed that P-cadherin was directly associated with the expression of the breast stem markers CD44, CD49f, and aldehyde dehydrogenase 1 in the basal subtype. Moreover, cell population enriched for P-cadherin expression comprised increased in vitro mammosphere-forming efficiency and capacity to grow colonies in three-dimensional cultures as well as greater tumorigenicity. Importantly, an association was found with stem-/progenitor-like phenotypes of the breast, including the luminal progenitor population, CD49f(+) CD24(+). Additionally, P-cadherin expression conferred resistance to x-ray-induced cell death, sustaining a role for this molecule in another stem cell property. In summary, we demonstrated, for the first time, that P-cadherin mediates stem cell properties, which could be explored in order to better define the CSC phenotype of basal-like breast tumors and the cell-of-origin of this malignancy.

A Monoclonal Antibody against Human Notch1 Ligand–Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem–like Cells

Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation.

P3-17-04: CXCR1/2 Regulates Human Breast Cancer Stem Cell Activity Via EGFR/HER2−Dependent and -Independent Pathway.

Abstract Background Increasing evidence suggests that breast cancers are sustained by breast cancer stem cells (CSCs). Interleukin-8 (IL-8) via its cognate receptors, CXCR1 and CXCR2, and HER2 regulate CSC activity. Cytokine receptors can trans-activate members of the epidermal growth factor receptor (EGFR) family in normal and malignant tissue. The aims of this study were to determine (1) the effect of the IL-8-CXCR1/2 signalling pathway on human breast CSC activity, (2) the effect of CXCR1/2 inhibition in combination with EGFR/HER-2 inhibition on CSC activity and (3) the interaction of CXCR1/2 and EGFR/HER2 in breast cancer. Methods: Human breast cancer cell lines used were HER2−18 and SKBR3 (both HER2 over-expressing). Primary human breast cancer cells were extracted from metastatic samples (pleural effusion n=4; ascites n=5; local recurrence n=1) and invasive breast cancers (n=1). CSC activity was investigated in vitro using the mammosphere assay. Mammosphere forming efficiency (MFE) was determined by dividing the number of mammospheres >60μm in diameter divided by the number of cells seeded and expressed as a percentage. Data is represented as mean MFE ± standard error of mean normalised to control. Statistical significance (P) was determined using Mann-Whitney U test (2-tailed). Inhibitors used were SCH563705 (a CXCR1/2 inhibitor; 100nM), Lapatinib (an EGFR/HER-2 inhibitor; 1–10μM) and Gefitinib (an EGFR inhibitor; 1μM). Results: IL-8 (100ng/ml) increased HER2−18 mean MFE from 1±0.05% to 1.20±0.04%; 19.5% increase; P=0.0061. This effect was reduced by SCH563705 (from 1.20±0.04% to 0.70±0.05; 41.7% reduction; P=0.0001) and Lapatinib (from 1.20±0.04% to 0.51±0.04; 57.5% reduction; P <0.0001). Combination treatment resulted in a greater reduction in mean MFE compared to either SCH563705 (P<0.0001) or Lapatinib (P=0.026). IL-8 (100ng/ml) increased mean MFE of primary human breast cancer cells from 1±0.03% to 1.43±0.06%; 42.9% increase; P<0.0001 (n=11 patient samples). This effect was reduced by SCH563705 from 1.43±0.06% to 0.96±0.04%; 32.9% reduction; P<0.0001. In HER2 over-expressing patient samples (n=4), IL-8-induced increase in MFE was reduced by SCH563705 (from 1.43±0.06% to 0.83±0.05; 42.0% reduction; P<0.0001) and Lapatinib (from 1.43±0.06% to 0.91±0.05%; 36.4% reduction; P<0.0001). Combination treatment resulted in a greater reduction in mean MFE compared to either SCH563705 (P=0.0019) or Lapatinib (P<0.0001). In HER2−non-over-expressing samples (n=6), IL-8-induced increase in MFE was reduced by SCH563705 (from 1.45±0.10% to 1.06±0.05; 27.0% decrease; P=0.0005) and Lapatinib (from 1.45±0.10% to 1.15±0.07%; 20.7% decrease; P=0.0145). Combination treatment had no additional effect. Western blot analysis of the HER2−18 and SKBR3 cell lines showed that IL-8 induced phosphorylation of HER-2, AKT and MAPK within 10 minutes; these effects were inhibited by Lapatinib and Gefitinib. Conclusions: IL-8/CXCR1/2 regulates the activity of human breast CSCs. Lapatinib inhibited IL-8 signalling suggesting that CXCR1/2 signal transduction involves an EGFR/HER2−dependent pathway. Targeting CXCR1/2 adds to the efficacy of targeting HER2 in HER2−over-expressing human breast CSCs through inhibition of an EGFR/HER2−independent pathway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-04.

P3-16-02: Targeting Tumor Initiating Cells with siRNA/Nanotherapy in Triple Negative Breast Cancer.

Abstract Residual human breast tumor cells after conventional therapies are enriched in tumor-initiating cells (TIC) characterized by CD44+/ CD24-/low/lineage- with self-renewal capacities. Our gene expression analyses in those cells and in breast cancer cells propagated as mammospheres (MSs) reveal an epithelial-mesenchymal-transition (EMT) signature (400 genes) mainly found in claudin-low molecular subtype human breast tumors. We performed lentiviral shRNA knockdown of that signature in MS. Critical shRNAs were found to decrease the TIC subpopulation. Among those genes, we found STAT3 (signal transducer and activator of transcription 3), NTN4 (netrin 4), and RPL39L (ribosomal protein L39-like). Here, we used a multiscale siRNA delivery system targeting those three genes in order to clarify the effect of silencing those genes over the self-renewal capacity on MSs with claudin-low features. To this end, duplex small interfering RNA (siRNA) against STAT3, NTN4, RPL39L, and scrambled siRNA as control, were introduced into neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC), and using mesoporous microscale biodegradable silicon particles as carriers. This multistage siRNA delivery system has been reported as a good approach for sustained gene silencing. Claudin-low-like human breast cancer cell lines (SUM159 and BT549) were plated in 24-well ultralow attachment plates with mammary epithelial growth medium (MEGM) (5,000 cells/well). Both cell lines were then treated with 1μg/well/6 wells of silicon particles loaded with DOPC nanoliposomes/siRNA. The primary MSs were allowed to grow for 3 days. MFs were counted by day 3 with a GelCount colony counter (Oxford Optronix, Oxford, UK). Mammosphere-Forming Efficiency (MSFE) was calculated by dividing the number of MSs by the number of seeded cells. In addition, established MSs were serially passaged by dissociation, and single cells were replated on fresh 24-well ultra-low attachment plates to form secondary mammospheres, which were counted after 3 days. One-way ANOVA and Tukey test were performed. A p value less than 0.05 was considered as significant. Our results show that silencing STAT3, NTN4, and RPL39L significantly reduces the MSFE in both primary (1%, 0.9%, and 1.7% respectively) (Figure 1) and secondary MSs (0.49%, 0.51%, and 0.45% respectively) (Figure 2) when compared to the scrambled control (2.4% and 1 % respectively) in BT549 cells. For SUM159 cells, we did not find any change in primary MSs for STAT3 (2.2%) and NTN4 (2.1%), even we found a higher percentage of MSFE in those cells treated with RPL39L siRNA (3.9%) when compared to the scrambled control (2.1%) (Figure 3). Nevertheless, a lesser MSFE were observed in those cells treated with STAT3 (0.9%, not significant), NTN4 (0.8%) and RPL39L (0.78%) in comparison to control (1.1%) (Figure 4). In conclusion, knocking-down of EMT-related genes (STAT3, NTN4, and RPL39L) decreases significantly the self-renewal capacity in mammospheres derived from claudin-low-like human breast tumor cells, being BT549 cells more sensitive than SUM159 cells to that silencing with siRNA loaded in DOPC nanoliposomes into silicon particles as carriers. Acknowledgments: We thank Fundacion Alfonso Martin Escudero for the personal support of Dr. Sergio Granados-Principal. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-02.

P1-04-04: Focal Adhesion Kinase Plays a Major Role in the Regulation of Human Ductal Carcinoma In Situ (DCIS) Stem Cell Activity.

Abstract Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that has been implicated in the regulation of normal mammary stem cells and cancer stem cells (CSCs) within mouse models. FAK is frequently overexpressed in human breast cancers but its role in human CSCs is not known. We aimed to investigate the role of FAK in human ductal carcinoma in situ (DCIS) stem cells (SCs). We have used PF573228 to inhibit FAK activity in SUM225 (HER2−overexpressing) and MCF10DCIS.com (HER2−normal) DCIS cell lines. PF573228 inhibits the autophosphorylation of FAK at tyrosine 397 causing inactivation. Western blot analysis confirmed PF573228 (0.1-5 μM) caused a dose dependent inhibition of FAK activity in both cell lines. The in vitro mammosphere colony assay was used to measure CSC activity in both DCIS cell lines and primary DCIS samples (n=5) (LREC#01/012). Mammosphere-forming efficiency (MFE) was calculated as percentage colony formation (>60μm) in the presence or absence of PF573228 (0.1-5μM). MFE was reduced in both the SUM225 cell line (p=0.02) and in the MCF10DCIS.com cell line (p=0.04) by PF573228 at a dose concentration of ≥0.1 μM (see Table 1). PF573228 (1 μM) also reduced mammosphere formation in human primary DCIS cells (n=5) by 18.1±5.75% when compared to control (p=0.03). To measure the effect of PF573228 on DCIS SC self-renewal capacity we performed secondary generation mammosphere colony assays. Primary generation mammospheres that had been treated with PF573228 (0.1-5μM) were dissociated into a single cell suspension and reseeded out into mammosphere culture conditions with no additional PF573228. The mammosphere regeneration ratio (MRR) was calculated as the proportion of secondary mammospheres formed relative to the number of primary mammospheres. MRR was reduced in both the SUM225 (p=0.005) and MCF10DCIS. com (p=0.001) cell lines after treatment with ≥0.5μM or ≥1.0μM PF573228 respectively (see Table 2). Preliminary data in human primary DCIS cells (n=1) also shows a reduction in MRR, from 0.56 to 0.14 with 0.5μM PF573228. These results suggest that CSC self-renewal capacity is reduced by FAK inhibition. We conclude that FAK signalling is important in human DCIS SC activity measured by mammosphere growth and regeneration. Targeting FAK in the treatment of DCIS may reduce disease recurrence and improve patient outcome. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-04.

P2-01-13: Impact of Erlotinib on MSC-Mediated TIC Expansion and EMT.

Abstract Background: Recently, we have demonstrated that mesenchymal stem cells (MSC) and MSC secreted factors (MSC-CM) have a profound effect on tumor initiating cells (TIC) enriched mammosphere formation and latency of tumor xenografts formation from breast cancer cell lines. Furthermore these interactions increased the expression of epithelial mesenchymal transition (EMT)-associated proteins which are associated with tumor cell invasion and metastasis as well as the TIC phenotype. (Klopp, A. H. et al., 2010, PLoS One. 5, e12180). Our data suggest that the presence of MSC in the tumor microenvironment may increase metastases by conferring stem progenitor cell biology on more differentiated non-metastatic cells. In addition, preliminary data suggested MSC-CM upregulated EGFR signaling in breast cancer cells. Therefore, we hypothesized that inhibiting EGFR signaling with erlotinib (tyrosin kinase inhibitor) can suppress MSC-mediated TIC expansion and EMT. Methods & Results: In order to demonstrate that erlotinib inhibits MSC-CM promoted expansion of TIC, we cultured breast cancer cells lines (SUM149, SUM159, SUM190, MDA-IBC3 and MCF-7) in anchorage independent conditions with MSC-CM and treated them with increasing concentrations of erlotinib. The efficiency of mammosphere formation was examined after 5 days. We found that erlotinib inhibited MSC mediated increase in mammosphere formation in triple negative cell lines SUM149 and SUM159, and HER2−positive cell lines SUM190 and MDA-IBC3, but not in ER-positive, erlotinib resistant MCF-7 cells. Furthermore, we evaluated the impact of erlotinib on cell cultures grown with breast cancer patient-derived fluids, such as seroma and malignant pleural effusions. We observed that the effect of erlotinib on mammospheres formation was attenuated by both types of patient fluids. Discussion: Patients with triple negative breast cancer have the highest rates of metastases and no available targeted therapies for treatment. EGFR is expressed in a significant proportion of triple negative breast cancers, and recent clinical and preclinical studies suggest that EGFR may contribute to the metastasis or aggressiveness of triple negative breast cancer. Here we demonstrate that host and environmentally-derived factors are critical for determining resistance to erlotinib. In vivo studies regarding the ability of erlotininb to prevent MSC-enhanced TIC survival and metastases are underway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-13.

P1-04-01: The Mechanism of Anti-Breast Cancer TICs Effect of Pyrvinium Pamoate Is through WNT/beta-Catenin Signaling.

Abstract We have previously shown that pyrvinium pamoate can decrease breast cancer TICs in vitro and shrink the tumor size in vivo. Although pyrvinium pamoate has been shown to target beta-catenin through activating CK-1alpha in a vitro model, the mechanism of its anti-breast cancer TICs effect is unknown. Herein, we use a constitutively active WNT/beta-catenin signaling construct EBETAP (ref) to determine if the anti-breast TIC effect of pyrvinium pamoate is through WNT/beta-catenin signaling. Using aldefluor expression and mammosphere formation efficiency as TIC surrogate assays, we found that TICs of SUM-159 transfected with EBETAP construct are resistant to pyrvinium pamoate treatment compared to control cells. Moreover, microarray analysis reveals a series of genes and signaling downstream of WNT-catenin were down-regulated in SUM-159 cells treated with pyrvinium pamoate. In summary, mechanism of anti-breast cancer TICs effect of pyrvinium pamoate is through WNT/beta-catenin signaling. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-01.

P3-17-02: Targeting the Autophagy Pathway for Drug Resistance of Breast Tumor-Initiating Cells.

Abstract Background: Our clinical data and experimental evidence reveal that the tumor-initiating cells (TICs) in the original tumor are intrinsically resistant to conventional chemotherapy and radiation therapy and greatly enriched in residual breast cancers after such treatments. We have published a gene expression signature of such breast TICs, and our pathway analysis on the gene signature suggests that the activation of autophagy pathway may be an intrinsic characteristic of the TICs. This motivates us to further investigate the role of the autophagy pathway in TICs self-maintenance and their resistance to hormonal and chemo therapies, as well as their response to TIC-targeted therapies. Methods: A collective 84 well-documented autophagy pathway genes were used to compare the activation of autophagy pathway in different microarray datasets, 1) flow-sorted CD44+/CD24−/low cancer cells vs. all other cells (representing 20 patients), and cancer-derived MSs vs. corresponding primary bulk tumors (representing 17 patients), 2) before vs. after letrozole and doxetaxel treatments (representing 30 patient pairs), 3) before vs. after letrozole treatment (representing 176 patient pairs), and 4) before vs. after lapatinib treatment (representing 115 patient pairs). We applied the Significance Analysis of Microarrays (SAM) algorithm to analyze the expression data of 211 normalized probes for the 84 genes. Low-density RTPCR array for the 84 genes was used to confirm the differential expressed genes on tumor tissues of 18 letrozol treated patient pairs. Two human-cancer-in-mouse triple-negative xenograft tumor lines were treated with Notch pathway inhibitor alone or in combining with docetaxel. The tumor growth, mammosphere formation efficiency, and the expression of autophagy marker proteins were evaluated. Results: Overall, the enrichment analysis of the 84-gene set in all the above microarray datasets showed that the autophagy genes are significantly enriched in the differential expressed genes. In particular, 28 out of the 84 autophagy genes are significantly up-regulated in the TICs populations while 5 other autophagy genes are down-regulated with the false discovery rate (FDR) less than 0.05. For the letrozole and doxetaxel treatment microarray analysis, 34 out of the 84 autophagy genes are significantly up-regulated in the after-treatment group (p<0.01), and only 3 out of the 84 autophagy genes are down-regulated (FDR<0.05). Interestingly, significantly more autophagy genes (15 out of the 84) are down-regulated in the after-lapatinib treatment group (p<0.05), and less genes (13 out of 84) are up-regulated after lapatinib treatment, thus confirming previous observations that lapatinib may affect TICs. We confirmed these findings in two human cancer xenograft studies of Notch pathway inhibitor (GSI) to target TICs, which showed significantly reduced expressions of LC3 and p62, two autophagic markers. In both xenograft models, GSI reduced mammosphere formation efficiency compared to vehicle control (p<0.05). Conclusion: Activation of the autophagy pathway in TICs is a promising target to combat the drug resistance of breast cancer to conventional systemic therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-02.

P1-03-02: The Norton-Simon Hypothesis and Cancer Stem Cells: How Cancer Stem Cells May Explain the Effectiveness of Dose-Dense Chemotherapy.

Abstract Background: Systemic therapies are effective initially in controlling and reversing tumor growth; however, residual cancers will invariably re-grow despite this initial response. We have published data from paired human breast cancer samples that standard therapy every three weeks kills dividing daughter cells but not tumor-initiating cells (TICs), so that samples obtained after therapy are enriched for CD44+/CD24−/low putative “tumor-initiating” or “cancer stem” cells, indicating that standard treatment regimens are missing the critical targets, TICs. Interestingly, we have recent data in human breast tumors that indicate that TICs may in fact be chemosensitive initially, with a decrease in TICs observed within two days of chemotherapy, but shortly thereafter, TICS are actually induced by chemotherapy. Materials and Methods: To evaluate TICs response to chemotherapy, mice with human breast tumor xenograft lines BCM-2665a and BCM-2147 were treated with vehicle, 10-, or 33-mg/kg docetaxel, and then tumors were collected for TIC assays and molecular analysis at both 48 and 72h after treatment. Using Affymetrix gene expression microarrays and reverse phase protein array (RPPA) analysis with 119 different validated antibodies, we identified pathways involved in regulation of TICs. Results: According to flow cytometric analysis for TIC markers and mammosphere (MS) formation efficiency, BCM-2665 TICs were reduced by docetaxel treatment compared to vehicle-treated at 48h post 10 mg/kg docetaxel (4-fold decrease) and 48 and 72h post 33 mg/kg docetaxel compared to control (14- and 2-fold decrease, respectively). Although the BCM-2147 TICs did not significantly decrease at any time points or doses tested, they were clearly induced at 72h post-treatment compared to control. Additionally, BCM-2665 TICs were increased within 72h post 10 mg/kg docetaxel, indicating that these time points are ideal for defining the mechanisms responsible for induction of TICs. Ingenuity Pathway Analysis of Affymetrix microarray data for both BCM-2665 and BCM-2147 revealed induction of inflammatory pathways, suggesting leukocyte infiltration associated with induction of TICs. Furthermore, RPPA analysis confirmed gene expression changes from the microarray data, and implicated apoptosis and inflammatory pathways. Sixteen of 28 proteins significantly changed with activation of CSC are involved in development of leukocytes. Discussion: These findings are consistent with the Norton-Simon Hypothesis in that chemotherapy regimens given more frequently may in fact eliminate TICs, thereby explaining the proven increased effectiveness of dose-dense chemotherapy. Based on when TICs became chemoresistant, we are comparing dose-dense treatment (4 mg/kg docetaxel every 3 days) to a traditional single dose of 32 mg/kg, in an effort to eliminate the tumor cells that cause tumor recurrence. Furthermore, our analysis of gene expression at both the RNA and protein level implicated the immune cells as TICs inducers. Since our immunocompromised mice lack T- and B- cells but have active macrophages, macrophages are indicated as inducers of TICs. We are focusing our current efforts at identifying how immune cells activate TICs and thus enhance tumorigenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-02.

Abstract A45: Targeting breast cancer stem cells using cancer preventive rexinoids

Abstract Background: Recent research in breast biology supports the cancer stem cell hypothesis which asserts that malignancies arise in tissue stem cells through dysregulation of self-renewal. The cancer stem cell hypothesis has important implications for early detection, prevention and treatment of breast cancer. If breast cancers arise from the transformation of normal stem cells, it may be possible to target stem cells for the prevention of cancer. We have previously shown that rexinoids prevent the development of ER-negative breast cancer in transgenic mouse models. Previous studies have demonstrated a role of retinoid signaling in the regulation of normal breast stem cell self-renewal and differentiation. Bexarotene and LG100268 are rexinoids that selectively bind RXR receptors. Based on the effects of these molecularly targeted agents, we hypothesize that rexinoids prevent breast cancer development by suppressing the growth and transformation of the breast stem cell population. Methods: We performed in vivo and in vitro studies to address our hypothesis. Cell proliferation assays were used to determine whether rexinoids can inhibit the growth of normal, pre-malignant and malignant breast cell lines (HMECs, MCF10A, DCIS.COM, SUM225, HCC1937 and HCC38 cell lines). To determine whether these agents affect stem cells, we performed mammosphere assays and FACS analysis after treatment with vehicle or rexiniods. We next conducted in vivo studies using MMTV-Wnt transgenic mice treated with LG100268 (50mg/kg) or vehicle, and studied the stem cell properties of mammary epithelial cells from these mice. The stem cell population in mammary glands was quantified by staining of CD24, CD49f and using FACS analysis and mammosphere assays. Results: Our results showed that the rexionids LG100268 and bexarotene inhibit the growth of breast cell lines only at a high concentrations (the IC50 (concentration that reaches a 50% growth inhibiton) in most of the tested cell lines exceeded 10uM). Results of in vitro mammosphere assays showed that 100nM LG100268 reduced mammosphere formation efficiency by more than 40% in all breast cancer cell lines. Results from our in vivo experiments showed that LG100268 reduced the mammary epithelial stem cell population by 57% as defined by the CD24+/CD49f++ population identified by FACS and by 47% as measured by in vitro mammosphere assays. Conclusion: These results from in vitro and in vivo studies demonstrate that the rexinoid LG100268 is a potent mammary stem cell inhibitor. This study shows that rexinoids suppress stem cell expansion and suggest that mammary stem cell inhibitors will be useful breast cancer preventive agents. This work is supported by a grant from the Breast Cancer Research Foundation (to PHB) and from an NIH/NCI RO1 grant (CA078480) (to PHB). Citation Information: Cancer Prev Res 2011;4(10 Suppl):A45.

Effect of fibroblasts on breast cancer cell mammosphere formation and regulation of stem cell-related gene expression

The purpose of this study was to investigate the regulatory effects of breast cancer fibroblasts (BCFs) vs. normal mammary fibroblasts (NMFs) on mammosphere formation and stem cell-related gene expression in breast cancer cells. Breast cancer cells (MCF-7) were cultured in suspension to generate primary and secondary mammospheres. The proportion of CD44+/CD24low/- cells was assessed by flow cytometry (FCM), and Wnt1, Notch1, β-catenin, CXCR4, SOX2 and ALDH3A1 gene expression was detected by quantitative real-time PCR. The fibroblasts from either breast cancer tissue or normal mammary tissue were purified from tissue specimens and co-cultured with breast cancer cells. The mammosphere formation efficacy was approximately 180/10,000 MCF-7 cells. FCM analysis showed that, compared to the 2.1% positive expression in the MCF-7 monolayer culture cells, the expression of CD44+/CD24low/- in MCF-7 mammosphere cells was significantly elevated to 10.4% (P<0.01). The proportion of the CD44+/CD24low/- subpopulation of the cells in mammospheres was nearly 5-fold higher than that of general MCF-7 cells. Compared with MCF-7 monolayer culture cells, mammosphere cells showed significantly (P<0.01) enhanced expression of Wnt1 [fold-change (FC), 2.25], Notch1 (FC, 2.45), β-catenin (FC, 1.72), CXCR4 (FC, 4.68), SOX2 (FC, 4.25) and ALDH3A1 (FC, 5.38). When BCFs were co-cultured with MCF-7 cells under mammosphere culture conditions, the length of time of mammosphere formation decreased, the volume of the mammo-spheres increased and the mammosphere-forming efficiency (MFE) was higher than that of NMFs and the control group. Both the BCF and NMF groups showed enhanced gene expression for the following genes: Wnt1 (FC, 3.18 and 1.27, respectively), β-catenin (FC, 1.75 and 1.22, respectively), Notch1 (FC, 2.09 and 1.31, respectively), CXCR4 (FC, 2.77 and 1.33, respectively), SOX2 (FC, 2.77 and 1.80, respectively) and ALDH3A1 (FC, 5.23 and 1.85, respectively). Cancer fibroblast cells can promote the MFE and up-regulate stem cell-related gene expression in breast cancer cells.

Abstract 3340: Expansion of human breast cancer stem /progenitor cell subpopulation through histone deacetylase inhibitors

Abstract We recently demonstrated that valproic acid, an histone deacetylase (HDAC) inhibitor, radiosensitized breast cancer cells grown on plastic with serum while it radioprotected cells grown in stem cell promoting culture conditions (Debeb et al., 2010) and recently, it has been shown that differentiated cancer cells can de-differentiate into the cancer stem cell phenotype (Meyers et al., 2009; Gupta et al., 2009). Based on these data, we hypothesized that HDAC inhibitors can increase the cancer stem cell population via dedifferentiation of differentiated cancer cells. To examine our hypothesis, Sum159 breast cancer cells were FACS-sorted based on ALDH activity and subsequently treated with one of two known HDAC inhibitors, valproic acid or SAHA. After sorting, ALDH-negative cells were treated either with valproic acid, SAHA, or vehicle. After a week, the percentage of ALDH-positive cells (passage 0, P0) was examined with flow cytometry while the remaining cells were passaged and incubated with and without valproic acid or SAHA for a week and the percentage of ALDH-positive cells again evaluated (P1). This was repeated for the third time (P2). Samples from each generation were also used to assay for, mammosphere formation efficiency, cell cycle analysis as well as to assess RNA and protein expression. In P0 cells, a 3-fold increase in ALDH positive cells was seen in valproic acid-treated cells (35.6% vs.12.6%) and a 1.5-fold increase in SAHA-treated cells (41% vs. 28%) compared to vehicle-treated controls. Moreover, HDAC inhibitors significantly increased primary and secondary mammosphere formation by 3.1 to 13.4-fold. Also, while the percentage of cells in the G0/G1 phase is increased with treatment, the percentage of cells in the S-phase of the cell cycle is significantly reduced. These effects were maintained through multiple passages. Furthermore, the mRNA and protein expression of Beta-catenin and EMT associated genes like vimentin, fibronectin and n-cadherin was significantly increased with treatment in initial and passaged cells. Further functional endpoint studies are being conducted in-vivo to validate these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2011-3340

Abstract LB-100: The Norton-Simon hypothesis and cancer stem cells: how cancer stem cells may explain the effectiveness of dose-dense chemotherapy

Abstract Systemic therapies are effective initially in controlling and reversing tumor growth; however, residual cancers will invariably re-grow despite this initial response. We have published data from paired human breast cancer samples that standard therapy every three weeks kills dividing daughter cells but not tumor-initiating cells (TICs), so that samples obtained after therapy are enriched for CD44+/CD24−/low putative “tumor-initiating” or “cancer stem” cells. These data indicate that standard treatment regimens are missing the critical targets, TICS; thus, we are trying to identify novel therapeutic combinations and regimens that will eliminate these TICS that ultimately cause breast tumor recurrence and metastasis. Interestingly, we have recent data in human breast tumors that indicate that TICs may in fact be initially chemosensitive, with a decrease in TICs observed within three days of chemotherapy, but shortly thereafter, TICS are actually induced by chemotherapy. To evaluate TIC response to chemotherapy, mice with xenografted human breast tumors were treated with docetaxel (20 mg/kg) or vehicle, and then tumors were collected and dissociated for TICs assays either 3d or 14d after treatment. According to multiple assays for TICs, TICs were reduced compared to vehicle-treated at 3d post-docetaxel, and increased at 14d. This was evident in both primary and secondary mammosphere (MS) formation. Flow cytometric analyses for TICs markers (CD44+/CD24−/low and ALDH+) further corroborated these data. These results indicate TICs were initially responsive but became chemoresistant, or even activated by chemotherapy. To further define TICs response to chemotherapy, mice with xenografted human breast tumors were treated with vehicle, 10 mg/kg docetaxel, or 33 mg/kg docetaxel, and then tumors were collected and dissociated for TICs assays either 48h or 72h after treatment. Although BCM-2147 TICs did not significantly decrease at any time points or doses tested, they were induced by 72h post-treatment compared to control (ALDH+ 2- to 4-fold increased). In contrast, according to TIC markers and MS formation efficiency, BCM-2665 TICS were reduced by docetaxel compared to vehicle at 48h post 10 mg/kg docetaxel (4-fold decrease), and 48h and 72h post 33 mg/kg docetaxel compared to control (14- and 2-fold decrease, respectively). Furthermore, BCM-2665 ALDH+ cells showed 6-fold increased proliferation 72h post cisplatin compared to control, indicating that chemotherapy may inducing proliferation of TICs. When TICs are induced to proliferate, they may be more susceptible to traditional proliferative targets such as chemotherapy. These findings are consistent with the Norton-Simon Hypothesis in that chemotherapy regimens given more frequently may in fact eliminate TICs, thereby explaining the proven increased effectiveness of dose-dense chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-100. doi:10.1158/1538-7445.AM2011-LB-100

Mesenchymal stem cells promote mammosphere formation and decrease E-cadherin in normal and malignant breast cells.

IntroductionNormal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures.ObjectiveWe tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.ResultsWe found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.ConclusionsMSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.