CancerTherapy: Preclinical Preferential Killing of Breast Tumor Initiating Cells by N,N-Diethyl-2- [4-(Phenylmethyl)Phenoxy]Ethanamine/Tesmilifene Tao Deng,1Jeff C. Liu,1Kathleen I. Pritchard,2,3 Andrea Eisen,2,3 and Eldad Zacksenhaus1, 3,4, 5 Abstract Purpose: N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE; tesmilifene) is thought to potentiate the antineoplastic effect of cytotoxic drugs. In a phase III randomized trial for metastatic breast cancer using doxorubicin with or without DPPE, addition of the latter resulted in a significant improvement in overall survival and a trend toward a difference in progression-free survival but, paradoxically, no difference in objective tumor response. Here we tested the hypothesis that DPPE targets breast tumor-initiating cells (TICs). Experimental Design: Human breast TICs from pleural effusions were identified as CD44+:CD24-/low cells by flow cytometry and functionally by their ability to form nonadherent spheres in culture. Mouse mammary TICs from two different models of breast cancer were identified as cells capable of initiating spheres in culture and secondary tumors following transplantation into the mammary gland of syngeneic mice. Results: We show that at physiologically attainable concentrations, treatment with DPPE alone reduced tumorsphere formation and viability of CD44+:CD24-/low breast cancer cells.The kinetics of killing varied for the different breast tumor cells and required continuous exposure to the drug. Whereas doxorubicin killed CD44+:CD24-/low and CD44-:CD24+ cells equally well, DPPE induced apoptosis preferentially in CD44+:CD24-/low cells. Treatment of Her2/Neu+ mammary tumor cells with DPPE in vitro efficiently killed TICs, as determined by flow cytometry and transplantation assays; DPPE further cooperated with doxorubicin to completely eradicate tumorigenic cells. Conclusions: Our results show that continuous treatment with DPPE alone directly targets breast TICs, and provide rationale to test for cooperation between DPPE and known drugs with efficacy toward breast cancer subtypes. Breast cancer mortality is declining in Western countries (e.g., doxorubicin) and the taxanes (e.g., docetaxel; ref. 3). primarily due to improvements in breast cancer screening and However, at best, only 50% of women treated with these agents in the adjuvant treatment of early stage disease (1, 2). will respond to therapy (4). Thus, there is an urgent need for Metastatic breast cancer remains an incurable illness for novel approaches to combat metastatic breast cancer. which few treatments have actually been shown to prolong There is growing evidence that cancer is organized in survival. The most active chemotherapeutic agents in the a hierarchy in which relatively rare tumor-initiating cells treatment of metastatic breast cancer include the anthracyclines (TICs; also known as cancer stem cells) are capable of disseminating cancer, whereas the majority of tumor cells have lost this potential (5–9). This hierarchy has been first Authors’ Affiliations: 1Toronto General Research Institute-University Health documented in acute myeloid leukemia (10, 11). More 2 3 4 Network; SunnybrookOdette Cancer Centre; and Medicine, Medical recently, TICs (cancer stem cells) were identified in breast Biophysics, and 5Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (12) and brain (13) cancers as well as other tumor types Received 7/3/08; revised 9/8/08; accepted 9/9/08. (14–21). Breast TICs express early epithelial markers such as Grant support: YMBiosciences, Inc., a matching grant from the Canadian Institute the cell surface marker CD44, whereas nontumorigenic cells for Health Research, the National Cancer Institute of Canada-Terry Fox Breast express CD24 (12). CD44+:CD24-/low cells, but not CD24+ Cancer Group Grant 13005, and the Ontario Institute for Cancer Research. The costs of publication of this article were defrayed in part by the payment of page cells, sorted from malignant pleural effusions give rise to new charges. This article must therefore be hereby marked advertisement in accordance tumors when injected into the fat pad of immunocompromised with 18 U.S.C. Section 1734 solely to indicate this fact. NOD/SCID mice (12). Note: Supplementary data for this article are available at Clinical Cancer Research Mouse models of cancer provide the power of mouse Online (http://clincancerres.aacrjournals.org/). genetics, the availability of ample samples of genetically Requests for reprints: Eldad Zacksenhaus, Division of Cell and Molecular Biology,Toronto General Research Institute-University Health Network, 67 College defined tumors and syngeneic transplantation assays. Several Street, Room 407,Toronto, Ontario, Canada M5G 2M1. Phone: 416-340-4800, ext. groups have recently identified TICs in mouse models of breast 5106; Fax: 416-340-3453; E-mail: [email protected]. Andrea Eisen, cancer (22–25). Our group has shown that TICs derived from Preventive Oncology,Toronto SunnybrookRegional Cancer Centre, 2075 Bayview Her2/Neu+ as well as Wnt1-induced mammary tumors are Avenue, Toronto, ON M4N 3M5, Canada. Phone: 416-480-4617; E-mail: andrea. [email protected]. capable of forming spheres under nonadherent conditions in a F 2009 American Association for Cancer Research. defined medium containing epidermal growth factor and doi:10.1158/1078-0432.CCR-08-1708 fibroblast growth factor (23). The tumorsphere-forming units www.aacrjournals.org 119 Clin Cancer Res 2009;15(1) January 1, 2009 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2009 American Association for Cancer Research. CancerTherapy: Preclinical prednisone in the treatment of hormone refractory prostate Translational Relevance cancer (39). Understanding the mechanism by which DPPE affects chemotherapy and overall survival may lead to Previous models implicated N,N-diethyl-2-[4-(phenyl- improved therapy (39). Using four different breast cancer methyl)phenoxy]ethanamine (DPPE; tesmilifene) in the models, we now show that treatment with DPPE alone potentiation of cytotoxicity by antineoplastic drugs. In a preferentially kills TICs. phase IIIrandomized trial for metastatic breast cancer using doxorubicin with or without DPPE, addition of the latter resulted in a significant improvement in overall survival Materials and Methods and a trend toward a difference in progression-free survival but, paradoxically, no difference in objective tumor re- Patient recruitment and consent. Subjects with metastatic breast sponse. We have now shown that continuous exposure to cancer and malignant pleural effusions were recruited from the clinical practices of a large, tertiary care cancer center under an Research pharmacologically attainable doses of DPPE effectively Ethics Board–approved protocol. All patients provided written kills tumor-initiating cells in four different models of breast informed consent. Pleural fluid samples were obtained by standard cancer. Our results suggest that DPPE-based therapies thoracocentesis. should involve continuous or frequent administration of Enrichment of lineage-depleted breast cancer cells from pleural the drug. Furthermore, more potent DPPE derivatives effusions. Pleural effusions were obtained and processed within 2 to with better specificity may be developed using the tumor- 4 h after thoracocentesis. The samples were centrifuged at 1,000 rpm initiating cell ^ specific assays presented in our study. for 10 min and cell pellets were washed with HBSS plus 2% Finally, using these assays, combinatorial drug regimens heat-inactivated fetal bovine serum (HIBS) and 2 mmol/L EDTA. with DPPE plus other antineoplastic agents with efficacy Enrichment of epithelial cells was achieved using a negative selection kit A Â 7 toward breast cancer subtypes should be assessed. StemSep (StemCell Technologies, Inc.). Briefly, 100 L per 5 10 cells of a cocktail containing biotinylated antibodies against human hematopoietic cells (CD2, CD14, CD16, CD38, CD45, and CD66) and glycophorin A were added. After 30-min incubation on ice, 60 AL (TFU) were indistinguishable by several criteria from TICs. of magnetic colloid were added and incubated for another 30 min on Specifically, TFUs and TICs were identified in the same fraction ice. Finally, the cells were passed trough magnetic columns and the of sorted cells, and cultured tumorspheres retained the supernatant containing epithelial cells was collected. The purified cells differentiation capacity, cell surface markers, and tumorigenic were cultured either as adherent monolayer with DMEM plus 10% fetal potential of freshly isolated TICs. Thus, TFUs can be used as a bovine serum or under nonadherent conditions (see below) plus surrogate for TICs in vitro. penicillin/streptomycin. Implicit in the cancer stem cell model is that targeted killing Mice and PCR-based genotyping. MMTV-Wnt1 (40) and MMTV-Neu of TICs, rather than the bulk of nontumorigenic cells, may be (41) transgenic mice, both on pure FvB background, were maintained in accordance with the Canadian Animal Care Council. The mice were curative (5, 6, 26). However, TICs are not only rare but, in some genotyped by PCR analysis of DNA extracted from tail biopsies instances, are also shown to be more resistant to radiation and using the following primers: Wnt1, forward 5¶-GGACTTGCTTCTCTTCT- cytotoxic therapy (27–31). The identification of agents that CATAGCC and reverse 5¶-CCACACAGGCATAGAGTGTCTGC; Neu, enhance the specificity of antineoplastic drugs toward TICs