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Immunoselection of Breast and Ovarian Cancer Cells with Trastuzumab and Natural Killer Cells: Selective Escape of CD44^high/CD24^low/HER2^low Breast Cancer Stem Cells

¹ Department for Obstetrics and Gynecology, ² Interdisciplinary Center for Clinical Research (IZKF), ³ KFO 124 and ⁴ Department of Neurology, University of Würzburg, Würzburg, Germany; and ⁵ Department of Neurology, University of Regensburg, Regensburg, Germany; and ⁶ Department of Neurology, University Hospital Aachen, Aachen, Germany

Requests for reprints: Jörg Wischhusen, Department of Gynecology, University of Würzburg, Josef-Schneider-Strasse 4, D-97080 Würzburg, Germany. Phone: 49-931-201-25291; Fax: 49-931-201-25406; E-mail: Wischhusen_J{at}klinik.uni-wuerzburg.de.

Key Words: human • antibody-dependent cell-mediated cytotoxicity • natural killer cells • trastuzumab • cancer stem cells • tumor immunology

Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Because natural killer (NK) cell–mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is thought to contribute to the therapeutic effects of trastuzumab, we have established a cell culture system to select for ADCC-resistant SK-OV-3 ovarian cancer and MCF7 mammary carcinoma cells. Ovarian cancer cells down-regulated HER2 expression, resulting in a more resistant phenotype. MCF7 breast cancer cells, however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44^highCD24^low "cancer stem cell–like" phenotype and expressed significantly less HER2 compared with non–stem cells. Likewise, the CD44^highCD24^low population was also found to be more immunoresistant in SK-BR3, MDA-MB231, and BT474 breast cancer cell lines. When immunoselected MCF7 cells were then re-expanded, they mostly lost the observed phenotype to regenerate a tumor cell culture that displayed the initial HER2 surface expression and ADCC-susceptibility, but was enriched in CD44^highCD24^low cancer stem cells. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. Thus, we provide evidence that the induction of ADCC by trastuzumab and NK cells may spare the actual tumor-initiating cells, which could explain clinical relapse and progress. Moreover, our observation that the "relapsed" in vitro cultures show practically identical HER2 surface expression and susceptibility toward ADCC suggests that the administration of trastuzumab beyond relapse might be considered, especially when combined with an immune-stimulatory treatment that targets the escape variants. [Cancer Res 2009;69(20):8058–66]