Reduction of CD44⁺/CD24⁻ breast cancer cells by conventional cytotoxic chemotherapy

Summary

Breast cancer cells with the CD44⁺/CD24⁻ phenotype have been associated with stem cell properties. To analyze effects of cytotoxic chemotherapy on these cells, we examined a series of 50 breast carcinomas before and after neoadjuvant chemotherapy with epirubicin/cyclophosphamide using double immunofluorescence. Before treatment, an average of 4.4% of the tumor cells displayed a CD44⁺/CD24⁻ phenotype. However, after chemotherapy, the frequency of CD44⁺/CD24⁻ cells dropped to 2% (P = .008). To test this unexpected finding, we analyzed a second collective of 16 patients that preoperatively had received either 4 cycles of doxorubicin/pemetrexed, followed by 4 cycles of docetaxel or 4 cycles of doxorubicin/cyclophosphamide, followed by 4 cycles of docetaxel with similar results (8.7% CD44⁺/CD24⁻ cells on average before and 1.1% after chemotherapy). In addition, no association was observed between the frequency of CD44⁺/CD24⁻ cells and the response to chemotherapy or patient survival. However, patients with tumors containing high numbers of CD44⁺/CD24⁻ cells more frequently developed bone metastases in the course of disease. In conclusion, our findings challenge the proposed role of CD44⁺/CD24⁻ cells as cancer stem cells in tumor resistance to chemotherapy as they apparently are not selected by conventional cytotoxic agents.

Introduction

Over the past years, the concept of tumor stem cells has gained major attention. Initial observations that large amounts of unselected tumor cells are needed to induce growth of novel tumors following xenograft transplantation suggested that only a small fraction of cells (ie, tumor stem cells) yielded sufficient regenerative capacity [1]. This finding was reproduced with tumors spontaneously arising in mice rendering potential influences of an anti-host immune response less likely. Furthermore, the number of cells needed for successful transplantation was reduced by repeated passaging within animals indicating a potential selection for tumor stem cells [2].

In human primary malignancies, the best data supporting the stem cell concept exist for leukemia in which individual tumor cells containing surface markers (CD34⁺/CD38⁻) similar to normal bone marrow stem cells have been identified. Transplantation of these CD34⁺/CD38⁻ cells into immunodeficient mice led to the initiation and sustained growth of the neoplastic infiltrate in vivo while the bulk mass of non-CD34⁺/CD38⁻ cells could not regrow the leukemia [3], [4]. As most leukemias show at least some tendency towards maturation into (atypical) blood cells or their precursors, this finding resembles the situation in normal bone marrow with stem cells regenerating the normal peripheral blood cells.

In solid tumors, stem cells have been a lot more elusive, for most organs or tissues no nonneoplastic adult stem cell population expressing specific surface markers has been demonstrated. For normal human mammary gland epithelia, a stem cell or precursor phenotype with high expression levels of cytokeratin 5/6 without cytokeratin 8/18 or smooth muscle actin has been suggested [5], but these data have not been reproduced by other authors [6] and no corresponding subset of cells has been reported for the majority of breast carcinomas. Recently, a subpopulation of human breast cancer cells expressing the CD44 surface antigen and showing little or no CD24 expression has been associated with stem cell qualities. Using cells derived from either primary tumors or pleural effusions that had been propagated in immunocompromised mice, Al Haji et al [7] could demonstrate that cells displaying a CD44⁺/CD24⁻/low phenotype had the capacity to regrow tumors following transplantation into recipient mice at relatively low numbers while cells with a different phenotype were nontumorigenic. In addition, breast cancer cells grown as floating clusters (so called mammospheres) displayed a CD44⁺/CD24⁻ phenotype, had the capacity to adhere and differentiate towards epithelial or myoepithelial cells after addition of fetal bovine serum and were tumorigenic following injection into SCID mice [8].

Tumor stem cells are not only characterized by an increased regenerating ability, but are also believed to preferentially escape conventional cytotoxic chemotherapy and represent the origin of tumor recurrences. In the present study, we have examined the frequency of CD44⁺/CD24⁻ breast cancer cells in primary breast carcinomas treated using neoadjuvant chemotherapy.