CD44 +/CD24 - tumor cells are reported to contain cancer stem cells in breast cancer. The main purpose of the present study is to develop an immunohistofluorescence method that can quantitatively analyze CD44 +/CD24 - tumor cell distribution in breast cancer tissue and help better define the role of CD44 +/CD24 - tumor cells in breast cancer. The samples used were from 21 primary breast cancer patients who underwent neoadjuvant chemotherapy and 17 cases with sentinel lymph nodes that had lymph node micrometastasis. CD44 +/CD24 - tumor cells were distinguished at a single cell level using improved triple-staining immunohistofluorescence and a simulated laser capture microdissection method. The percentage of CD44 +/CD24 - cells significantly increased following neoadjuvant chemotherapy treatment (0.93% and 2.78%, before and after, respectively, P=0.0043). The percentage of CD44 +/CD24 - cells was also significantly high in micrometastatic sentinel lymph nodes (0.49% and 1.91%, primary tumors and lymph nodes, respectively, P=0.0246). The CD44 +/CD24 - tumor cell distribution was heterogeneous in both breast cancer tissue and lymph node metastasis. In a xenograft model using immunodeficient mice, the hedgehog signaling inhibitor cyclopamine repressed the tumorigenicity of CD44 +/CD24 - cells. Our results suggest that this semi-quantitative immunohistochemical analysis is valuable for detecting a small population of cells in cancer tissues and that the hedgehog signaling pathway inhibitor cyclopamine is useful for regulating the CD44 +/CD24 - tumor cells in breast cancer.
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