Flow cytometric analysis for detection of tumor-initiating cells in feline mammary carcinoma cell lines

A small population of cells known as tumor-initiating cells (TICs), which have the capacity to self-renew, differentiate, and form tumors at high frequency, has a potential role in tumor initiation, aggression, and recurrence. In human breast cancers, TICs are identified by surface markers, such as CD44 and CD24, and an aldefluor assay based on aldehyde dehydrogenase activity (ALDH+) using flow cytometry. However, the usefulness of surface markers CD44 and CD24 and ALDH activity in feline mammary carcinomas remains largely elusive. We attempted to identify CD44+CD24− and ALDH+ cells using 8 feline mammary carcinoma cell lines, including FKNp, which was obtained from a primary lesion, and the capacity to generate tumor nodules was analyzed in immunodeficient mice injected with ALDH+ FKNp-derived cells. The CD44+CD24− and ALDH+ cells were detected in all cell lines derived from feline mammary carcinomas. Xenograft transplantation into immunodeficient mice demonstrated that as few as 1 × 102 ALDH+ cells could initiate tumor growth in 1 out of 4 mice, while 1 × 103 ALDH+ cells initiated tumor growth in 5 out of 6 mice. However, 1 × 103 ALDH− cells failed to initiate tumors in all the tested mice. ALDH+-derived tumors contained both ALDH+ and ALDH− cells, indicating that ALDH+ FKNp-derived cells had higher tumorigenicity than ALDH− cells. These results suggest that TICs may exist in feline mammary carcinomas, and further characterization of CD44+CD24− and ALDH+ cells is needed to define novel therapies targeted against TICs. This study provides the foundation for elucidating the contribution of TICs in tumorigenesis.