Identification and Characterization of Cancer Stem Cells in Mouse Malignant Melanoma

Recent evidences suggest that tumors arise from a small subpopulation of cells، the cancer stem cells (CSCs) or tumor initiating cells. CSCs are able to resist the conventional methods of cancer therapy due to existence of ABC transporters on their surface. This leads to CSC resistance and maintenance resulting in post-treatment relapse and metastasis. Therefore، precise identification and characterization of these cells as a target for new therapeutic regimens is the goal of numerous studies. This study، with the intent to design a new method of immunotherapy for targeting cancer stem cells in mouse malignant melanoma، initially characterized the cancer stem cells in this malignancy. In order to identify the CSCs we induced a melanoma tumor using the B16F10 cell line in C57BL/6 mice. The tumor bulk was dissociated by an enzymatic method and homogeneous tumor cells were sorted using anti-CD44 and anti-CD24 antibodies. The sorted tumor cell subpopulations were compared according to their ability to form cell spheres in serum free medium (SFM). We determined the tumor formation ability of all cell subpopulations by transplanting serial dilutions of B16-F10 and all sorted cells sub-populations into C57/BL6 mice. The results showed that although all separated cell subpopulations and B16-F10 cells formed non-adherent spheroids in SFM in the presence of B-27، but the CD24+ cells presents a significantly higher ability to produce spheroids. The B16F10 cell line، CD44+CD24- and CD44-CD24- cells showed equal potencies in tumor induction (1 in 21730 cells). The CD44-CD24+ cells tumor induction potency was 1 in 17426 and this ability for the double positive cells (CD44+CD24+) was 1 in 11295. Collectively،the double positive (CD24+CD44+) cells were more potent in both spheroid formation and tumorogenicity. Hence they might be the CSC population of mouse melanoma.