Anti-tumor Effects of Canine and Human Adipose Tissue-derived Mesenchymal Stem cells in a Xenograft Mouse Model for Melanoma

Abstract

Mesenchymal stem cells (MSCs) show promising potential for use in regenerative medicine and a variety of diseases because of their capacity of self-renewal and multipotential differentiation. Especially, MSCs have been identified as promising therapeutic tools in cancer treatment since they possess a powerful capacity for tumor-directed migration and incorporation, highlighting their potential as an optimal vehicle for delivering anticancer agents. This dissertation is composed of two parts. The first part of the study was performed to investigate the anti-tumor effect of human adipose tissue-derived mesenchymal stem cells (AT-MSCs) on human melanoma. The growth-inhibitory effect of AT-MSC-conditioned medium (AT-MSC-CM) on A375SM and A375P melanoma cells was evaluated using a cell viability assay. The results showed that AT-MSC-CM significantly inhibited the viability of A375SM and A375P cells. To investigate whether AT-MSC-CM inhibit melanoma cell proliferation by altering cell cycle distribution and inducing apoptosis, A375SM and A375P cells were analyzed by flow cytometry after treatment with AT-MSC-CM for 72 h. AT-MSC-CM induced G0/G1 cell cycle arrest and apoptosis in A375SM and A375P melanoma cells. Western blot assays showed that cyclin D1 levels were decreased and caspase-3, caspase-7 and PARP protein levels were increased in melanoma cells cultured with AT-MSC-CM. To evaluate an anti-tumor effect of AT-MSCs on melanoma in vivo, CM-DiI-labeled AT-MSCs were circumtumorally injected to the tumor-bearing nude mice, and change in the tumor size was monitored. Treatment with AT-MSCs suppressed tumor growth in the mice and fluorescence analysis revealed that AT-MSCs migrated efficiently to the tumor tissues. The second part of the dissertation was designed to test the hypothesis that the stem cell-based gene therapy combined with low-dose cisplatin would improve therapeutic efficacy against canine melanoma. The IFN-β transduced canine AT-MSCs (cAT-MSC-IFN-β) inhibited the growth of LMeC canine melanoma cells in vitro in direct as well as indirect co-culture systems. Flow cytometric cell cycle analysis showed that the proportion G0/G1 phase LMeC cells co-cultured with cAT-MSC-IFN-β was higher than that of the controls. G1 arrest occurred concurrently with a reduction in the percentage of S phase cells. In animal experiments using BALB/c nude mouse xenograft model which was established by injecting LMeC cells subcutaneously, the combined treatment of cAT-MSC-IFN-β and low-dose cisplatin significantly reduced tumor volume compared to the control and single agent treatment groups. Fluorescent microscopic analysis of the tumor section provided evidence for homing of cAT-MSC-IFN-β to the tumor site and TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) assay showed that the combination treatment of cAT-MSC-IFN-β and low-dose cisplatin induced high levels of cell apoptosis. In conclusion, cell therapy using AT-MSCs demonstrated an anti-tumor effect on canine and human melanomas, suggesting a possible therapeutic option for this type of cancer. These findings may provide valuable information warranting further explorations of the application of the combined therapy for various tumors including malignant melanoma.