A31

Abstract

Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) are multipotent progenitor cells used to function as cellular delivery vehicles for antitumor agents (Miletic et al., 2007; Gunnarsson et al., 2010; van Eekelen et al., 2010; Studeny et al., 2002). The use of mesenchymal stem cells for the delivery of anticancer drugs based on the hypothesis that stem cells migrate predominantly into tumor tissue. However, the property of MSCs migrate to the tumor is not clear demonstrated and requires further experimental confirmation. The aim of our study is to identify how tumor growth affects on MSCs distribution after systemic administration on a model melanoma B16F10. Materials and methods: There were two groups of mice in this study: healthy + intravenously transplantation MSCs (n = 5), B16F10 + intravenously transplantation MSCs (3 days later tumor inoculation) (n = 5). Preparation of MSCs for transplantation: the isolation of MSCs from bone marrow of male C57BL/6 mice was performed using the methods described in work of Esposito M. (Bazan et al., 2004). To investigate their capacity for mesodermal differentiation adipogenic and osteogenic differentiation was carried out. To phenotype cell-surface antigens, MSCs were stained with FITC, PE or APC-conjugated antibodies specific for the following mice antigens CD45-FITC, CD90-FITC, CD34-PE, CD73- PE, and CD105-APC. Stained cells were analysed using FACS Calibur flow cytometer (Becton Dickinson, USA). One hour, 3, 7 and 14 days after cell transplantation (3, 7, 10, 17 days after tumor inoculation, respectively), the animals were sacrified by cervical dislocation. The blood, tumor, lymph nodes, sentinel lymph nodes (regional lymph nodes), lung, liver, spleen, bone marrow, brain, heart from each animal were immediately excised and processed for qPCR analysis. Fur further validatuion of PCR measurements, fluorescence microscopy of a section of bone marrow of mice bearing melanoma B16F10 was performed to detect Hoechst 33342 labeled transplanted cells. Results: MSCs isolated from bone marrow possess ability to differentiate into osteocytes and adipocytes, plastic adherence and expression of cluster of differentiation (CD) markers such as CD105, CD73, and CD90 in >95% of the culture with absent expression of markers including CD34 and CD45. In order to study the effect of the melanoma B16F10 growth on the distribution of transplanted MSCs, we have shown the similarities and differences in the distribution of MSCs in the body of healthy animals and animal bearing tumor. 50–65% of Y-positive MSCs detected in lungs as healthy mice, as mice bearing melanoma B16F10 after one hour of MSCs administrated. We found Hoechst labeled MSCs in capillaries of interalveolar septa by fluorescent microscopy. In other examined organs, Y-positive cells were not found. Tumor tissue contained the maximum number of transplanted MSCs 3 days after cell transplantation. In 3 days after MSCs administration heart and brain of mice bearing melanoma B16F10 contained significantly greater number of Y-positive cells compared to those of healthy mice. In 7 and 14 days after transplantation, bone marrow of mice bearing tumor contained more than 2000 Y-positive cells/100,000, while in the bone marrow of healthy animals, onlysingle cellswere detected. Conclusion: Melanoma B16F10 affects on pattern of mesenchymal stem cell distribution, which depends on stage of tumor growth.