A29

Abstract

Cancer cells attractive and activate many non-tumoral cells, including bone marrow-derived cells, as a result stimulate migration of this cells. Many studies demonstrate that tumor causes active redistribution of bone marrow cells during specific stages of cancer progression and metastasis. However, to date how the bone marrow derived cells at primary sites is hijacked to support tumor growth and how tumor progression influence of bone marrow cells distribution is not understood. The most complete picture of the impact of tumor development on bone marrow cells migration and distribution required to better understanding the process of forming a tumor microenvironment. The objective of the present study was to investigate the influence of growth and progression of melanoma B16F10 on unfractionated bone marrow cells (BMCs) distribution. Materials and Methods: There were two groups of mice in this study: healthy + intravenously transplantation BMCs (n = 5), B16F10 + intravenously transplantation BMCs (3 days later tumor inoculation) (n = 5). Whole bone marrow was harvested from male C57Bl6 mice and transplanted into female recipient C57BL6 mice bearing melanoma B16F10 or healthy female mice. After 7 and 14 days of cell transplantation (10, 17 days after tumor inoculation, respectively), the animals were sacrificed by cervical dislocation. The blood, tumor, lymph nodes, sentinel lymph nodes (regional lymph nodes), lung, liver, spleen, bone marrow, brain, heart from each animal was immediately excised and processed for qPCR analysis. To further validated PCR measurements was performed fluorescence microscopy section of bone marrow of mice bearing melanoma B16F10 to detection Hoechst 33342 labeled transplanted cells. Results: In 10 days after tumor inoculation both healthy mice and mice bearing melanoma B16F10, 80–90% of total detected cells were identified in spleen and bone marrow. Accumulation of BMCs was mainly observed in spleen of healthy animals (mean 4424/100,000, standard deviation 1005/100,000), whereas spleen of mice bearing melanoma B16F10 contained significantly smaller number of cells (mean 947/100,000, standard deviation 203/100,000) (p < 0.001). Accumulation of BMCs was observed mainly in the bone marrow of mice bearing melanoma B16F10 (mean 5554/100,000, standard deviation 925/100,000), whereas bone marrow of healthy mice contained significantly lower number of transplanted BMCs (mean 2193/100,000, standard deviation 921/100,000) (p < 0.001). Tumor contained a small number of Y-positive BMCs (mean 135/100,000, standard deviation 13/100,000). Significant differences in the quantitative distribution of BMCs in other examined organs were not found. In 14 days after cells transplantation(17 days after tumor inoculation) 80% of cells was detected in lymph nodes of healthy mice (mean 3190/100,000, standard deviation 756/100,000). 10% Y-positive BMCs into bloodstream of healthy mice was also found, whereas transplanted BMCs were not found in the lymph nodes and bloodstream of mice bearing tumor. At this time it was revealed that bone marrow of mice bearing melanoma B16F10 contained BMCs in 50 times greater than in bone marrow of healthy mice (mean 2309/100,000, standard deviation 695/100,000 and mean 42/100,000, standard deviation 10/100,000 respectively). Tumor of mice bearing melanoma B16 contained large amount of Y-positive BMCs (mean 569/100,000, standard deviation 262/100,000). The liver, spleen, lung and heart did not contain significant amount of Y positive BMCs. Fluorescence microscopy provided clear evidence of Hoechst 33,342 labeled transplanted cells migrated to the bone marrow of mice bearing melanoma B16F10 and further validated our PCR measurements. Only a small number of BMCs migrated and engrafted to bone marrow of healthy mice, while bone marrow of mice with melanoma B16F10 contain significant number of transplanted BMCs 10 and 17 days after BMCs administration. Conclusion: Thus, it is shown that melanoma B16F10 regulate distribution of BMCs with a preferred orientation in the host bone marrow.