Bone marrow-derived cells are recruited by the melanoma tumor with endothelial cells contributing to tumor vasculature.

PURPOSE: Tumor expansion is dependent on neovascularization, a process that requires sustained new vessel formation. Although the critical role of angiogenesis by endothelial sprouting in this process, controversy still prevails on whether angiogenesis involving bone marrow-derived endothelial cells, does contribute to this process. This study aims to evaluate the recruitment of bone marrow-derived cells by the melanoma tumor, including endothelial cells, and if they contribute to angiogenesis. METHODS: A chimeric mouse model of GFP bone marrow was used to induce melanoma tumors derived from murine B16-F10 cell line. These tumors were evaluated for the presence of myeloid cells (CD11b), T lymphocytes (CD3, CD4 and CD8) and endothelial cells (VEGFR2 and CD31) derived from bone marrow. RESULTS: Mice transplanted with GFP+ cells showed significant bone marrow chimerism (90.9 ± 0.87 %) when compared to the GFP transgenic mice (90.66 ± 2.1 %, p = 0.83) demonstrating successful engraftment of donor bone marrow stem/progenitor cells. Analysis of the murine melanoma tumor showed the presence of donor cells in the tumors (3.5 ± 1.7 %) and interestingly, these cells represent endothelial cells (CD31+ cells; 11.5 ± 6.85 %) and myeloid cells (CD11b+ cells; 80 ± 21 %), but also tumor-infiltrating lymphocytes (CD8+ T cells, 13.31 ± 0.2 %; CD4+ T-cells, 2.1 ± 1.2 %). Examination of the tumor endothelium by confocal microscopy suggests the presence of donor CD31+/GFP+ cells in the wall of some blood vessels. CONCLUSION: This study demonstrates that bone marrow-derived cells are recruited by the murine melanoma tumor, with myeloid cells and CD4 and CD8 T lymphocytes migrating as antitumor immune response, and endothelial cells participating of the tumor blood vessels formation.

Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector.

Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/µg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.