A new humanized in vivo model of KIT D816V+ advanced systemic mastocytosis monitored using a secreted luciferase

Systemic mastocytosis are rare neoplasms characterized by accumulation of mast cells in at least one internal organ. The majority of systemic mastocytosis patients carry KIT D816V mutation, which activates constitutively the KIT receptor. Patient with advanced forms of systemic mastocytosis, such as aggressive systemic mastocytosis or mast cell leukemia, are poorly treated to date. Unfortunately, the lack of in vivo models reflecting KIT D816V+ advanced disease hampers pathophysiological studies and preclinical development of new therapies for such patients. Here, we describe a new in vivo model of KIT D816V+ advanced systemic mastocytosis developed by transplantation of the human ROSAKIT D816V-Gluc mast cell line in NOD-SCID IL-2R g−/− mice, using Gaussia princeps luciferase as a reporter. Intravenous injection of ROSAKIT D816V-Gluc cells led, in 4 weeks, to engraftment in all injected primary recipient mice. Engrafted cells were found at high levels in bone marrow, and at lower levels in spleen, liver and peripheral blood. Disease progression was easily monitored by repeated quantification of Gaussia princeps luciferase activity in peripheral blood. This quantification evidenced a linear relationship between the number of cells injected and the neoplastic mast cell burden in mice. Interestingly, the secondary transplantation of ROSAKIT D816V-Gluc cells increased their engraftment capability. To conclude, this new in vivo model mimics at the best the features of human KIT D816V+ advanced systemic mastocytosis. In addition, it is a unique and convenient tool to study the kinetics of the disease and the potential in vivo activity of new drugs targeting neoplastic mast cells

Additional file 4: of Protein kinase C inhibitor Gรถ6976 but not Gรถ6983 induces the reversion of E- to N-cadherin switch and metastatic phenotype in melanoma: identification of the role of protein kinase D1

PKD1 knockdown induces the expression of E-cadherin and inhibits the expression of N-cadherin in M4T2 metastatic melanoma cells. M4T2 cells were transfected or not with either specific PKD1-targeting (siPKD1) or control non-targeting (siControl) siRNAs according to the manufacturer protocol (Santa Cruz Biotechnology, sc-36245 and sc-37007, respectively). Three days after transfection, these cells were analyzed by western blot for PKD1, E-cadherin, N-cadherin and actin expression. (PPTX 102 kb

Additional file 2: of Protein kinase C inhibitor Gö6976 but not Gö6983 induces the reversion of E- to N-cadherin switch and metastatic phenotype in melanoma: identification of the role of protein kinase D1

Effect of Gö6976 on the expression of E- and N-cadherins and the anchorage-independent growth in I5 primary melanoma cell line. A. I5 primary melanoma cells were seeded in 6-well plates at the density of 50,000 cells per well. After three days of culture, cells were treated with 1 μM Gö6976, 1 μM Gö6983 or DMSO for 24 h. Cells were then lysed and proteins analyzed by western blot using anti-E-cadherin, anti-N-cadherin or anti-actin antibodies. T1 melanoma cells were used as positive control for E-cadherin expression. B. I5 primary melanoma cells were seeded in methylcellulose (1000 cells per plate) containing 1 μM Gö6976 or 1 μM Gö6983. The colonies were counted after 18 days of culture. (PPTX 135 kb

Additional file 3: of Protein kinase C inhibitor Gö6976 but not Gö6983 induces the reversion of E- to N-cadherin switch and metastatic phenotype in melanoma: identification of the role of protein kinase D1

Correlation between PKD1 expression and mesenchymal features in primary (T1 and I5) and metastatic (G1, M2 and M4T2) melanoma cells. The relationship between PKD1 expression and mesenchymal features in melanoma cells was estimated using Pearson’s correlation analysis applied on the data from western blot, MTT, methylcellulose and wound healing assays that are presented in Figs. 1, 6 and 7a. (PPTX 51 kb