Molecular characterization of quiescent melanoma cells
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ABSTRACT:
PROVIDER: PRJNA730416 | ENA |
REPOSITORIES: ENA
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Project description:Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice, rapidly declining after B-RAF inhibitor treatment. CTCs were shed early from localized tumors and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled comparison of RNA sequencing profiles with the matched primary tumor. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. In patients with metastatic melanoma, CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF targeted therapy. Together, the capture of CTCs and their molecular characterization provide insight into the hematogenous spread of melanoma. We adapted a microfluidic platform, the HbCTC-Chip (Stott et al., 2010, Pubmed ID: 20930119), to capture melanoma CTCs derived from mouse tumors, using panels of antibodies against melanoma-specific cell surface markers, followed by staining for melanoma antigens and optimized on-chip fluorescent imaging. We used a tamoxifen inducible BRAF(CA/+)/PTEN(flox/flox) melanoma mouse model (Dankort et al., 2009, Pubmed ID: 19282848) derived from a C57BL/6 background. Such mice received focal subcutaneous injection of tamoxifen (Sigma) (50ul at 5mg/ml in 50% EtOH suspension) at the left flank at 6-7 weeks after birth. Blood samples were collected from five mice with high tumor burden following tamoxifen injection. Blood from each mouse was split and processed through the CTC-chips functionalized with anti-CSPG4/MCAM antibody and control IgGs, respectively. Matched primary (from the tamoxifen injection site) and metastatic (from upper or lower back) tumors were harvested from the same mouse and immediately flash-frozen in liquid nitrogen. RNA extraction, single molecular sequencing and determination of Digital Gene Expression was as in Yu et al., 2012 (Pubmed ID: 22763454). One of the five mice yielded no CTC or IgG data. In addition, skin was taken from a sixth mouse, which was a complete wild type C57BL/6 mouse (without the BRAF/PTEN transgenes).The skin was taken after euthanasia of the animal and was processed as the tissue from the matched primary and metastatic tumors.
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Project description:The adult kidney replaces lost cells in-vivo via proliferation of cells functioning as committed clonal progenitors. Here we combined the generation of single cell derived clonal cultures from human adult kidney with transcriptomic analysis for molecular characterization of in-vitro clonal behavior at inception and after propagation. We discovered two types of clones; rapidly proliferating de-differentiated fibroblast-like (FL) originating from the proximal tubule and stably proliferating cuboidal epithelial-like (EL) originating from distal segments that efficiently propagate with one cell giving rise to 3.3*10(6) cells. Segment-specific clonal heterogeneity included molecular characteristics of cell-cycle, epithelial-mesenchymal transition (high NCAM1, Serpine1) and oxidative phosphorylation in FL-clones. In contrast, the more quiescent EL-clones harboring markers of mature kidney epithelia (high CD24, CDH1/E-cad, EpCAM) activate the BMP pathway. Thus, the human adult kidney harbors progenitor cell function in which segment identity and the level of epithelial maturation dictate clonal behavior and sustained renal cell formation in culture.
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