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Gene Expression Profiles of tumor sorted fractions demarcating cancer stem cell heirarchy using PKH dye in ovarian cancer xenograft tumor

ABSTRACT: Our earlier report states isolation of ovarian cancer stem cell heirarchy using membrane labeling PKH26/67 dyes (Kusumbe and Bapat Cancer Research. 2009) in a xenograft model. We earlier reported that ovarian cancer is a stem cell disease with isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). A4T cells were labelled with PKH26 membrane labeling dye, flourescence intensity was quantified and set as 100% dye retaining cells (PKHhi) with flow cytometery. 2.5x10^6 P65 A4PKH26 labeled cells were injected s.c in NOD/SCID mice, while unlabeled cells were also injected for generation of control tumors . Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Tumor cells were acquired on BD FACS Aria II across PE channel of blue laser. Cells retaining dye intensity as that of preinjected cells were gated as PKHhi while cells exhibiting complete dye depletion were gated as PKHneg using unlabeled tumor cells as control; while cells showing intermediate label intensity were gated as PKHlo. Details of the flow cytometry analysis and sorting are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009).

ORGANISM(S): Homo sapiens

PROVIDER: GSE74851 | GEO | 2016/01/01

SECONDARY ACCESSION(S): PRJNA301680

REPOSITORIES: GEO

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Gene Expression Profiles of tumor sorted fractions demarcating cancer stem cell heirarchy using PKH dye in ovarian cancer xenograft tumor

Project description:Our earlier report states isolation of ovarian cancer stem cell heirarchy using membrane labeling PKH26/67 dyes (Kusumbe and Bapat Cancer Research. 2009) in a xenograft model. We earlier reported that ovarian cancer is a stem cell disease with isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). A4T cells were labelled with PKH26 membrane labeling dye, flourescence intensity was quantified and set as 100% dye retaining cells (PKHhi) with flow cytometery. 2.5x10^6 P65 A4PKH26 labeled cells were injected s.c in NOD/SCID mice, while unlabeled cells were also injected for generation of control tumors . Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Tumor cells were acquired on BD FACS Aria II across PE channel of blue laser. Cells retaining dye intensity as that of preinjected cells were gated as PKHhi while cells exhibiting complete dye depletion were gated as PKHneg using unlabeled tumor cells as control; while cells showing intermediate label intensity were gated as PKHlo. Details of the flow cytometry analysis and sorting are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009). PKHhi PKHlo and PKHneg were sorted with 99% purity and 1x10^5 cells from each fraction was subjected to RNA isolation, labeling and microarray analysis. RNA of each fraction was labeled with Cy3 and designated as A4PKHhi, A4PKHlo and A4PKHneg. Three samples of each were chosen for hybridization. Custom Agilent Human whole Gene Expression Microarray 8x60K designed by Genotypic Technology Private Limited.(AMADID: 27114);Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)

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