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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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Project description:Mesenchymal stem cells (MSCs) therapy for sepsis has been extensively studied in the past decade, however, the treatment regimen and mechanism of action of MSCs remain elusive. Here, we attempted to understand the efficacy and mechanism of action of MSCs on rescuing mice with sepsis. A mouse model of sepsis was produced by cecal ligation and puncture (CLP). Allogeneic adipose-derived MSCs (ADSCs) were administered by intravenous infusion at 6 h after CLP, and dose-related effects of ADSCs on these mice were determined by survival rate, histopathological changes, biochemical and coagulation parameters, bacterial load, and plasma levels of endotoxin and inflammatory cytokines. The tissue distribution of intravenously infused ADSCs in septic mice was investigated by pre-labeling ADSCs with the lipophilic membrane dye PKH26. RNA sequencing analysis was performed to assess the transcriptional changes in peripheral blood mononuclear cells (PBMCs) and the liver. A significant therapeutic effect of ADSCs at a dose of 2×107 cells/kg in septic mice was evidenced by a remarkable reduction in mortality (36% vs 9% survival rate), blood bacterial burden, systemic inflammation, and multiple organ damage. In contrast, ADSCs at a lower dose (1×107 cells/kg) failed to achieve any beneficial outcomes, while ADSCs at a higher dose (4×107 cells/kg) caused more early death within 24 hours after CLP, retaining a steady survival rate of 21% thereafter. PKH26-labeled ADSCs were predominantly localized in the lungs of septic mice after intravenous infusion, with only a smaller proportion of PKH26-positive signals appearing in the liver and spleen. RNA-sequencing analysis identified that insufficient phagocytic activity of PBMCs in addition to a hyperactivation of the hepatic immune response was responsible for the ineffectiveness of low-dose ADSCs therapy, and acute death caused by high-dose ADSCs infusion was associated with impaired coagulation signaling in PBMCs and exacerbated hepatic hypoxic injury. Our findings demonstrate a dose-specific effect of ADSCs on the treatment of sepsis due to dose-related interactions between exogenous stem cells and the host’s microenvironment. Therefore, a precise dosing regimen is a prerequisite for ADSCs therapy for sepsis.

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

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