Isolation and Characterization of Cancer Stem Cells In Vitro

Gedye, Craig; Ailles, Laurie

doi:10.1007/978-1-62703-128-8_12

Cited by 13 publications

(11 citation statements)

References 81 publications

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“…While we validated each individual enzyme in isolation, in everyday practice these are often used in combination, e.g. collagenase and hyaluronidase in tissue digestion [30]. In the preliminary screen, some epitopes showed almost complete abrogation of detection, including common antigens such as CD1d, CD4, CD25, CD27 and CD62P, a particular concern for the study of heterogeneity and immunology in solid tumors.…”

Section: Discussionmentioning

confidence: 98%

Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

et al. 2014

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Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.

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Section: Discussionmentioning

confidence: 98%

Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

et al. 2014

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“…Remaining samples were dissociated into single cell suspensions. For dissociation, tumours were minced with sterile scalpels and digested with 1X collagenase/hyaluronidase (Stem Cell Technologies) and DNase I (125 U/mL, Invitrogen) in defined serum-free media (see below) plus antibiotics at 37 °C for a maximum of two hours with frequent gentle trituration as previously described 39 . Red blood cells were lysed by applying 1 mL of ammonium chloride lysis buffer (Gibco) to the cell pellet for 5 minutes on ice, followed by an immediate wash in DMEM + 10% foetal bovine serum (FBS).…”

Section: Methodsmentioning

confidence: 99%

“…Established cell lines were cultured at 37 °C, 5% CO 2 in high-glucose DMEM (Wisent, St. Bruno, Canada) supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, and passaged with 0.05% trypsin in 2 mM EDTA in PBS, pH 7.4 (Wisent). Routine Mycoplasma surveillance (MycoAlert, Lonza, Basel, Switzerland) and cell line identity verification by STR profiling (AmpFLSTR® Identifiler®, TCAG, SickKids Toronto) were performed as described previously 39 .…”

Section: Methodsmentioning

confidence: 99%

Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma

Gedye

Sirskyj

Lobo

et al. 2016

Sci Rep

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Rare cancer stem cells (CSC) are proposed to be responsible for tumour propagation and re-initiation and are functionally defined by identifying tumour-initiating cells (TICs) using the xenotransplantation limiting dilution assay (LDA). While TICs in clear cell renal cell carcinoma (ccRCC) appeared rare in NOD/SCID/IL2Rγ−/− (NSG) mice, xenografts formed more efficiently from small tumour fragments, indicating the LDA underestimated ccRCC TIC frequency. Mechanistic interrogation of the LDA identified multiple steps that influence ccRCC TIC quantitation. For example, tissue disaggregation destroys most ccRCC cells, common assays significantly overestimate tumour cell viability, and microenvironmental supplementation with human extracellular factors or pharmacological inhibition of anoikis increase clonogenicity and tumourigenicity of ccRCC cell lines and primary tumour cells. Identification of these previously uncharacterized concerns that cumulatively lead to substantial underestimation of TICs in ccRCC provides a framework for development of more accurate TIC assays in the future, both for this disease and for other cancers.

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“…Except where noted, cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA). The CSCs were derived as described in the literature [17,18] . Briefly, serial dilutions were made of U87 cells in neural stem cell (NSC) media containing DMEM/F12 1:1 media, B27 serum-free supplement (1×), penicillin (10,000 IU/mL), streptomycin (10,000 μg/mL), 20 ng/mL fibroblast growth factor (FGF), 50 ng/mL epidermal growth factor (EGF), HEPES 1 M solution, and 5 mg/mL heparin (Sigma-Aldrich, St. Louis, MO).…”

Section: Csc Derivation and Characterizationmentioning

confidence: 99%

Gene silencing of HIF-2α disrupts glioblastoma stem cell phenotype

Nusblat¹,

Tanna²,

Roth³

2020

CDR

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Aim: Improved treatment strategies are desperately needed for eradicating cancer stem cells (CSCs), which drive malignancy and recurrence in glioblastoma multiforme. Hypoxic regions within the tumor microenvironment help maintain and promote the proliferation of CSCs. Here, we explored the effects of silencing hypoxia inducible factor-2α (HIF-2α) because of its specificity for CSCs within the hypoxic environment. Methods: Cancer stem cell neurospheres were formed by enriching from both the glioblastoma cell line U87 and from brain tumor stem cells isolated directly from human brain tumors. Silencing of human HIF-2α was performed using both commercial and in-house transfection of a validated short interfering RNA, with all results compared to an established non-silencing control short interfering RNA. Silencing of HIF-2α was established by Western blotting, and phenotypic effects were assayed by cell migration assays, cell viability measurements, and immunofluorescence staining of differentiation markers. Results: Transfection with either our previously reported pH-sensitive, cationic amphiphilic macromolecule-based delivery system or Lipofectamine was similarly effective in silencing HIF-2α. The chemotherapeutic resistance and neurosphere formation were reduced when HIF-2α was silenced. Migratory capacities in the presence of macrophage conditioned media were modulated. HIF-2α silencing was complementary to temozolomide treatment in producing phenotypic rather than cytotoxic effects. Conclusion: HIF-2α silencing under hypoxia inhibited CSC phenotypes while promoting differentiated cell phenotypes and is complementary to existing DNA alkylating treatments in inhibiting glioma CSC activity.

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Isolation and Characterization of Cancer Stem Cells In Vitro

Cited by 13 publications

References 81 publications

Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma

Gene silencing of HIF-2α disrupts glioblastoma stem cell phenotype

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