Flow cytometric techniques for detection of candidate cancer stem cell subpopulations in canine tumor models
Comparative Oncology and Stem Cell Research Group, The Royal (Dick) School of Veterinary Studies and Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, Edinburgh, UK Flow Cytometry Facility, Ashworth Laboratories, Institute of Immunology and Infection Research, The University of Edinburgh, School of Biological Sciences, Kings' Buildings, Edinburgh, UK MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh, UK.Veterinary and Comparative Oncology (Impact Factor: 2.73). 10/2011; 10(4). DOI: 10.1111/j.1476-5829.2011.00293.x
The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of 'CSC'. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell-associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker-defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context-dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria.
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ABSTRACT: A small population of cells known as tumor-initiating cells (TICs), which have the capacity to self-renew, differentiate, and form tumors at high frequency, has a potential role in tumor initiation, aggression, and recurrence. In human breast cancers, TICs are identified by surface markers, such as CD44 and CD24, and an aldefluor assay based on aldehyde dehydrogenase activity (ALDH(+)) using flow cytometry. However, the usefulness of surface markers CD44 and CD24 and ALDH activity in feline mammary carcinomas remains largely elusive. We attempted to identify CD44(+)CD24(-) and ALDH(+) cells using 8 feline mammary carcinoma cell lines, including FKNp, which was obtained from a primary lesion, and the capacity to generate tumor nodules was analyzed in immunodeficient mice injected with ALDH(+) FKNp-derived cells. The CD44(+)CD24(-) and ALDH(+) cells were detected in all cell lines derived from feline mammary carcinomas. Xenograft transplantation into immunodeficient mice demonstrated that as few as 1×10(2) ALDH(+) cells could initiate tumor growth in 1 out of 4 mice, while 1×10(3) ALDH(+) cells initiated tumor growth in 5 out of 6 mice. However, 1×10(3) ALDH(-) cells failed to initiate tumors in all the tested mice. ALDH(+)-derived tumors contained both ALDH(+) and ALDH(-) cells, indicating that ALDH(+) FKNp-derived cells had higher tumorigenicity than ALDH(-) cells. These results suggest that TICs may exist in feline mammary carcinomas, and further characterization of CD44(+)CD24(-) and ALDH(+) cells is needed to define novel therapies targeted against TICs. This study provides the foundation for elucidating the contribution of TICs in tumorigenesis.Veterinary Immunology and Immunopathology 08/2013; 156(1-2). DOI:10.1016/j.vetimm.2013.08.006 · 1.54 Impact Factor
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ABSTRACT: Basal-like tumours constitute 2-18% of all human breast cancers (HBCs). These tumours have a basal myoepithelial phenotype and it has been hypothesized that they originate from either myoepithelial cells or mammary progenitor cells. They are heterogeneous in morphology, clinical presentation, outcome and response to therapy. Canine mammary carcinomas (CMCs) have epidemiological and biological similarities to HBCs, are frequently biphasic and are composed of two distinct neoplastic populations (epithelial and myoepithelial). The present study evaluates the potential of CMCs as a natural model for basal-like HBCs. Single and double immunohistochemistry was performed on serial sections of 10 normal canine mammary glands and 65 CMCs to evaluate expression of cytokeratin (CK) 8/18, CK5, CK14, α-smooth muscle actin (SMA), calponin (CALP), p63 and vimentin (VIM). The tumours were also evaluated for Ki67 and human epidermal growth factor receptor (HER)-2 expression. A hierarchical model of cell differentiation was established, similar to that for the human breast. We hypothesized that progenitor cells (CK5(+), CK14(+), p63(+) and VIM(+)) differentiate into terminally-differentiated luminal glandular (CK8/18(+)) and myoepithelial (CALP(+), SMA(+) and VIM(+)) cells via intermediary luminal glandular cells (CK5(+), CK14(+) and CK8/CK18(+)) and intermediary myoepithelial cells (CK5(+), CK14(+), p63(+), SMA(+), CALP(+) and VIM(+)). Neoplastic myoepithelial cells in canine complex carcinomas had labelling similar to that of terminally-differentiated myoepithelial cells, while those of carcinomas-and-malignant myoepitheliomas with a more aggressive biological behaviour (i.e. higher frequency of vascular/lymph node invasion and visceral metastases and higher risk of tumour-related death) were comparable with intermediary myoepithelial cells and had significantly higher Ki67 expression. The majority of CMCs examined were negative for expression of HER-2. The biphasic appearance of CMCs with involvement of the myoepithelial component in different stages of cell differentiation may help to define the role of myoepithelial cells in the mammary carcinogenetic process and the heterogeneous nature of basal-like HBCs.Journal of Comparative Pathology 06/2014; 151(2). DOI:10.1016/j.jcpa.2014.04.013 · 1.14 Impact Factor
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ABSTRACT: Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.Veterinary and Comparative Oncology 07/2014; DOI:10.1111/vco.12110 · 2.73 Impact Factor
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