title: Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.
abstract: The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples.
authors: Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic.
authors: Radek
authors: R
authors: Fedr
authors: Zuzana
authors: Z
authors: Pernicová
authors: Eva
authors: E
authors: Slabáková
authors: Nicol
authors: N
authors: Straková
authors: Jan
authors: J
authors: Bouchal
authors: Michal
authors: M
authors: Grepl
authors: Alois
authors: A
authors: Kozubík
authors: Karel
authors: K
authors: Souček
mesh_terms: D000071916:AC133 Antigen; D015703:Antigens, CD / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry / Q000379:methods*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D066298:In Vitro Techniques; D039503:Integrin alpha6 / Q000378:metabolism; D008297:Male; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D010455:Peptides / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D014410:Tumor Stem Cell Assay / Q000379:methods*
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D018960:Hyaluronan Receptors; D039503:Integrin alpha6; C000606023:PROM1 protein, human; D010455:Peptides; C499380:TACSTD2 protein, human