Abstract
The characterization of cancer stem cell (CSC) subpopulation, through the comparison of the gene expression signature in respect to the native cancer cells, is particularly important for the identification of novel and more effective anticancer strategies. However, CSC have peculiar characteristics in terms of adhesion, growth, and metabolism that possibly implies a different modulation of the expression of the most commonly used housekeeping genes (HKG), like b-actin (ACTB). Although it is crucial to identify which are the most stable HKG genes to normalize the data derived from quantitative Real-Time PCR analysis to obtain robust and consistent results, an exhaustive validation of reference genes in CSC is still missing. Here, we isolated CSC spheres from different musculoskeletal sarcomas and carcinomas as a model to investigate on the stability of the mRNA expression of 15 commonly used HKG, in respect to the native cells. The selected genes were analysed for the variation coefficient and compared using the popular algorithms NormFinder and geNorm to evaluate stability ranking. As a result, we found that: 1) Tata Binding Protein (TBP), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ), Peptidylprolyl isomerase A (PPIA), and Hydroxymethylbilane synthase (HMBS) are the most stable HKG for the comparison between CSC and native cells; 2) at least four reference genes should be considered for robust results; 3) the use of ACTB should not be recommended, 4) specific HKG should be considered for studies that are focused only on a specific tumor type, like sarcoma or carcinoma. Our results should be taken in consideration for all the studies of gene expression analysis of CSC, and will substantially contribute for future investigations aimed to identify novel anticancer therapy based on CSC targeting.
Highlights
Different populations of cells form malignant tumors
The stemness-like features for all the cancer stem cells (CSC) cultures included in this study were previously characterized [16,27], with the exception of cscMG-63, cscMDA-MB-231, and cscACHN for which the ability to growth as floating aggregates (Fig 1), and the mRNA expression for KLF4, c-Myc, Nanog, and OCT3/4 stemness markers [28] were here confirmed
The stemness genes were normalised by ACTB, one of the most commonly used housekeeping genes (HKG)
Summary
Native tumor cell lines and CSC culturesRS cell line (RD), OS cell lines (MG-63, HOS, Saos-2), ES cell line (A-673), BC cell line (MDA-MB-231) and RC cell line (ACHN), were purchased from American Type Culture Collection (ATCC,Manassas, VA, USA), and cultured in Iscove’s modified Dulbecco’s medium (IMDM, Gibco), plus 20 U/mL penicillin, 100 mg/mL streptomycin, and 10% heatinactivated fetal bovine serum (FBS) (complete IMDM) at 37°C in a humidified 5% CO2 atmosphere. Native tumor cell lines and CSC cultures. Tissue samples were subjected to mechanical mincing, followed by enzymatic digestion, to obtain single cells that were seeded in complete IMDM until the formation of a monolayer. All native tumor cell cultures were maintained in anchorage-independent conditions in DMEM:F12 medium with progesteron (20 nM), putrescin (10 mg/mL), sodium selenite (30 nM), apo-transferrin (100 μg/mL), and insulin (25 μg/mL) (Sigma-Aldrich, St. Louis, MO) in low-attachment flasks (Nunc, Penfield, NY) (sphere-forming assay). We obtained the CSC culture by maintaining the spheroid in anchorage-independent conditions in specific cell media, adding the growth factors EGF and bFGF every 3–4 days (twice at week). Spheroid cultures were amplified by treating the primary CSC culture with trypsin, followed by gentle mechanical dissociation, and by re-plating single-cell suspension to obtain the second spheroid culture. Those cultures that were able to form spheres and that expressed stem cell-related markers were considered
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