Abstract
CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.
Highlights
Cancer stem-like cells (CSCs) are defined as a subpopulation of tumour cells that have both tumour-initiating ability and the ability to reconstitute the cellular heterogeneity typical of their tumours of origin [1]
As these influence cell behaviour, it was of interest initially to determine whether differences in isoform expression patterns exist between the Epithelial-to-mesenchymal transition (EMT) (CD44highESAlow) and non-EMT (CD44highESAhigh) CSC subpopulations that we have previously identified in squamous cell carcinoma (SCC) [32]
In the present study we found, using flow cytometry, that the total levels of CD44 expression detected for cells isolated by non-enzymatic methods were similar for EMT and non-EMT cells
Summary
Cancer stem-like cells (CSCs) are defined as a subpopulation of tumour cells that have both tumour-initiating ability and the ability to reconstitute the cellular heterogeneity typical of their tumours of origin [1]. Initially CSCs were thought typically to represent a relatively small fraction of the total number of tumour cells, subsequent reports suggest that their number may vary quite widely both within a given type of tumour and between differing types of tumours [10,11]. The fraction of cells found to initiate tumours on transplantation is influenced markedly by the method and site of transplantation and by the type of recipient mouse [10] It is unclear how the composition of test populations is influenced by the method used for their isolation, and there has been little investigation of the effects of differing methods of cell isolation on the levels of CSC markers subsequently detectable. Many cell isolation protocols use the proteolytic enzymes trypsin [10,16] and collagenase [3,12,15], yet it is not known whether these enzymes degrade cell surface molecules used to isolate CSCs
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