Abstract
BackgroundCervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer Stem cells (CSC) have been implicated in treatment resistance and metastases development; hence understanding their significance is important.MethodsPrimary culture from tissue biopsies of invasive cervical cancer and serial passaging was performed for establishing cell lines. Variable Number Tandem Repeat (VNTR) assay was performed for comparison of cell lines with their parental tissue. Tumorsphere and Aldefluor assays enabled isolation of cancer stem cells (CSC); immunofluorescence and flow cytometry were performed for their surface phenotypic expression in cell lines and in 28 tissue samples. Quantitative real-time PCR for stemness and epithelial-mesenchymal transition (EMT) markers, MTT cytotoxicity assay, cell cycle analysis and cell kinetic studies were performed.ResultsFour low-passage novel cell lines designated RSBS-9, − 14 and − 23 from squamous cell carcinoma and RSBS-43 from adenocarcinoma of the uterine cervix were established. All were HPV16+. VNTR assay confirmed their uniqueness and derivation from respective parental tissue. CSC isolated from these cell lines showed CD133+ phenotype. In tissue samples of untreated invasive cervical cancer, CD133+ CSCs ranged from 1.3–23% of the total population which increased 2.8-fold in radiation-resistant cases. Comparison of CD133+ with CD133− bulk population cells revealed increased tumorsphere formation and upregulation of stemness and epithelial-mesenchymal transition (EMT) markers with no significant difference in cisplatin sensitivity.ConclusionLow-passage cell lines developed would serve as models for studying tumor biology. Cancer Stem Cells in cervical cancer display CD133+ phenotype and are increased in relapsed cases and hence should be targeted for achieving remission.
Highlights
Cervical cancer is a major cause of cancer-related mortality in women in the developing world
[1] Advanced stage at presentation and resistance to standard chemoradiation are important factors leading to a high mortality and in turn may be attributed to residing cancer stem cells [Cancer Stem cells (CSC)] which, by definition are a selfrenewing population of cells within a tumor capable of initiation and maintenance of a tumor
Using flow cytometry and immunofluorescence studies, we established that CD133 is a surface phenotypic marker of cancer stem cells in cervical carcinoma of both the squamous cell carcinoma and adenocarcinoma subtypes
Summary
Cervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer stem-like cells were first identified to exist by Feng et al [4] and subsequently by others [5,6,7,8] Identification of their surface phenotype is the initial step towards their isolation and enrichment for subsequent biological studies. Our initial step was to generate short-term primary cultures from tissue biopsies of invasive cervical cancer. In this process, we were able to establish 4 lowpassage cell lines, all from Indian ethnicity. The clinical significance of the CSCs was evaluated in biopsies from 29 cases of invasive cervical cancer
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