Abstract
BackgroundCancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line.Materials and MethodsA549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed.ResultsTGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable.ConclusionsThe induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers.
Highlights
Two important hypothesis have been postulated in the genesis, formation, growth, and metastasis of epithelial cancer: the role of Cancer Stem Cells (CSCs) or Tumour Initiating Cells (TICs) and the involvement of the so called Epithelial-Mesenchymal Transition (EMT).Cancer stem cells have been defined as ‘‘a cell within a tumour that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumour’’ [1]
Twist and b-catenin m-RNA transcripts were up-regulated in TGFb-1 treated LC31 cell line confirming EMT
In order to investigate the effect of TGFb-1 on LC31 and A549 cell lines, we treated them with 2 ng/ml of TGFb-1
Summary
Cancer stem cells have been defined as ‘‘a cell within a tumour that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumour’’ [1] These two definitive biological properties are what make the CSCs the prime candidate for initiation of relapse. CSCs hypothesis asserts that only a small subset of cells within a tumour is able of both tumour initiation and sustainment [2,3] These cells express stemness markers, are able to form floating spheres in serum-free medium, a property associated with stem cells, and are able to differentiate in an aberrant cell phenotype constituting tumour heterogeneity [4]. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line
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