Epithelial cancers could be described as abnormal organs composed of cells with different phenotype and functional properties. It has been suggested that only a small fraction of cells defined as “cancer stem cells” (CSCs) is capable of initiating and maintaining a new tumor and that local microenvironment could be involved in modulation of stemness properties and metastatic colonization by induction of the epithelial-mesenchymal transition (EMT). To investigate how signals from stromal fibroblasts can influence stemness properties we used a primary lung adenocarcinoma cell line (LT73) containing a subpopulation of CD133+ CSCs (0.1%) and its derivative devoid of CD133+ cells (LT73CD133neg) which remains stable during short-term culturing. Medium conditioned by primary fibroblasts cell lines (CM) resulted in increase of CD133 positive cells, more pronounced in LT73CD133neg, indicating positive regulation of the stem cell pool and de novo generation of CSCs. Accordingly, expression of stemness related genes OCT4, NANOG and GA6 was increased (fold change 1.5-3). In vivo experiments also confirmed that tumors generated by injection of CM-treated cells had greater size compared to controls and maintained increased stemness features. Moreover stimulation with CM, associated to generation of CD133+ cells, was also often accompanied by gene expression changes consistent to EMT activation. To verify the importance of fibroblasts in providing the right cues also for successful colonization of the lungs, LT73 cells were injected in the tail vein of SCID mice. After 2 months LT73 cells co-injected with fibroblasts were present in the lungs in larger numbers (4 fold increase) compared to control mice indicating a possible role for fibroblasts in the preparation of the pre-metastatic niche
Finally to establish whether the EMT process was required for acquisition of a stem like phenotype in presence of microenvironmental stimuli, we overexpressed in LT73 wt cell line miR200c which has been shown to prevent TGFbeta-induced EMT. Preliminary experiments showed that, although miR200c was able to partially prevent the EMT process by contrasting downregulation of the epithelial marker CDH1, its overexpression was insufficient to abrogate upregulation of the mesenchymal markers SNAI2, VIM, FN1 and de novo generation of stem-like cells (CD133+) after TGFbeta treatment. This indicates that even partial activation of the EMT process could be sufficient to modulate stemness features.
Together these data demonstrate that stimuli from fibroblasts could de novo generate CD133+ cells modulating stem cells phenotype probably through EMT process and that the proficient cross-talk between fibroblasts and cancer stem cells could also be relevant for metastatic colonization.
Citation Format: Francesca Andriani, Tiziana Caputo, Giulia Bertolini, Federica Facchinetti, Erika Baldoli, Massimo Moro, Roberto Caserini, Gabriella Sozzi, Luca Roz. Microenvironment stimuli elicited by fibroblasts contribute to epithelial mesenchymal transition and acquisition of stemness phenotype in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4867. doi:10.1158/1538-7445.AM2014-4867