Endothelial cell/Schwann cell communication in the tumor microenvironment: understanding treatment response in non-small cell lung cancer
Résumé
Non-small cell lung carcinoma (NSCLC) remains one of the deadliest cancers worldwide with due, among other, to drug resistance. The tumor microenvironment is an essential compartment for understanding the mechanisms of its resistance. Endothelial (EC) and Schwann cells (SC) are of particular interest. EC can be involved in endothelial-to-mesenchymal transition (EndoMT) to confer mesenchymal properties to EC while SC reprogramming was associated with poor prognosis by inducing epithelial-to-mesenchymal transition. This aim of this study was to develop three-dimensional (3D) spheroid models to evaluate tumor-stroma interactions and the possible phenotypic changes in EC and SC. Mono- and multicellular spheroids were generated using A549, H23, HUVEC or SC. Each spheroid was seeded at 10,000 cells/well in ultra-low-attachment plates and cultured for 4-7 days. For multicellular spheroid, the ratio was 1 :1 (NSCLC/EC) and 2 :1 :1 (NSCLC/EC/SC). Growth was assessed by daily monitoring of spheroid volume. Viability was determined using Cell-Titer-Glo 3D assay and Live/Dead staining. EndoMT markers were analyzed by Western blot (vWF, VEGFR2, CD31 SM22, vimentin) or flow cytometry using Cell Tracers and these same antibodies. RNA-Seq will be performed to identify gene expression profiles associated with EndoMT. Spheroids formed from A549 and H23 cells showed optimal viability and stability after 4 days. Live/dead analysis revealed a heterogeneous distribution of cells with a significant proportion of viable cells. The cells constituting the multicellular spheroids could be identified by specific labelling. Western blot and flow cytometry showed a decrease in endothelial markers and an increase in mesenchymal markers, suggesting a partial transition of EC. Integration of SC into spheroids is underway and will help to elucidate their interaction and impact on EC/tumor cell behaviour. Understanding the interactions between cells in the tumor stroma is an important goal to better define and optimise cancer treatments.
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