Speaker
Description
"Background: Tissue engineering methodologies have been used to generate in vitro models that emulate the characteristics of different types of cancer, including lung cancer. Lung cancer stands out for its great capacity for dissemination and mortality, based in part on the great capacity of cancer cells to develop drug resistance and in part on the complexity of tumor microenvironment (TME). TME includes fibroblasts, cancer-associated fibroblasts (CAFs), endothelial cells, and the extracellular matrix components. The interactions between they determine the characteristic behavior of lung cancer. We propose to generate a three-dimensional model for the in vitro study that allows us to better understand the interaction between CAFs, non-tumor fibroblasts and cancer cells. This system will allow not only to identify new possible pharmacological targets, but also to increase our knowledge regarding to the acquisition of drug resistance or the metastasis process.
Methods: Alginate microspheres with a diameter around 250 μm were produced by microfluidics. The surface was coated with two bilayers of gelatin/alginate polyelectrolyte multilayer using a layer-by-layer, LbL, procedure. A covalent cross-linking of the coating was performed with glutaraldehyde followed by a treatment with glycine to inactivate remnant unreacted aldehyde groups. CAFs and non-tumor fibroblasts were isolated from 4 human lung cancer tumors. Cells were characterized by flow cytometry according to CD29, CD31, CD44, CD45, CD73, CD105, CD146, and STRO-1 expression. The cells were cultured with microparticle in DMEM cultured medium supplemented with 10% fetal bovine serum, penicillin, streptomycin and fungizone for 72 hours under orbital shaking. Agarose-coated cell culture wells were used to prevent cell adhesion to the plastic of the culture plates. Cell adhesion and morphological characteristics of CAFs and non-tumor fibroblasts were studied by fluorescence staining of F-acting using phalloidin-rhodamine. Vimentin, FSP1, type I collagen, and N-cadherin expression were analyzed by immunofluorescence and real time RT-PCR.
Results: The isolated cells were positive for CD29, CD44, CD73, CD105 and CD146, and negative for CD31, CD45 and STRO-1. Significative differences in the expression of CD45 (18.45 ± 2.14 positive CAFs compared to 8.27 ± 1.15 positive non-tumoral fibroblasts) were found. After 72 hours of culture, the cells adhered to the microparticles. These cells showed a stellate morphology, with abundant actin stress fibers. Vimentin and FSP1 expression was significantly higher compared to that of cells cultured in the absence of microparticles. Likewise, a marked expression of type I collagen by the cells cultured on the microspheres was detected. No significant differences were observed between CAFs and non-tumor fibroblasts in relation to the proteins studied.
Conclusion: The manufactured microparticles represent a suitable support for the culture of stromal cells isolated from lung cancer tumor tissue. The cells that adhered to the microparticles acquired a more marked secretory mesenchymal phenotype compared to that cells cultured in conventional 2D systems. This system will allow the generation of co-culture systems of CAFs and non-tumor fibroblasts in different ratios in a controlled manner, being a suitable in vitro system for the study of the effects of different drugs on stromal cells of TME."
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