Introduction/background: Different hydrogels have recently been used to study the interactions between the different components of the tumor microenvironment (TME) in several types of cancer, including lung cancer. These studies try to emulate the characteristics of the tumor extracellular matrix. The pulmonary extracellular matrix is very complex. It varies in the different portions of the lung and includes variable amounts of collagen fibers of different diameters, elastic fibers, proteoglycans, and a wide variety of macromolecules. On the other hand, the tumor extracellular matrix has its own composition, different from that of the normal lung. Due to the difficulty in emulating the extracellular pulmonary matrix, strategies have been developed based on the use of decellularized tissue matrices that serve as a support for the culture of TME cellular components. Its application to the lung environment is difficult due to the due to the great variability in the composition of the extracellular matrixand the small size that most lung tumors tend to have. In this work we intend to generate a mixed gelatin-alginate hydrogel that includes elements of the pulmonary extracellular matrix to use it as a structural support for the study of interactions between cancer associated fibroblasts (CAFs), non-tumor fibroblasts and cancer cells.
Methods: 4% gelatin-2% alginate hydrogels were generated. Rat lung tumor tissue was decellularized using Triton X-100. After histological validation of the decellularization protocol, the tissue was further digested with pepsin, lyophilized and a homogeneous powder was obtained by grinding a mortar and liquid nitrogen. The powder was combined with the gelatin-alginate hydrogel. CAFs and non-tumor fibroblasts were isolated from 4 lung cancer tumors. The cells were characterized by flow cyt[MMR1] ometry, embedded in the manufactured hydrogel and cultured in DMEM culture medium supplemented with a 10% of fetal bovine serum, penicillin, streptomycin and fungizone for 72 hours in the presence or absence of TFG-beta 5 ng/ml. Morphological characteristic 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 andCD146 and negative for CD31, CD45 and STRO-1. Statistically significant differences in the expression of CD45 (18.45 ± 2.14 positive CAFs compared to 8.27 ± 1.15 positive non-tumor fibroblasts) were found. The cells cultured in the hydrogel showed a stellate morphology,with abundant actin stress fibers. The expression of vimentin and FSP 1 was significantly higher compared to cells cultured in conventional 2D culture systems. Likewise, a marked expression of type I collagen was detected by the cells cultured on the hydrogels. No significant differences were observed comparing CAFs and non-tumor fibroblasts in relation to the presence or absence of TGF-beta in the culture media.
Conclusion: The manufactured hydrogel represents a suitable support for the culture of stromal cells isolated from lung cancer tissue. This prototype will serve as a basic support to study different physical forces, biochemical factors or cellular elements conforming the TME.