Utilization of interstitial fluid extract (IFE) to model cancer in the context of old age in vitro.

Interstitial fluid is the biological fluid that occupies the spaces between cells within tissues, acting as a vital medium for the exchange of nutrients and waste products. It reflects not only the composition of specific microenvironment of an organ but is also reflective of the tumor milieu, including secreted factors and small molecules from both tumor and normal cells. As such, interstitial fluid captures a biologically relevant snapshot of the organ's microenvironment, whether cancer is present or not. Here we introduce interstitial fluid extract (IFE) and present a protocol for its isolation from the lungs of metastasis-bearing mice, followed by ex vivo treatment of cultured cells and high-throughput transcriptomic analysis. This approach is highly flexible, supporting a variety of downstream applications and adaptable to different experimental formats such as mouse strain, cancer cell type, and organ of interest. By comparing the molecular profiles of IFE-treated cells with those of cells sorted directly from metastasis-bearing lungs, the protocol helps validate the biological relevance of the IFE model. This approach is particularly valuable for studying in vivo-like conditions where direct in vivo experiments are challenging or not feasible, such as in the context of old age, offering a practical alternative for investigating complex tumor microenvironments and cellular responses. Overall design: 1)Global analysis of RNA levels to determine transcriptional reprogramming in human lung adenocarcinoma cell line A549 upon treatment with 10 % human serum derived from 6 pools of 30 young (<30) and 6 pools of 30 old (>60) healthy donors for 3 days. 2) Global analysis of RNA levels to determine transcriptional reprogramming in human lung adenocarcinoma cell line A549 upon treatment cortisol (0.5 µM) or treatment with vehicle (ethanol) for a total of 3 days. 3) Global analysis of RNA levels to determine transcriptional reprogramming in human lung adenocarcinoma cell line A549 harboring a) non-targeting shRNA (shNT) treated with 10 % human serum derived from 6 pools of 30 young (<30) healthy donors b) A549 shNT and A549 bearing shRNAs targeting the glucocorticoid receptor (shGR#1 and shGR#2) with treated with 10 % human serum derived from 6 pools of 30 old (>60) healthy donors. The treatments were performed for a total of 3 days.