Protocol for the utilization of murine interstitial fluid extract to model breast cancer in the context of old age in vitro.

Interstitial fluid reflects both the organ microenvironment and tumor milieu; however, its direct study in vivo remains technically challenging. Here, we present a protocol for using murine interstitial fluid extract from lungs of metastasis-bearing mice as a model to recapitulate the metastatic environment ex vivo. This protocol outlines steps for cell preparation, tail vein injection, ex vivo tissue processing, and incubation, followed by detailed procedures for interstitial fluid isolation, in vitro cell treatment, and RNA sequencing (RNA-seq) analysis.