Fresh tissue procurement and preparation for multicompartment and multimodal analysis of the prostate tumor microenvironment

Abstract

Background: Prostatic cancers include a diverse microenvironment of tumor cells, cancer-associated fibroblasts, and immune components. This tumor microenvironment (TME) is a known driving force of tumor survival after treatment, but the standard-of-care tissue freezing or fixation in pathology practice limit the use of available approaches/tools to study the TME's functionality in tumor resistance. Thus, there is a need for approaches that satisfy both clinical and laboratory endpoints for TME study. Here we present methods for clinical case identification, tissue processing, and analytical workflow that are compatible with standard histopathology while enabling molecular and functional interrogation of prostate TME components. Methods:We first performed a small retrospective review to identify cases where submission of alternate prostate tissue slices and a parallel live tissue processing protocol complement traditional histopathology and enable viable multicompartment analysis of the TME. Then, we tested its compatibility with commonly employed methods to study the microenvironment including quantification of components both in situ and after tissue dissociation. We also evaluated tissue digestion conditions and cell isolation techniques to aid various molecular and functional endpoints.Results: We identified Gleason Grade Group 3+ clinical cases where tumor volume was sufficient to allow slicing of unfixed tissue and distribution of alternating tissue slices to standard-of-care histopathology and viable multi-modal TME analyses. No single method was found that preserved cellular sub-types for all downstream readouts; instead, tissues were further divided so techniques could be catered to each endpoint. For instance, we show that incorporating the protease dispase into tissue dissociation improves viability for culture and functional analyses but hinders immune cell analysis by flow cytometry. We also found that flow activated cell sorting provides highly pure cell populations for quantitative reverse-transcription polymerase chain reaction and RNA-seq while isolation using antibody-labeled paramagnetic particles facilitated functional coculture experiments.

Establishment of tumor microenvironment-preserving organoid model from patients with intracranial meningioma

“…We also used a liquid media culture system instead of embedding MNOs into Matrigel, resulting in an easy-to-handle, cost-efficient, and time-saving model. Notably, reduced processing time due to our optimized protocol might increase the succession rate of organoid culture because the viability of cells in fresh tissue is more vulnerable than stabilized and isolated contexts [ 42 ]. Although we used simplified protocols, cryopreservation and recovery cycles did not retard proliferation of MNOs, indicating their value as a research model system.…”