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

• Published in: The Prostate Vol. 82; no. 7; pp. 836 - 849
• Main Authors: Vitek, Ross A., Huang, Wei, Geiger, Peter G., Heninger, Erika, Lang, Joshua M., Jarrard, David F., Beebe, David J., Johnson, Brian P.
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01-05-2022; John Wiley and Sons Inc
• Subjects: Cancer-Associated Fibroblasts - pathology; Cell culture; DNA-directed RNA polymerase; Fibroblasts; Flow cytometry; Freezing; functional analyses; Histopathology; Humans; Male; molecular analyses; Original; Polymerase chain reaction; prostate; Prostate - pathology; Prostate cancer; Prostatic Neoplasms - pathology; Questioning; Tissue and Organ Procurement; tissue processing; Tumor cells; Tumor microenvironment; Tumor Microenvironment - physiology; molecular analyses; tissue processing; functional analyses; prostate; tumor microenvironment
• ISSN: 0270-4137; 1097-0045
• DOI: 10.1002/pros.24326

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.
Conclusions
The identification of candidate cases and use of these techniques enable translational research and the development of molecular and functional assays to facilitate prostate TME study without compromising standard‐of‐care histopathological diagnosis. This allows bridging clinical histopathology and further interrogation of the prostate TME and promises to advance our understanding of tumor biology and unveil new predictive and prognostic markers of prostate cancer progression.