Vital Ex Vivo Tissue Labeling and Pathology-Guided Micropunching to Characterize Cellular Heterogeneity in the Tissue Microenvironment

Johnson, Brian P.; Vitek, Ross A; Geiger, Peter; Huang, Wei; Jarrard, David F.; Lang, Joshua M.; Beebe, David J.

doi:10.2144/000114626

Cited by 5 publications

(8 citation statements)

References 23 publications

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“…These results displayed significant variation in tissue viability and cellular composition from patient sample to patient sample indicating that single downstream measurements of post‐digestion viability and cellular composition can be skewed by upstream quality of the source tissue. This whole‐mount tissue slicing method can also be used to identify functional heterogeneity and pinpoint focal areas of interest for further study as previously described 20 . Yet, these in situ tissue‐level analyses are often omitted in traditional tissue processing protocols and TME studies.…”

Section: Discussionmentioning

confidence: 99%

“…Before imaging, slices were stained in screw‐top vials with fluorescent dyes. Slices were stained as previously described with viability markers (Cellometer ViaStain AOPI Staining Solution) and fluorescent antibodies (Hoechst, CD45 – FITC, EpCAM (epithelial cell adhesion molecule) – PE, CD49a – Alexa Fluor™ 647) 20 . Reagent specifics are listed in Table .…”

Section: Methodsmentioning

confidence: 99%

“…Tissue sectioning was done as previously described. 20 Briefly, a small portion (10%) of each tissue sample (acquired in accordance with the UW‐Madison Institutional Review Board) was superglued to the cutting stage of a VF‐300 Compresstome (Precisionary Instruments). Then, tissue was covered in low melt agarose, immersed in sterile media, and sectioned to 100 μm thickness.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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“…The TS method preserves histological features and allows for spatial examination of endpoints such as immunohistochemistry, in situ hybridization, ISH or spatial transcriptomics. The method of MDT (described above) ( 14 ) and pathology guided micropunching (PGM) ( 30 ) sample smaller areas (260-500 µM), reducing heterogeneity of the specimens, but also provide limited tissue for endpoint analyses. The relatively short length of culture for androgen adaptation studies is a limitation to both PDEs and TS.…”

Section: Limitations and Challenges Of Prostate Ts And Ex V...mentioning

confidence: 99%

Harnessing the Utility of Ex Vivo Patient Prostate Tissue Slice Cultures

Perez

Nonn

2022

Front. Oncol.

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Patient-derived prostate tissue explant cultures are powerful research tools that offer the potential for personalized medicine. These cultures preserve the local microenvironment of the surrounding stroma but are not without limitations and challenges. There are several methods and processing techniques to culture tissue ex vivo, that include explant tissue chunks and precision-cut tissue slices. Precision-cut tissue slices provide a consistent distribution of nutrients and gases to the explant. Herein we summarize the prostate tissue slice method, its limitations and discuss the utility of this model, to investigate prostate biology and therapeutic treatment responses.

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“…Here, sophisticated microscopic techniques such as (lattice) light sheet and spectral microscopy [77], and further elaboration of multiphoton imaging hold a great potential. Similarly, multi-marker analysis of small sample volumes, tissue microdissection combined with single-cell -Omics, innovative live-cell tissue labelling [78], and robust bioinformatics pipelines may be key technologies for efficient data readouts.…”

Section: Limitationsmentioning

confidence: 99%

3D organ models—Revolution in pharmacological research?

Weinhart

Hocke

Hippenstiel

et al. 2019

Pharmacological Research

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A B S T R A C T 3D organ models have gained increasing attention as novel preclinical test systems and alternatives to animal testing. Over the years, many excellent in vitro tissue models have been developed. In parallel, microfluidic organ-on-a-chip tissue cultures have gained increasing interest for their ability to house several organ models on a single device and interlink these within a human-like environment. In contrast to these advancements, the development of human disease models is still in its infancy. Although major advances have recently been made, efforts still need to be intensified. Human disease models have proven valuable for their ability to closely mimic disease patterns in vitro, permitting the study of pathophysiological features and new treatment options. Although animal studies remain the gold standard for preclinical testing, they have major drawbacks such as high cost and ongoing controversy over their predictive value for several human conditions. Moreover, there is growing political and social pressure to develop alternatives to animal models, clearly promoting the search for valid, cost-efficient and easy-to-handle systems lacking interspecies-related differences.In this review, we discuss the current state of the art regarding 3D organ as well as the opportunities, limitations and future implications of their use.

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Vital Ex Vivo Tissue Labeling and Pathology-Guided Micropunching to Characterize Cellular Heterogeneity in the Tissue Microenvironment

Cited by 5 publications

References 23 publications

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

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

Harnessing the Utility of Ex Vivo Patient Prostate Tissue Slice Cultures

3D organ models—Revolution in pharmacological research?

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