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Vital ex vivo tissue labeling and pathology-guided micropunching to characterize cellular heterogeneity in the tissue microenvironment.
- Source :
-
BioTechniques [Biotechniques] 2018 Jan 01; Vol. 64 (1), pp. 13-19. Date of Electronic Publication: 2018 Jan 01. - Publication Year :
- 2018
-
Abstract
- Cellular heterogeneity within the tissue microenvironment may underlie chemotherapeutic resistance and response, enabling tumor evolution; however, this heterogeneity it is difficult to characterize. Here, we present a new approach-pathology-guided micropunching (PGM)-that enables identification and characterization of heterogeneous foci identified in viable human and animal model tissue slices. This technique consists of live-cell tissue labeling using fluorescent antibodies/small molecules to identify heterogeneous foci (e.g., immune infiltrates or cells with high levels of reactive oxygen species) in viable tissues, coupled with a micropunch step to isolate cells from these heterogeneous foci for downstream molecular or vital functional analysis. Micropunches obtained from epithelial or stromal fibroblast foci in human prostate tissue show 6- to 12-fold enrichment in transcripts specific for EpCam/cytokeratin 8 and vimentin/a-smooth muscle actin/integrin 1-α, respectively. Transcriptional enrichment efficiency agrees with epithelial and stromal laser capture microdissection samples isolated from human prostate. Micropunched foci show a loss of cellular viability in the periphery, but centrally localized cells retained viability before and after dissociation and grew out in culture.
- Subjects :
- Animals
Cell Survival
Fibroblasts pathology
Gene Expression Profiling methods
Humans
Male
Prostate metabolism
Prostatic Neoplasms genetics
Prostatic Neoplasms pathology
RNA genetics
RNA isolation & purification
Reactive Oxygen Species analysis
Transcriptome
Tumor Microenvironment
Fluorescent Antibody Technique methods
Laser Capture Microdissection methods
Prostate pathology
Subjects
Details
- Language :
- English
- ISSN :
- 1940-9818
- Volume :
- 64
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- BioTechniques
- Publication Type :
- Academic Journal
- Accession number :
- 29384072
- Full Text :
- https://doi.org/10.2144/000114626