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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.

Vital ex vivo tissue labeling and pathology-guided micropunching to characterize cellular heterogeneity in the tissue microenvironment