Johnson Brian P, Vitek Ross A, Geiger Peter G, Huang Wei, Jarrard David F, Lang Joshua M, Beebe David J
Department of Biomedical Engineering, University of Wisconsin, Madison, WI.
Carbone Cancer Center, University of Wisconsin, Madison, WI.
Biotechniques. 2018 Jan 1;64(1):13-19. doi: 10.2144/000114626.
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.
组织微环境中的细胞异质性可能是化疗耐药性和反应的基础，促使肿瘤发生演变；然而，这种异质性很难进行表征。在此，我们提出一种新方法——病理学引导的微量打孔法（PGM），该方法能够识别和表征在存活的人类和动物模型组织切片中发现的异质性病灶。这项技术包括使用荧光抗体/小分子对活细胞组织进行标记，以识别存活组织中的异质性病灶（如免疫浸润或具有高水平活性氧的细胞），再结合微量打孔步骤，从这些异质性病灶中分离细胞，用于下游分子或重要功能分析。从人前列腺组织的上皮或基质成纤维细胞病灶获得的微量打孔样本分别显示，EpCam/细胞角蛋白8和波形蛋白/α-平滑肌肌动蛋白/整合素1-α特异性转录本富集了6至12倍。转录富集效率与从人前列腺分离的上皮和基质激光捕获显微切割样本一致。微量打孔获得的病灶在周边显示细胞活力丧失，但中央定位的细胞在解离前后均保持活力，并在培养中生长。
