Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment
Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment
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DOI：
10.4161/cbt.5.6.2676
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发表时间：
2006-06-01
影响因子：
3.6
通讯作者：
Coukos, George
中科院分区：
文献类型：
--
作者：
Buckanovich, Ronald J.;Sasaroli, Dimitra;Coukos, George
使用微阵列进行表达谱分析已经成为询问肿瘤生物学的重要工具。然而，整个肿瘤RNA的分析反映了肿瘤和宿主细胞，使得难以剖析肿瘤微环境中特定细胞区室内的分子事件。我们开发并优化了一种简单，快速的技术，结合免疫组织化学和激光捕获显微切割（免疫LCM），从肿瘤微环境中纯化特定的细胞群，然后进行RNA分离和扩增用于微阵列分析。使用这种方法，我们能够阐明原位表达谱的纯肿瘤细胞和肿瘤内皮细胞从卵巢肿瘤活跃的免疫浸润。该技术不仅提高了分析分离细胞群的特异性，消除了周围细胞表达的基因，而且提高了分析的灵敏度，允许检测在整个肿瘤阵列中未检测到的低表达基因。与培养的肿瘤细胞系中鉴定的主要是代谢的途径相比，原位肿瘤细胞的途径分析鉴定了会聚于NF-κ B的信号传导途径的不同活化。对肿瘤血管细胞的分析揭示了大多数已知的泛内皮和肿瘤内皮特异性标志物，并揭示了骨髓单核细胞谱系特异性基因。我们建议，免疫LCM加上转录谱是一个方便的工具，解剖复杂的生物系统，如肿瘤微环境中的分子和细胞事件。
Expression profiling using microarrays has become an essential tool for interrogating tumor biology. However, profiling of whole tumor RNA reflects both tumor and host cells, making it difficult to dissect molecular events within specific cellular compartments in the tumor microenvironment. We developed and optimized a simple, rapid technique combining immunohistochemistry and laser-capture microdissection (immuno-LCM) to purify specific cell populations from the tumor microenvironment followed by RNA isolation and amplification for microarray analysis. Using this methodology, we were able to elucidate the in situ expression profile of pure tumor cells and tumor endothelial cells from ovarian tumors with brisk immune infiltrates. This technique not only increased the specificity of profiling isolated cell populations, eliminating genes expressed by surrounding cells, but also increased the sensitivity of analysis, allowing for the detection of low expression genes that were not detected in whole tumor arrays. Pathway analysis of tumor cells in situ identified distinct activation of signaling pathways converging on NF-kappa B, as compared to pathways identified in cultured tumor cell lines, which were primarily metabolic. Profiling of tumor vascular cells revealed most known panendothelial and tumor endothelial-specific markers, and unveiled genes specific to the myeloid-monocytic lineage. We propose that immuno-LCM coupled with transcriptional profiling is a convenient tool for dissecting molecular and cellular events in complex biological systems such as the tumor microenvironment.