Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment.
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-kappaB, 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.
Buckanovich
Ronald J
RJ
Center for Research on Reproduction and Women's Health, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Sasaroli
Dimitra
D
O'brien-Jenkins
Ann
A
Botbyl
Jeffrey
J
Conejo-Garcia
Josè R
JR
Benencia
Fabian
F
Liotta
Lance A
LA
Gimotty
Phyllis A
PA
Coukos
George
G
D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic
D018076:DNA, Complementary