Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1–1.5 h.
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Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).
Chung, G.G., Kielhorn, E.P. & Rimm, D.L. Subjective differences in outcome are seen as a function of the immunohistochemical method used on a colorectal cancer tissue microarray. Clin. Colorectal Cancer 1, 237–242 (2002).
Chung, C.H., Bernard, P.S. & Perou, C.M. Molecular portraits and the family tree of cancer. Nat. Genet. 32 (Suppl.), 533–540 (2002).
Wulfkuhle, J.D. et al. Proteomic approaches to the diagnosis, treatment, and monitoring of cancer. Adv. Exp. Med. Biol. 532, 59–68 (2003).
Wulfkuhle, J.D. et al. Proteomics of human breast ductal carcinoma in situ. Cancer Res. 62, 6740–6749 (2002).
Liotta, L.A. & Kohn, E.C. The microenvironment of the tumour-host interface. Nature 411, 375–379 (2001).
Liotta, L.A. & Kohn, E.C. Stromal therapy: the next step in ovarian cancer treatment. J. Natl. Cancer Inst. 94, 1113–1114 (2002).
Imbeaud, S. & Auffray, C. 'The 39 steps' in gene expression profiling: critical issues and proposed best practices for microarray experiments. Drug Discov. Today 10, 1175–1182 (2005).
Imbeaud, S. et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 33, e56 (2005).
Liotta, L.A. & Stracke, M.L. Tumor invasion and metastases: biochemical mechanisms. Cancer Treat. Res. 40, 223–238 (1988).
Iyengar, P. et al. Adipocyte-derived collagen VI affects early mammary tumor progression in vivo, demonstrating a critical interaction in the tumor/stroma microenvironment. J. Clin. Invest. 115, 1163–1176 (2005).
Geho, D.H., Bandle, R.W., Clair, T. & Liotta, L.A. Physiological mechanisms of tumor-cell invasion and migration. Physiology (Bethesda) 20, 194–200 (2005).
Stracke, M.L., Murata, J., Aznavoorian, S. & Liotta, L.A. The role of the extracellular matrix in tumor cell metastasis. In Vivo 8, 49–58 (1994).
Wada, K. et al. Requirement of cell interactions through adhesion molecules in the early phase of T cell development. Cell. Immunol. 170, 11–19 (1996).
Chan, S. et al. The use of laser capture microdissection (LCM) and quantitative polymerase chain reaction to define thyroid hormone receptor expression in human 'term' placenta. Placenta 25, 758–762 (2004).
Vogt, P.M. et al. Significant angiogenic potential is present in the microenvironment of muscle flaps in humans. J. Reconstr. Microsurg. 21, 517–523 (2005).
Wulfkuhle, J.D. et al. Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays. Proteomics 3, 2085–2090 (2003).
Simone, N.L. et al. Laser capture microdissection: beyond functional genomics to proteomics. Mol. Diagn. 5, 301–307 (2000).
Sheehan, K.M. et al. Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma. Mol. Cell Proteomics (2005).
Petricoin, E.F. et al. Mapping molecular networks using proteomics: a vision for patient-tailored combination therapy. J. Clin. Oncol. (2005).
Emmert-Buck, M.R. et al. Laser capture microdissection. Science 274, 998–1001 (1996).
Bonner, R.F. et al. Laser capture microdissection: molecular analysis of tissue. Science 278, 1481–1483 (1997).
Kolble, K. The LEICA microdissection system: design and applications. J. Mol. Med. 78, B24–B25 (2000).
Micke, P., Ostman, A., Lundeberg, J. & Ponten, F. Laser-assisted cell microdissection using the PALM system. Methods Mol. Biol. 293, 151–166 (2005).
Schutze, K. et al. Cut out or poke in–the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease. Genet. Anal. 14, 1–8 (1997).
Schutze, K., Posl, H. & Lahr, G. Laser micromanipulation systems as universal tools in cellular and molecular biology and in medicine. Cell. Mol. Biol. 44, 735–746 (1998).
Schermelleh, L. et al. Laser microdissection and laser pressure catapulting for the generation of chromosome-specific paint probes. Biotechniques 27, 362–367 (1999).
Kraft, T. Compact, robust lasers suit biotechnology applications. Biophotonics Intl. July, 44–46 (2004).
Buckanovich, R.J. et al. Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment. Cancer Biol. Ther. published online 9 June (2006).
Nakazono, M., Qiu, F., Borsuk, L.A. & Schnable, P.S. Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 15, 583–596 (2003).
Xiang, C.C. et al. Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling. Nucleic Acids Res. 32, e185 (2004).
Namimatsu, S., Ghazizadeh, M. & Sugisaki, Y. Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method. J. Histochem. Cytochem. 53, 3–11 (2005).
Fink, L., Kwapiszewska, G., Wilhelm, J. & Bohle, R.M. Laser-microdissection for cell type- and compartment-specific analyses on genomic and proteomic level. Exp. Toxicol. Pathol. (2006).
Belluco, C. et al. Kinase substrate protein microarray analysis of human colon cancer and hepatic metastasis. Clin. Chim. Acta 357, 180–183 (2005).
Gillespie, J.W. et al. The role of tissue microdissection in cancer research. Cancer J. 7, 32–39 (2001).
Simone, N.L. et al. Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection. Am. J. Pathol. 156, 445–452 (2000).
Gulmann, C. et al. Proteomic analysis of apoptotic pathways reveals prognostic factors in follicular lymphoma. Clin. Cancer Res. 11, 5847–5855 (2005).
Grubb, R.L. et al. Signal pathway profiling of prostate cancer using reverse phase protein arrays. Proteomics 3, 2142–2146 (2003).
Elliott, K. et al. Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides. Forensic Sci. Int. 137, 28–36 (2003).
Emmert-Buck, M.R. et al. Molecular profiling of clinical tissues specimens: feasibility and applications. J. Mol. Diagn. 2, 60–66 (2000).
Chen, X. et al. Gene expression patterns in human liver cancers. Mol. Biol. Cell 13, 1929–1939 (2002).
DiMartino, D. et al. Laser microdissection and DNA typing of cells from single hair follicles. Forensic Sci. Int. 146 (Suppl.), S155–S157 (2004).
Anslinger, K., Bayer, B., Mack, B. & Eisenmenger, W. Sex-specific fluorescent labelling of cells for laser microdissection and DNA profiling. Int. J. Legal Med. published online 18 March (2006) (DOI:10.1007/S00414-005-0065-7).
Nawshad, A., LaGamba, D., Olsen, B.R. & Hay, E.D. Laser capture microdissection (LCM) for analysis of gene expression in specific tissues during embryonic epithelial-mesenchymal transformation. Dev. Dyn. 230, 529–534 (2004).
Klitgaard, K. et al. Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization. Biotechniques 39, 864–868 (2005).
Ranjit, N. et al. A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy. Int. J. Parasitol. 36, 701–710 (2006).
Raab, T. et al. FaQR, required for the biosynthesis of the strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone, encodes an enone oxidoreductase. Plant Cell 18, 1023–1037 (2006).
Hartmann, S. et al. Genetic imprinting during impaired spermatogenesis. Mol. Hum. Reprod. (2006).
Brown, M.R. et al. Allelic loss on chromosome arm 8p: analysis of sporadic epithelial ovarian tumors. Gynecol. Oncol. 74, 98–102 (1999).
Chandrasekharappa, S.C. et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 276, 404–407 (1997).
Chuaqui, R., Silva, M. & Emmert-Buck, M. Allelic deletion mapping on chromosome 6q and X chromosome inactivation clonality patterns in cervical intraepithelial neoplasia and invasive carcinoma. Gynecol. Oncol. 80, 364–371 (2001).
Sabah, M., Cummins, R., Leader, M. & Kay, E. Loss of p16INK4A expression is associated with alleic imbalance/loss of heterozygosity of chromosome 9p21 in microdissected synovial sarcomas. Virchows Arch. 447, 842–848 (2005).
Takeshima, Y. et al. Heterogeneous genetic alterations in ovarian mucinous tumors: application and usefulness of laser capture microdissection. Hum. Pathol. 32, 1203–1208 (2001).
Kaserer, K. et al. Construction of cDNA libraries from microdissected benign and malignant thyroid tissue. Lab. Invest. 82, 1707–1714 (2002).
Leethanakul, C. et al. Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries. Oral Oncol. 36, 474–483 (2000).
Kinnecom, K. & Pachter, J.S. Selective capture of endothelial and perivascular cells from brain microvessels using laser capture microdissection. Brain Res. Brain Res. Protoc. 16, 1–9 (2005).
Pagedar, N.A. et al. Gene expression analysis of distinct populations of cells isolated from mouse and human inner ear FFPE tissue using laser capture microdissection—a technical report based on preliminary findings. Brain Res. (2006) (DOI:10.1016/j.brainres.2006.01.0S7).
Jin, L. et al. Detection of fusion gene transcripts in fresh-frozen and formalin-fixed paraffin-embedded tissue sections of soft-tissue sarcomas after laser capture microdissection and rt-PCR. Diagn. Mol. Pathol. 12, 224–230 (2003).
Yao, F. et al. Microarray analysis of fluoro-gold labeled rat dopamine neurons harvested by laser capture microdissection. J. Neurosci. Methods 143, 95–106 (2005).
Paweletz, C.P. et al. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 20, 1981–1989 (2001).
Liotta, L.A. et al. Protein microarrays: meeting analytical challenges for clinical applications. Cancer Cell 3, 317–325 (2003).
Ornstein, D.K. et al. Characterization of intracellular prostate-specific antigen from laser capture microdissected benign and malignant prostatic epithelium. Clin. Cancer Res. 6, 353–356 (2000).
Jones, M.B. et al. Proteomic analysis and identification of new biomarkers and therapeutic targets for invasive ovarian cancer. Proteomics 2, 76–84 (2002).
Martinet, W. et al. Western blot analysis of a limited number of cells: a valuable adjunct to proteome analysis of paraffin wax-embedded, alcohol-fixed tissue after laser capture microdissection. J. Pathol. 202, 382–388 (2004).
Haqqani, A.S. et al. Characterization of vascular protein expression patterns in cerebral ischemia/reperfusion using laser capture microdissection and ICAT-nanoLC-MS/MS. FASEB J. 19, 1809–1821 (2005).
Melle, C. et al. Detection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling. Proteomics 6, 2600–2608 (2006).
Paweletz, C.P., Liotta, L.A. & Petricoin, E.F., III . New technologies for biomarker analysis of prostate cancer progression: laser capture microdissection and tissue proteomics. Urology 57, 160–163 (2001).
Fend, F. et al. Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis. Am. J. Pathol. 154, 61–66 (1999).
Murakami, H., Liotta, L. & Star, R.A. I.F.-L.C.M. laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication. Kidney Int. 58, 1346–1353 (2000).
Rupp, C. et al. Laser capture microdissection of epithelial cancers guided by antibodies against fibroblast activation protein and endosialin. Diagn. Mol. Pathol. 15, 35–42 (2006).
Mojsilovic-Petrovic, J. et al. Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses. J. Neurosci. Methods 133, 39–48 (2004).
Hunter, F. et al. Rhodamine-RCA in vivo labeling guided laser capture microdissection of cancer functional angiogenic vessels in a murine squamous cell carcinoma mouse model. Mol. Cancer 5, 5 (2006).
Prieto, D.A. et al. Liquid Tissue: proteomic profiling of formalin-fixed tissues. Biotechniques (Suppl.): 32–35 (2005).
Tangrea, M.A. et al. Expression microdissection: operator-independent retrieval of cells for molecular profiling. Diagn. Mol. Pathol. 13, 207–212 (2004).
Gillespie, J.W. et al. Evaluation of non-formalin tissue fixation for molecular profiling studies. Am. J. Pathol. 160, 449–457 (2002).
Mouledous, L. et al. Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples. Proteomics 3, 610–615 (2003).
Wong, M.H. et al. Genetic mosaic analysis based on Cre recombinase and navigated laser capture microdissection. Proc. Natl. Acad. Sci. USA 97, 12601–12606 (2000).
Arcturus Bioscience, Inc. User Guide Veritas Microdissection Instrument. Version A. (Arcturus Bioscience, Inc., Mountain View, California, USA, 2004).
Agar, N.S., Halliday, G.M., Barnetson, R.S. & Jones, A.M. A novel technique for the examination of skin biopsies by laser capture microdissection. J. Cutan. Pathol. 30, 265–270 (2003).
Roy, S. et al. Transcriptome analysis of the ischemia-reperfused remodeling myocardium: temporal changes in inflammation and extracellular matrix. Physiol. Genomics (2006), in press.
Upson, J.J. et al. Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection. J. Cell. Physiol. 201, 366–373 (2004).
Obiakor, H. et al. A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ. Anal. Biochem. 306, 55–62 (2002).
Suarez-Quian, C.A. et al. Laser capture microdissection of single cells from complex tissues. Biotechniques 26, 328–335 (1999).
Keays, K.M. et al. Laser capture microdissection and single-cell RT-PCR without RNA purification. J. Immunol. Methods 302, 90–98 (2005).
Poznanovic, S. et al. Differential radioactive proteomic analysis of microdissected renal cell carcinoma tissue by 54 cm isoelectric focusing in serial immobilized pH gradient gels. J. Proteome Res. 4, 2117–2125 (2005).
Ai, J. et al. Proteome analysis of hepatocellular carcinoma by laser capture microdissection. Proteomics 6, 538–546 (2006).
Ornstein, D.K. et al. Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines. Electrophoresis 21, 2235–2242 (2000).
Lee, J.R. et al. Differential protein analysis of spasomolytic polypeptide expressing metaplasia using laser capture microdissection and two-dimensional difference gel electrophoresis. Appl. Immunohistochem. Mol. Morphol. 11, 188–193 (2003).
Li, C. et al. Accurate qualitative and quantitative proteomic analysis of clinical hepatocellular carcinoma using laser capture microdissection coupled with isotope-coded affinity tag and two-dimensional liquid chromatography mass spectrometry. Mol. Cell. Proteomics 3, 399–409 (2004).
Baker, H. et al. Proteome-wide analysis of head and neck squamous cell carcinomas using laser-capture microdissection and tandem mass spectrometry. Oral Oncol. 41, 183–199 (2005).
de Groot, C.J. et al. Peptide patterns of laser dissected human trophoblasts analyzed by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Proteomics 5, 597–607 (2005).
The authors gratefully thank J. Milia, A. Malekafzali, D. Choiniere, M. Bellamy and T. Taylor for consistently informative discussions regarding LCM.
The authors declare no competing financial interests.
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Espina, V., Wulfkuhle, J., Calvert, V. et al. Laser-capture microdissection. Nat Protoc 1, 586–603 (2006). https://doi.org/10.1038/nprot.2006.85
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