Laser capture microdissection of epithelial cancers guided by antibodies against fibroblast activation protein and endosialin.
Transcriptional profiling of cancer biopsies is used extensively to identify expression signatures for specific cancer types, diagnostic and prognostic subgroups, and novel molecular targets for therapy. To broaden these applications, several challenges remain. For example, the integrity of RNA extracted even from small tissue samples has to be insured and monitored. Moreover, total tumor RNA may hide the marked histologic heterogeneity of human cancers. A principle approach to this heterogeneity has been provided by laser capture microdissection performed on antibody-stained tissue sections (immuno-LCM; iLCM). In this study, we have established a procedure to assess the quality of RNA obtained from tissue sections, coupled with immunostaining using antibodies to different tumor stromal markers, and subsequent iLCM to selectively capture the cancer stroma compartments. The procedure was applied to 53 frozen specimens of human epithelial cancers. Sections were stained for histopathological evaluation, and RNA was isolated from adjacent serial sections. RNA quality was assessed by the Agilent-Bioanalyzer (Agilent, Palo Alto, CA) and by multiplex RT-PCR. Two thirds of the specimens were found to yield good to excellent RNA quality. For microdissection of the tumor stroma with reactive fibroblasts and tumor blood vessels, a rapid incubation protocol with antibodies against fibroblast activation protein (FAP) and against endosialin was developed to ensure RNA integrity for subsequent iLCM. Using these procedures, RNA from distinct tumor compartments can be isolated, analyzed, amplified, and used for transcription profiling.
Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria.
Christian
C
Rupp
Helmut
H
Dolznig
Christina
C
Puri
Norbert
N
Schweifer
Wolfgang
W
Sommergruber
Norbert
N
Kraut
Wolfgang J
WJ
Rettig
Dontscho
D
Kerjaschki
Pilar
P
Garin-Chesa
D000912:Antibodies, Neoplasm / Q000276:immunology; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D002277:Carcinoma / Q000175:diagnosis* / Q000473:pathology; D010450:Endopeptidases; D005629:Frozen Sections; D018093:Gelatinases / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D042282:Microdissection / Q000379:methods*; D009363:Neoplasm Proteins / Q000032:analysis* / Q000276:immunology; D012334:RNA, Neoplasm / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000032:analysis* / Q000276:immunology; D000099167:Fibroblast Activation Protein Alpha
D000912:Antibodies, Neoplasm; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D010450:Endopeptidases; D018093:Gelatinases; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D012334:RNA, Neoplasm; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha; C078203:CD248 protein, human