In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection.
Hypoxia is a key determinant of tumor aggressiveness, yet little is known regarding hypoxic global gene regulation in vivo. We used the hypoxia marker EF5 coupled with laser-capture microdissection to isolate RNA from viable hypoxic and normoxic regions of 9L experimental gliomas. Through microarray analysis, we identified several mRNAs (including the HIF targets Vegf, Glut-1, and Hsp27) with increased levels under hypoxia compared with normoxia both in vitro and in vivo. However, we also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. Intriguingly, the mRNA levels of a substantial number of immunomodulatory and DNA repair proteins including CXCL9, CD3D, and RAD51 were found to be downregulated in hypoxic areas in vivo, consistent with a protumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic versus normoxic tumor regions. Moreover, CD8(+) T cells, which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. This is the first study to analyze the influence of hypoxia on mRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors.
Department of Radiation Oncology, University of Pennsylvania, Pennsylvania, USA.
Diane
D
Marotta
Jayashree
J
Karar
W Timothy
WT
Jenkins
Monika
M
Kumanova
Kevin W
KW
Jenkins
John W
JW
Tobias
Donald
D
Baldwin
Artemis
A
Hatzigeorgiou
Panagiotis
P
Alexiou
Sydney M
SM
Evans
Rodolfo
R
Alarcon
Amit
A
Maity
Cameron
C
Koch
Constantinos
C
Koumenis
D000818:Animals; D015687:Cell Hypoxia / Q000235:genetics; D017341:Etanidazole / Q000031:analogs & derivatives; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D055551:HSP27 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D006845:Hydrocarbons, Fluorinated; D008297:Male; D042282:Microdissection; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D051135:Rad51 Recombinase / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D011916:Rats, Inbred F344; D005466:Fluorocarbons
D017341:Etanidazole; D055551:HSP27 Heat-Shock Proteins; D006845:Hydrocarbons, Fluorinated; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D051135:Rad51 Recombinase; C486114:Hspb1 protein, rat; C096011:2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide; D005466:Fluorocarbons; C495155:Rad51 protein, rat
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