Detection of brain tumor invasion and micrometastasis in vivo by expression of enhanced green fluorescent protein.
OBJECTIVE
To determine whether fluorescence from human brain tumor cells transfected with the enhanced green fluorescent protein (EGFP) gene in vitro and xenotransplanted into the brain of nude mice would permit the detection of brain tumor invasion and metastasis in vivo.


METHODS
Daoy medulloblastoma cells were transfected with a long terminal repeat-based retroviral vector containing the EGFP gene. Stable EGFP-expressing clones were isolated and stereotactically injected into the frontal cortex of nude mice. Four weeks later, whole brain sections were examined using fluorescence microscopy, immunohistochemistry, and routine hematoxylin and eosin staining for the visualization and detection of tumor cell invasion and metastasis.


RESULTS
We demonstrate that EGFP-transduced Daoy cells maintain stable high-level EGFP expression in the central nervous system during their growth in vivo. EGFP fluorescence clearly demarcated the primary tumor margins and readily allowed for the visualization of distant micrometastases and local invasion on the single-cell level. Small metastatic and locally invasive foci, including those immediately adjacent to the tumor's leading invasive edge, were virtually undetectable by routine hematoxylin and eosin staining and immunohistochemistry. EGFP expression also persisted in vitro after cell reculture from brain tissue extracts.


CONCLUSIONS
We show, for the first time, that EGFP-transduced human brain tumor cells can be visualized by fluorescence microscopy after intracerebral implantation. This method is superior to routine hematoxylin and eosin staining and immunohistochemistry for the detection and study of physiologically relevant patterns of brain tumor invasion and metastasis in vivo.
MacDonald
T J
TJ
Department of Pediatrics, Neil Bogart Memorial Laboratories, Childrens Hospital of Los Angeles, 90027, USA.
Tabrizi
P
P
Shimada
H
H
Zlokovic
B V
BV
Laug
W E
WE
D000818:Animals; D001932:Brain Neoplasms / Q000737:chemistry / Q000473:pathology*; D018076:DNA, Complementary / Q000235:genetics; D004801:Eosine Yellowish-(YS); D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006416:Hematoxylin; D006801:Humans; D007124:Immunoenzyme Techniques; D008164:Luminescent Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D008527:Medulloblastoma / Q000737:chemistry / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D008979:Moloney murine leukemia virus / Q000235:genetics; D009361:Neoplasm Invasiveness / Q000175:diagnosis*; D009362:Neoplasm Metastasis / Q000175:diagnosis*; D009363:Neoplasm Proteins / Q000032:analysis; D009368:Neoplasm Transplantation; D011956:Receptors, Cell Surface / Q000032:analysis; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D013238:Stereotaxic Techniques; D020079:Terminal Repeat Sequences; D014162:Transfection; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured / Q000637:transplantation
D018076:DNA, Complementary; D008164:Luminescent Proteins; D009363:Neoplasm Proteins; C524898:PLAUR protein, human; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004801:Eosine Yellowish-(YS); D006416:Hematoxylin