Visualization of xenotransplanted human rhabdomyosarcoma after transfection with red fluorescent protein.
OBJECTIVE
Discosoma sp red fluorescent protein (DsRed2) is a newly developed marker for in vivo labeling studies in different biologic systems. After vector transfection, DsRed2 is expressed in mammalian cells and can be detected by fluorescence microscopy. The aims of this study were to establish a DsRed2-transfected human rhabdomyosarcoma (RMS) cell line and to perform a xenotransplantation on nude mice to use imaging as a tool for further basic research studies on this neoplasm.


METHODS
The human alveolar RMS cell line Rh30 was transfected with the pDsRed2-N1 vector by lipofection. The DsRed2-positive cells were sorted out by fluorescence-activated cell sorting analysis 96 hours after transfection and selected in culture with G418. Expression of DsRed2 messenger RNA was assessed using single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Transfected and parental cells were characterized cytologically, cytogenetically, immunohistochemically, and in vivo after subcutaneous injection in NMRI (nu/nu) nude mice.


RESULTS
After vector transfection, a pure and stable DsRed2-positive cell line was established by monoclonal growth of the cells. Reverse transcriptase polymerase chain reaction revealed constant expression of DsRed2 messenger RNA in fluorescencing cells. There was no difference between transfected and parental cells by means of cell morphology and desmin expression. Clonal cells (1 x 10(6)) were used for xenotransplantation. Tumors were visualized noninvasively through the skin of the mice using specific emission and excitation filters. Tumor vascularization and vessel growth could be discriminated from tumor tissue using this imaging system.


CONCLUSIONS
This is the first report on successful transfection of an RMS cell line with red fluorescent protein followed by xenotransplantation into nude mice. This model can serve as an imaging tool for in vivo studies investigating tumor biology and metastases of human RMS.
Seitz
Guido
G
Department of Pediatric Surgery, University Children's Hospital, 72076 Tübingen, Germany. guido.seitz@med.uni-tuebingen.de
Warmann
Steven W
SW
Fuchs
Jörg
J
Mau-Holzmann
Ulrike A
UA
Ruck
Peter
P
Heitmann
Heike
H
Hoffman
Robert M
RM
Mahrt
Jens
J
Müller
Gerhard A
GA
Wessels
Johannes T
JT
D000818:Animals; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D005260:Female; D006801:Humans; D049408:Luminescent Agents; D008164:Luminescent Proteins; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D009368:Neoplasm Transplantation; D012208:Rhabdomyosarcoma / Q000175:diagnosis*; D014162:Transfection; D000097573:Red Fluorescent Protein
D049408:Luminescent Agents; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein