Isolation of mouse stromal cells associated with a human tumor using differential diphtheria toxin sensitivity.
Tumor vascularization is accompanied by the migration of stromal cells, including endothelial cells, smooth muscle cells, and fibroblasts, into the tumor. The biological contributions of stromal cells to tumor vascularization have not been well-defined, partly due to the difficulty of culturing stromal cells in the presence of large numbers of fast-growing tumor cells. To address this problem, a strategy was devised to kill tumor cells but not stromal cells. Advantage was taken of the observation that diphtheria toxin (DT) kills human but not rodent cells. Human melanoma (MMAN) tumor cells were injected subcutaneously into nude mice. The tumors were excised, homogenized, and treated with 50 ng/ml DT for 24 hours. Elimination of melanoma cells by DT treatment was demonstrated by lack of detectable levels of microphthalmia, a transcription factor that is a marker for melanoma cells. The murine stromal cells were viable and found to be mostly smooth muscle cells. These cells constituted about 1.5% of the MMAN tumor. RNase protection assays using a specific murine vascular endothelial growth factor probe confirmed the murine origin of the stromal cells. This method allows rapid isolation of stromal cells and should facilitate biochemical and genetic analysis of tumor-stromal interactions.
Arbiser
J L
JL
Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia, USA. jarbise@emory.edu
Raab
G
G
Rohan
R M
RM
Paul
S
S
Hirschi
K
K
Flynn
E
E
Price
E R
ER
Fisher
D E
DE
Cohen
C
C
Klagsbrun
M
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D004167:Diphtheria Toxin / Q000494:pharmacology*; D016228:Endothelial Growth Factors / Q000235:genetics; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines / Q000235:genetics; D008297:Male; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000378:metabolism; D012878:Skin Neoplasms / Q000378:metabolism* / Q000473:pathology; D017154:Stromal Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D014183:Transplantation, Heterologous; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000199:Actins; D004167:Diphtheria Toxin; D016228:Endothelial Growth Factors; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors