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(American Journal of Pathology. 1999;155:723-729.)
© 1999 American Society for Investigative Pathology

Technical Advance

Isolation of Mouse Stromal Cells Associated with a Human Tumor Using Differential Diphtheria Toxin Sensitivity

Jack L. Arbiser^*, Gerhard Raab, Richard M. Rohan, Subroto Paul, Karen Hirschi, Evelyn Flynn, E. Roydon Price^¶, David E. Fisher^¶, Cynthia Cohen and Michael Klagsbrun^§

From the Departments of Dermatology^*
and Pathology,
Emory University School of Medicine, Atlanta, Georgia; the Departments of Surgery
and Pathology,^§
Children's Hospital, Harvard Medical School, Boston, Massachusetts; and the Department of Pediatric Hematology/Oncology,^¶
Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts

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.

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