A human melanoma xenograft in a nude rat responds to isolated limb perfusion with TNF plus melphalan.

Abstract:

BACKGROUND:Isolated limb perfusion (ILP) with tumor necrosis factor-alpha (TNF) and melphalan for advanced extremity malignancies achieves significant complete response rates. To study molecular mechanisms underlying this response, a nude rat ILP model with a human melanoma xenograft was developed. METHODS:NIH1286 human melanoma was grown subcutaneously in the hind limb of nude rats. Anesthetized rats underwent a 10-minute ILP via femoral vessels with hetastarch, heparin, and melphalan, TNF, or TNF plus melphalan. The tumors and ulcers were measured and viable tumor area was calculated. Post-ILP tumors were analyzed by electron microscopy for vascular damage and also by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI/MS/MS) for free melphalan levels. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide (MTT) assays were performed on NIH1286 cells and human dermal microvascular endothelial cells (HDMVEC) to test for direct cytotoxicity to TNF and melphalan. Post-ILP tumors sections were also examined by electron microscopy. RESULTS:The mean maximal decrease in viable tumor area after ILP for control, TNF, melphalan, and TNF + melphalan groups were 0%, 22 +/- 13%, 61 +/- 14%, and 100 +/- 0%, respectively. LC/APCI/MS/MS revealed no difference in the free tumor melphalan concentration between melphalan alone and TNF + melphalan groups. The percent cytotoxicity in MTT assays using TNF, melphalan, and TNF + melphalan against NIH1286 were 0%, 51-59%, and 74-81%, respectively, and against HDMVEC were 28%, 16-23%, and 6-13%, respectively. Electron microscopic analyses showed that addition of TNF to the perfusate caused erythrostasis in tumor blood vessels. CONCLUSION:We developed a human melanoma nude rat ILP model with tumor responses very similar to the human ILP trials. This model will allow further investigation of the synergistic mechanism of TNF and melphalan in human melanoma in a preclinical setting, and extension of this study to current clinical trials.

journal_name

Surgery

journal_title

Surgery

authors

Bauer TW,Gutierrez M,Dudrick DJ,Li J,Blair IA,Menon C,Fraker DL

doi

10.1067/msy.2003.113

subject

Has Abstract

pub_date

2003-04-01 00:00:00

pages

420-8

issue

4

eissn

0039-6060

issn

1532-7361

pii

S0039606003000084

journal_volume

133

pub_type

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