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


CONCLUSIONS
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.
Bauer
Todd W
TW
Division of Surgical Oncology, Department of Surgery, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA.
Gutierrez
Marcos
M
Dudrick
Daniel J
DJ
Li
Jiarong
J
Blair
Ian A
IA
Menon
Chandrakala
C
Fraker
Douglas L
DL
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D004195:Disease Models, Animal; D005260:Female; D006614:Hindlimb; D006801:Humans; D008545:Melanoma / Q000188:drug therapy*; D008558:Melphalan / Q000494:pharmacology*; D010477:Perfusion; D051381:Rats; D011923:Rats, Nude; D012878:Skin Neoplasms / Q000188:drug therapy*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating; D014409:Tumor Necrosis Factor-alpha; D008558:Melphalan