Abstract B14: A novel strategy for the treatment of melanoma
Authors: Xu Mai , Lockhart Albert C. , Myerson Robert J. , Zoberi Imran , Linette Gerald P. , Wang-Gillam Andrea 
Published: July 2015

Abstract
Introduction: Approximately 76,000 individuals are diagnosed with melanoma, and about 9,000 die as a result of the disease each year in the United States. Even though there are many options for standard treatment of melanoma such as surgical resection, radiotherapy, intraregional therapy with sclerosing agents, isolated limb perfusion, and immunotherapy, the mortality rate is still high. The high mortality reflects a lack of effective therapeutic methods for the treatment of melanoma. The purpose of this study is to develop an effective therapeutic approach for the treatment of melanoma, using mild/moderate (less-than 41oC) hyperthermia (HT) to actively modulate the delivery of the FDA approved anticancer drug nab-paclitaxel (human serum albumin (HSA) based paclitaxel-nanoparticle) to tumors. Physiologically, HSA serves as a natural carrier to deliver nutrients, including itself as an amino acid source to cells with metabolic and/or reproductive needs by binding to its receptor albondin on the cell surfaces. We and other investigators have reported that the rate of cellular metabolism increases as temperature rises to the mild/moderate temperature range of 38oC to 41oC, implying that heat increase cellular uptake of HSA to meet the demands of the higher metabolic rate. In the clinic, nab-paclitaxel has demonstrated preferential uptake in tumors with good solubility, stability and longer circulation time with limited toxicity. In this study, we used HT to deliver nab-paclitaxel to melanoma xenograft in athymic mice.
Materials and Methods: Human melanoma A375 and G361 cells were purchased from ATCC (American Type Culture Collection) and maintained in a growth medium of Dulbecco's Modified Eagle's Medium (A375) and McCoy's 5A medium (G361) supplemented with 10% human serum, 100 units/ml penicillin and 100 μg/ml streptomycin. Both melanoma cells were adapted to human serum containing medium by maintaining them in the medium for at least 6 months. Human fibroblast GM05399 cells were obtained from Coriell Cell Repositories (Coriell Institute, Camden, NJ). Near infrared (NIR) dye (IRDye 800CW) Protein Labeling Kits were purchased from Li-Cor Inc. Nude mice, 4 or 5-weeks-old, were bought from the National Cancer Institute at Frederick, MD. Immunofluorescence staining and flow cytometry methods were applied to measure HSA in tumor cells in vitro after heat treatment. To observe the biodistribution of nab-paclitaxel in vivo, we labeled nab-paclitaxel with NIR dye using an IRDye 800CW Protein Labeling Kit following the manufacturer's instructions. The integrity of the nab-paclitaxel after a single freezing and thawing cycle, and nab-paclitaxel-NIR dye conjugates were measured using Dynamic Light Scattering (DLS). Biodistribution and kinetic changes of the nab-paclitaxel-NIR dye conjugates were examined and monitored after injection of the conjugates and heat treatment with the Pearl Imager in melanoma xenograft carrying animals.
Results: (1). HSA accumulated in melanoma cells after heating at 41oC for as little as 0.5 hour; there was no increase in the amount of HSA in human normal fibroblasts after the same heat treatment. (2). Integrity of the nab-paclitaxel was not interrupted after a single freezing and thawing cycle, and by the labeling procedures with fluorescent dye. (3). Most interestingly nab-paclitaxel and its NIR dye conjugates are stable after heating at 41oC for 1 hr. (4). Mild/moderate HT actively modulated the delivery of nab-paclitaxel-NIR dye conjugates to tumors, accumulated and retained nab-paclitaxel-NIR conjugates in the tumor for at least 72 hrs.
Conclusion and Future Studies: HT has been previously used to improve blood circulation and increase cell membrane permeability for antitumor chemical delivery to tumors in the clinic. In this study, the accumulation of nab-paclitaxel in heated tumor cells appears to be receptor-mediated. Because of size constraints, nab-paclitaxel could not passively diffuse into tumor cells, even cells permeabilized by heat treatment. Therefore, our preliminary results have demonstrated that mild/moderate hyperthermia can actively facilitate delivery of nab-paclitaxel to tumors in vitro and in vivo. HT promoted retention of nab-paclitaxel-NIR conjugates in tumor xenograft for a prolonged period of time as compared to controls (without heat treatment). A study is currently underway to evaluate the therapeutic efficacy of HT combined with nab-paclitaxel in the treatment of melanoma in vivo. A study to assess whether whole body hyperthermia can modulate delivery of nab-paclitaxel to metastatic tumors is being considered. Magnetic Resonance-guided High Intensity Focused Ultrasound mediated HT in combination with nab-paclitaxel may be an effective approach for the treatment of melanoma.
Citation Format: Mai Xu, Albert C. Lockhart, Robert J. Myerson, Imran Zoberi, Gerald P. Linette, Andrea Wang-Gillam. A novel strategy for the treatment of melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B14.