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RESEARCH PROFILES
During a 2-year fellowship at the U.S. National Institute of Allergy and Infectious Diseases, W. David Culp encountered the question that he is now spending his graduate career trying to answer: Why do melanomas become harder to treat once they reach a certain stage? At the time, Culp was a recipient of a Postbaccalaureate Intramural Research Training Award and worked in an immunotherapy laboratory, where he tested treatments on a common mouse melanoma cell line called B16‑F10. “We found that we could treat young established tumors, but we could not treat older established tumors,” explains Culp. “Once the tumor got to a certain size, no matter what we did, immunotherapy seemed to fail.” To delve deeper into the problem, Culp wanted to study the melanoma cell line in mice to determine the protein changes that occur as tumors advance through successive stages. In his current work at the Karolinska Hospital and Institute (Sweden) and the U.S. National Eye Institute (NEI), he has done just that. In this issue of JPR (pp 1332– 1343), Culp and colleagues at the Karolinska Hospital and Institute, NEI, and the Tumor Research Institute report having performed a kinetic proteomics analysis of melanoma tumors in vivo at various stages. The researchers identified several proteins that are differentially expressed in melanomas as they become more difficult to treat. Other researchers had analyzed melanoma cell lines before, but Culp and colleagues wanted to study the progression of melanoma tumors in vivo. Culp explains that only by examining tumors in an animal can researchers begin to understand the complex interactions between tumors and their native environments. The researchers injected B16-F10 melanoma cells into mice and removed the tumor masses at 3, 5, 7, and 10 d af-
ter injection. On the basis of Culp’s previous research, he knew that day-3 tumors could be treated, but day-7 tumors could not. The researchers also chose a time point in the middle (day 5) and at a late stage (day 10). All of the tumor masses were composed of >90% tumor cells. Proteins from the tumors were
place by day 7. With the use of western blotting, the researchers confirmed that vascular endothelial growth factor (known as VEGF) is greatly increased from day 5 to day 7. Inflammatory cells invade the tumor by day 5, and necrosis is evident by day 7. A surprising result was that the expression of several forms of albumin was decreased between days 5 and 7. Albumin levels were exProteins (n = 44) 1.4 pected to increase because angio1.05 0.7 genesis was taking place, and albu0.35 min is a major component of blood. Day 5 The levels of cathepsin D, which Day 5 degrades albumin, increase from Day 5 Day 3 day 3 to day 5 and from day 5 to day Day 3 7. Culp explains that tumor cells Day 7 might degrade albumin so they can Day 7 use the protein as a source of nuDay 7 Day 10 trients or as a way to regulate presDay 10 sure within the tissue. Day 10 Another intriguing observaDay 10 tion was that differentially regu0.1 0.05 lated tumors didn’t follow one or two simple trends. “Spots weren’t The heat is on. A heat map shows the drastic switch in just increasing or decreasing; some the expression of 44 proteins between day-5 and day-7 were sporadic,” says Culp. The remelanomas. searchers identified 6 major trends of expression, including one in run on separate 2DE gels, and statistiwhich protein levels decreased from cal tests were used to determine whethday 3 to day 5, then increased from day er spots were significantly up- or down5 to day 10, and another in which proregulated. From day 3 to day 5, 46 spots tein levels increased slightly from day were differentially regulated; from day 3 to day 5, increased more substantial5 to day 7, 58 spots were d ifferentially ly from day 5 to day 7, then decreased. regulated; and from day 7 to day 10, Culp says that these trends emphasize 15 spots were differentially regulated. the dynamic nature of melanomas. Proteins in the spots were identified by With this initial analysis of melaMALDI TOFMS mass fingerprinting. noma tumor stages, the researchers Interestingly, when the researchers have already learned a lot. However, plotted the differentially regulated proCulp points out that the study examteins on a heat map in which up- and ined only highly abundant proteins in the tumors. “We want to try to identi down-regulated proteins were colorfy some of the low-abundance proteins coded, they found that a major change in protein expression occurred between within these tumors because I think day 5 and day 7. “I think [this result] rethat could tell us about what’s going on flects a dramatic shift in the biology within these tumors and what, if any, of the tumor at that time,” says Culp. of the constituents may have effects on One of the major phenotypic changes the immune system,” he says. “That’s at this stage is angiogenesis. By stainwhat we’re really interested in—trying tumors for CD31, a membrane proing to help our immunotherapeutic approaches.” tein on blood vessels, the researchers confirmed that vascularization takes —Katie Cottingham
Mice
Kinetic proteomics of melanoma tumors
1296 Journal of Proteome Research • Vol. 5, No. 6, 2006
© 2006 American Chemical Society
