References Cheng, Xin, Rong Luo, Guang Wang, Chang-jun Xu, Xin Feng, Ren-hao Yang, E. Ding, Yan-qing He, Manli Chuai, Kenneth Ka Ho Lee, and Xuesong Yang. "Effects of 2,5- Hexanedione on Angiogenesis and Vasculogenesis in Chicken Embryos." Reproductive Toxicology. Elsevier, 27 Dec Web Fall.. Kamura, Takashi, Hirohisa Yano, Naoyo Nishida, Toshiharu Kamura, and Masamichi Kojiro. "Angiogenesis in Cancer." Vascular Health and Risk Management. Dove Medical Press, Sept Web. 06 Feb "Public Health Statement for N-Hexane." Agency for Toxic Substances and Disease Registry. N.p., July Web. 06 Feb Acknowledgements I would like to thank Dr. Ronald Kaltreider for all of his help in designing this study, maintaining cell lines, and completing this study. I would also like to thank Dr. Jeffrey Thompson for generously supplying us with the U87 cancer cells we used in this study. Angiogenesis and tumorshttp://cisncancer.org/research/images/angiogenesis- rew.jpg Hexane-2D-B.png/200px-Hexane-2D-B.png n- Hexane n -Hexane Increases Blood Vessel Formation in a Human U87 Glioblastoma In Vivo Chicken Chorioallantoic Membrane (CAM) Culture System Ryan J. Carter and Ronald Kaltreider, Ph. D. Department of Biological Sciences, York College of Pennsylvania Introduction n -Hexane is commonly found in commercial cleaning agents in the printing, textile, furniture, and shoemaking industry as well as in construction, manufacturing plants, and the car manufacturing and mechanic industry Exposure is due to being employed in occupations where it is used, living near hazardous waste sites, or its manufacturing, processing, or storage facilities where you could breathe in n -Hexane vapors It is also found in gasoline, meaning nearly everyone is exposed to it over their lifetimes Research has indicated that n -Hexane exposure is directly linked to nerve disorders (Agency for Toxic Substances and Disease Registry 1999) No data has indicated a relationship between n -Hexane and cancer, however, a study by Cheng et al. (2014) suggested that n -Hexane exposure was uncovered to be associated with altered expression of VEGF as well as increase in vasculature, in other words an increase in angiogenesis, using gastrulating chicken embryos as a model Angiogenesis, which is the formation of new blood vessels derived from preexisting ones, plays a major role in many physiological processes but also is vital for the tumor cell proliferation process In order for the proliferation, as well as metastatic spread of cancer cells, there must be an adequate supply of oxygen and nutrients as well as the removal of waste products, which is provided by the induction of new growth in the vascular network formed through angiogenesis (Kamura et al. 2006) Research done by Cheng et al. (2014) led us to question if n- Hexane could increase angiogenesis in human U87 glioblastoma tumors using a CAM assay, where chicken embryos are used as a living model system, providing the first evidence of n-Hexane potentially enhancing the tumor cell proliferation process by enhancing angiogenesis Objectives & Hypothesis Determine the effects of n- Hexane at environmentally relevant doses on the vascular network in human U87 glioblastoma tumors We suspect to see an increase in the vascular network in CAMs where n- Hexane is applied in comparison to controls Materials & Methods Maintained U87 MG (ATCC HTB-14) glioblastoma cancer cell lines in extracellular matrix culture medium (DMEM) and split at 60-80% confluency Purchased 95% n- Hexane from Sigma-Aldrich, stored at room temperature, and diluted with PBS using 1:10 serial dilutions to get treatment concentrations White horn chicken eggs purchased from Moyer’s Chicks and upon arrival they were incubated at 98 F for the entire study On day 10, the CAMS were candled to establish viability and location of the air sac CAMs were put intro treatment groups where each had 2 CAMs (n=2) and were as follows: control receiving a carrier agent (DMSO), a low dose, intermediate dose, and high dose (0.001 M, 0.01 M, 0.1 M) of n -Hexane On day 11 shell surrounding the air sac was removed and we added 50,000 cells/ml (50 ul, 25% cells, 25 % extracellular matrix) U87 glioblastoma cancer cells to viable CAMs using a syringe for injection On day 13, n -Hexane (50 ul) was added in the desired concentrations to the appropriate CAMs at the tumor site using a micropipette On day 17, tissue (4x4 cm) was excised from each tumor, fixed in 4% paraformaldehyde, and blood vessel formation and density were visually determined in CAMs using a micro dissection-imaging microscope and the NIKON Digital DS-03 camera software. Results Figure 1. Micro dissection imaging of tumor tissue excised from each CAM taken at 1X. The top two images are the carrier agent control (A), the second two are the low dosage (0.001 M) (B), the third two are the intermediate dosage (0.01M) (C), and the bottom two are the high dosage (0.1M) (D). Results (cont’d.) The control showed no blood vessels in the tissue excised that were visually apparent (Figure 1, A) All doses of n -Hexane dramatically increased blood vessel formation and density Small, but very dense vasculature was present throughout the entirety of the tissue in the low dosage treatment in both CAMs (Figure 1, B). More apparent, large vessels and arteries were seen in one of the CAMs of the intermediate dosage treatment while the other showed little to none vasculature (Figure 1, C) Blood vessel formation was both very large and dense throughout the tissue of the high dosage treatment and in addition to the main arteries and vessels, the branched networks of vessels were also large in size and density (Figure 1, D) Conclusions The goal of this study was to determine the effects of n- Hexane on angiogenesis in U87 glioblastoma tumors Our results strongly supported our hypothesis in that vasculature would be increased by n -Hexane exposure Lack of vasculature in one of the CAMs in our intermediate dose, could be due to some type of experimental error Currently, by OSHA standard, permissible n- Hexane exposure in the work place is 500 parts per million parts of air (ppm) (Center for Disease Control 1978) Our low dose of M, being environmentally relevant, is equal to ppm,well under the permissible levels of n- Hexane by OSHA standard of 500 ppm Our study is the first to suggest n -Hexane could enhance the tumor cell proliferation process, and suggest that environmentally relevant doses of n -Hexane could pose a threat to individuals in that although it may not cause cancer, it could possibly play a role in the progression of cancer Future Studies Replicate this research using various cancer types, to determine if there is any specificity associated with n -Hexane and cancer types Determine n -Hexane levels in various areas across the United States, and then directly apply those concentrations to tumors in effort to see how angiogenesis is affected under those conditions. chicken-egg.jpg.jpg A B C D