Prostate SPORE Summer Research Experience

Name of Participant: Bradley Christman Year of Summer Research Experience: 2012 Please return to Jill Miller by September 4, 2012 at jsmiller@umich.edu. Describe why you were interested in working in a research lab. I was interested in working in a research lab this summer because I wanted to find out, first hand, what working and learning in a professional medical laboratory was like. I made visits to my mom’s lab for years as a kid and I was intrigued by how all those crazy contraptions were used. In college my coursework in biology and chemistry gave me a strong theoretical foundation. I became eager to apply the things I learned in the classroom to a scientific research project. It has been a great opportunity to work with Dr. Macoska and the talented members of her lab and I couldn’t have asked for a more rewarding and motivating summer. Describe what you learned and any skills you acquired. I acquired the skills and know how to do cell culture for 3 different types of cells, prepare medias, count cells via hemocytometer and Trypan Blue exclusion, use a centrifuge, create conditioned medias, plate for all types of proliferation assays, prevent contamination under the hood, use a spectrophotometer, properly freeze down/re-plate in cryopreservation, professionally assemble and analyze data, present data at weekly lab meetings, collaborate with other lab members, correctly aspirate and re-suspend cells, plate cells at specific concentrations, professional upkeep of my laboratory notebook, precise use of pipettes and meeting deadlines with cell culture based research projects. Describe accomplishments of the project. There were quite a few accomplishments from this project. I was able to effectively establish my results for cell proliferation assays of PC3 cancer cells and immortalized N1 cells in serum free and serum containing medias as standard and consistent. After my assays were deemed consistent I moved on to the preparation of the conditioned media by culturing N1 fibroblast cells then treating them with 2 and 4 ng/ml of TGFβ at 24 and 48 hr, successfully phenoconverting the treated N1 fibroblasts into fibrotic myofibroblasts. At the designated time periods this media was aspirated and saved for the proliferation assay. The results of the proliferation assay showed that the 2ng/ml 48 hr TGFβ treated N1 cell conditioned media caused a 25% increases in the proliferation of PC3 cancer cells and the 4ng/ml 48 hr TGFβ treated conditioned media caused a 12.9% increase in proliferation compared to the untreated control 48hr conditioned media. The 4ng/ml 24hr TGFβ treated N1 cells conditioned media caused a16.7% increase in the PC3 cancer cell proliferation and the 24hr 2 ng/ml TGFβ treated conditioned media caused a 11.5% increase in proliferation compared to the 24hr untreated conditioned media. The results support that PC3 cancer cells grown in conditioned media taken from TGFβ treated fibroblasts proliferate at a higher rate compared to those grown in untreated conditioned media. A WST assay was also completed to verify that any residual TGFβ left over in the conditioned media from the original experiment was not the cause of the increased PC3 cancer cell proliferation. I found that TGFβ on its own actually caused a slight decrease in cancer cell proliferation when at 2 and 4 ng/ml concentrations. Since TGFβ treated fibroblasts causes phenoconversion to fibrotic myofibroblasts, our data shows that the cell signals and macromolecules excreted from fibrotic cells into the media may cause cancer cells to proliferate at a higher rate. From my research I have two testable hypotheses for this increase in proliferation. One hypothesis is based on the increased levels of collagen excreted into the media and the other on the growth factors excreted. Collagen, which is over expressed and released into the ECM by myofibroblasts could anchor the cancer cells activating focal adhesion kinase (FAK), which activates enzymes involved in cell division. This mechanism has been shown in breast cancer cells. To test this hypothesis, FAK could be blocked to see its role in prostate cancer cell proliferation and motility. This connection between cancer cells and collagen could also potentially increase cancer cell motility via cell crawling along ECM filaments. Assays could be performed to determine what types of growth factors and molecules are found in the conditioned media. List any publications resulting from the project. None.

Are there any grant submissions planned as a result of this project? No.