The goal of my research was to provide a histological basis of TH-302 in pancreatic tumor cells from hypoxia markers and quantitative image analysis. I sought to determine what regions of the tumor TH-302 penetrates, to track where the drug accumulates, and to correlate these tracks in order to determine if there was uptake of the drug in areas of low vascular perfusion. My main motivation was to create a process to aid researchers in visualizing complex data about drug performance.
The human pancreatic cancer cell lines ASPC-1, Panc-1, BxPC-3, and MIA PACA were obtained from commercial sources (American Tissue Culture collection). Xenograft tumors, were generated by subcutaneously injecting 5 × 106 tumor cells in 100μl of phosphate-buffered saline (PBS) into the right hind limb of female immune-compromised NCI nu/nu mice, were provided as samples from other ongoing experiments.
Injection and Dissection
Sample sections, 10 μm thick, were cut from each tumor using a Microm HM500 cryostat microtome, and used for subsequent staining experiments.
Immunohistochemical Staining & Image Acquisition
Over the course of two summers I developed a novel process by which tumor sections would be stained with pimonidazole hydrochloride and Hoechst 33342 as well as antibodies. After keeping the sections over night I would analyze the sections with a fluorescent microscope equipped with a Coolsnap cooled charge-coupled device digital camera, a hand-controlled motorized stepping stage, and imaging software. Using my design and technical knowledge, I was able to collate the captured images and colorize them in such a way that they could be viusally interpreted in seconds. These images replaced dull excel spreadsheets filled with data points.
Autoradiographic images of the distribution of 14C-TH-302 were obtained using a Biospacelab BetaImager. These images were used to compare the uptake of 14C-TH-302 with the uptake of pimonidazole and Hoechst 33342