Abstract A22: Exosomal release of IAPs may contribute to chemoresistance and aggressiveness in pancreatic cancer.

Purpose: Pancreatic cancer is one of the most aggressive cancers, making it extremely difficult to treat. The few treatment options are limited due to the difficulty detecting pancreatic cancer. Chemotherapy offers a possible treatment, of which the antimetabolite agents gemcitabine (Gem) and 5-fluorouracil (5'FU) are used to treat this metastatic disease. Despite treatment, patients develop chemoresistance against the agents. Here, we evaluate five agents representing two antimetabolite families in order to determine whether they have the ability to decrease the expression of the inhibitor of apoptosis (IAP) proteins survivin and XIAP in comparison to Gem and 5'FU and whether the repertoire of proteins and mRNA confined to a shed membranous vesicle called an exosome changes with therapy, resulting in tumor microenvironment changes. Methods: Cell death was evaluated using Annexin V / PI and analyzed by flow cytometry. Cell number for death curves were acquired by flow cytometry. Data was collected by MACS Quant and analyzed using FlowJo. Protein expression of survivin and XIAP was determined by Western blotting. PCR was performed to determine mRNA expression of the IAPs. Exosomes were isolated by centrifugation and presence was confirmed by acetylcholinesterase activity. Results: Panc-1 cells treated with various antimetabolites showed a time and dose dependent cell death. Treatment with sublethal and lethal concentrations at 24, 48 and 72hrs were selected for all further studies. Modulation of survivin was more evident in comparison to XIAP across all treatments, in particular after 72 hrs. There is also a change in mRNA expression of the IAPs after treatment. Although no significant difference in the amount of exosomes shed in each treatment was detected, there was different exosomal survivin and XIAP mRNA and protein expression. Conclusions: Novel antimetabolite agents have proven their effectiveness in vitro in modulating survivin and XIAP protein levels and a concomitant enhanced apoptotic/ necrotic cellular death. Exosomal release of IAP mRNA and proteins into the tumor microenvironment may contribute to the aggressiveness of the cancer as well as the development of chemoresistance. Exosomal packaging of survivin, XIAP and other biomarkers may prove useful in selecting patient specific treatment regimens.