Abstract. Pancreatic ductal adenocarcinoma (pancreatic cancer, PDAC) is the classic example of a recalcitrant tumor that is extremely challenging to treat. Therapeutic strategies which can bypass the desmoplasia `fortress' and access hypoxic microenvironments without significantly affecting healthy cells would address the critical issues presented by PDAC physiology. Localized therapies are a critical component of PDAC treatment and there is strong interest in innovative ways to intensify radiation therapy (RT). A novel approach to enhancing the radiation dose delivered to tumors is to increase the radiation-interaction probability of the target tissues by delivering high atomic number (Z) nanoparticles (e.g., gold or hafnium oxide) to tumor cells. However, due to the exuberant desmoplasia characteristic of PDAC, the diffusion of even the smallest nanoparticles is limited by the dense stroma making delivery to cancer cells exceedingly challenging. Furthermore, recent evidence confirms that depleting the stroma may not be the answer since the stroma restrains and confines cancer cells to within the pancreas rather than promotes the growth of cancer cells. We contend that the ideal strategy is to penetrate the stroma without destroying it. Here we address this delivery barrier standing in the way of effective radiosensitization of PDAC tumors by employing a recently reported process of in situ gold biomineralization by mammalian cancer cells. This strategy will allow replacement of pre-synthetized radiosensitizing gold nanoparticles (GNPs) with ionic gold atoms thus achieving the smallest possible size of a therapeutic agent – a single ionic atom. Our hypothesis is that small gold ions (i) will uniformly distribute throughout the tumor as their diffusion is not likely to be impeded by the stroma, (ii) will be reduced to GNPs via the process of in situ biomineralization after specific uptake by cancer cells, and (iii) will radiosensitize the tumor while sparing adjacent normal tissue. This hypothesis is based on our preliminary data demonstrating a strong radiosensitization effect in PDAC cells by the intracellularly synthetized GNPs both in vitro and in vivo. In addition, normal pancreatic cells synthesized significantly fewer GNPs than cancer cells, consistent with recent reports showing higher efficiency of in situ gold biomineralization in cancer vs. non-cancer cells. Further, a long history of the clinical use of gold-salt based drugs in treatment of rheumatoid arthritis can provide a clear path towards clinical translation. In this project we have devised comprehensive mechanistic studies to evaluate and to optimize in situ biomineralization for efficient radiosensitization of PDAC. To this end we will carry out studies in three synergistic Aims: (1) to determine the mechanism of and to optimize conditions for intracellular synthesis of GNPs by PDAC cells; (2) to evaluate the determinants of radiosensitization efficacy in vitro and in vivo; an...