Abstract The use of targeted gold nanoparticles (AuNPs) has shown tremendous promise in the early detection and improved therapy of cancer. Successful development and implementation of these new theranostic approaches, however, depends on sufficient transvascular migration and specific accumulation of AuNPs to extravascular tumor tissue. This study utilized photoacoustic (PA) imaging to track - in three dimensions and through 24 hours - the extravasation and accumulation of targeted AuNPs in a subcutaneous murine model of prostate cancer. AuNPs (nanorods; 760-nm peak) targeting the Luteinizing-hormone-releasing hormone receptor were systemically injected into three mice bearing PC-3 human prostate cancer tumors; as a control, three additional mice were injected with PEGylated AuNPs. For all particle-tracking studies, multi-wavelength (680, 760, 800, 920, & 940 nm), volumetric (spherical volume with 25.6-mm diameter) PA imaging was conducted at pre-injection, post-injection, 15-min, 60-min, 240-min, and 24-hr time points; the imaging volume contained the tumor and the spleen (control). Following imaging, tumors were excised and inductively coupled plasma mass spectrometry (ICP-MS) of samples and two-photon imaging of histological sections was performed to confirm AuNP accumulation and diffusion, respectively. Volumetric PA perfusion assessment was then conducted by monitoring the wash-out of indocyanine green (ICG) in near real-time (0.25 Hz for 180 s post-injection). All PA imaging studies were performed on the Nexus 128 preclinical imaging system (Endra Inc., Ann Arbor, MI). Spectral unmixing of the multi-wavelength PA data was achieved to segment AuNPs and deoxy-/oxyhemoglobin (i.e. for local hypoxia assessment). Accumulation and extravasation data of the targeted AuNPs in the tumor were then compared to tumor perfusion data, PA-derived tumor hypoxia data, PEGylated AuNP accumulation data in the tumor, AuNP accumulation data in the spleen, and ICP-MS/histological analysis. Results of the study establish that volumetric PA imaging with multi-wavelength unmixing is able to track nanoparticle accumulation and extravasation throughout an in vivo tumor model. The location and relative degree of particle accumulation was also found to correlate with PA-based perfusion and vascularity assessment. This study demonstrates that volumetric PA imaging has the spatiotemporal resolution and sensitivity to track extravasation of AuNPs in an in vivo tumor model. This in vivo tracking ability - along with concurrent PA-based perfusion and hypoxia mapping - could be of significant benefit in understanding and improving nanoparticle targeting for the diagnosis and treatment of cancer. Citation Format: Richard Bouchard, Tatiana Wolfe, Michael Thornton, Timothy Morgan, Trevor Mitcham, Shanta Bhattarai, Jonathan Grant, Jihyoun Lee, John Hazle, Sunil Krishnan. Three-dimensional in vivo photoacoustic tracking of targeted nanoparticles in a pancreatic cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2051. doi:10.1158/1538-7445.AM2014-2051