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Introduction: Pancreatic cancer is characterized by early metastasis and resistance to chemotherapy. To elucidate the transcriptional reprogramming and drug resistance that emerges during the metastatic cascade, we set out to characterize the transcriptomic profiles and drug response of matched sets of cancer cell organoids derived from multiple sites (primary tumor, metastatic lesion, and circulating tumor cells) in tumor-bearing PKCY (p53L/wt, KrasLSL-G12D/wt, ptf1acre/wt, Rosa26LSL-YFP/LSL-YFP) mice. Methods: To establish primary and metastatic organoids, tumor lesions are excised, enzymatically digested, and plated in Matrigel. Previously described organoid cell culture media (containing R-spondin1, Wnt3A, Noggin, FGF10, and EGF) was used to maintain organoids in culture. To allow for the culture and analysis of circulating tumor cell (CTC) organoids, we developed a CTC isolation and culture protocol. Briefly, whole blood is collected from tumor-bearing mice using a terminal cardiac puncture into the right ventricle, followed by RBC lysis and plating in Matrigel. To allow for high-throughput drug screening, we developed a single organoid per 384-well approach where organoid growth and response to drug treatment can be quantified using automated fluorescent imaging. Results: We established organoid cultures from primary tumors and metastatic lesions (liver or lung) from 30 tumor-bearing PKCY mice, with a 100% success rate. In addition, we successfully established CTC organoid lines from 10 metastatic PKCY mice, with an overall success rate of 40% (10/25). The success rate was higher (60%) in mice harboring both macroscopic liver and lung metastasis, whereas mice harboring only liver or lung metastasis displayed a lower success rate (33% and 40%, respectively). We have established 5 matched sets of primary, metastatic (liver and/or lung), and CTC-derived organoids as wells as normal pancreatic ductal organoid lines from CY (ptf1acre/wt, Rosa26LSL-YFP/LSL-YFP) mice, serving as noncancer controls. Pancreatic origin of all organoids was confirmed with the YFP lineage-label. Preliminary drug testing of an 8-drug panel (gemcitabine, paclitaxel, 5FU, irinotecan/SN38, oxaliplatin, 5-azacytidin, SAHA, and EPZ6438) revealed several cases of increased drug resistance in organoids derived from metastatic lesions and CTCs relative to matched primary tumor organoids. To expand on this analysis, we are implementing a high-throughput drug screen evaluating a large library of small compound drugs and inhibitors and correlating the results to transcriptomic analysis. Conclusion: We developed an approach for mapping the pharmacotranscriptomic landscape of pancreatic cancer organoids derived from primary tumor, metastatic lesions, and CTCs. We are exploring our initial findings further using high-throughput drug screening, transcriptomic analysis, and orthotopic transplantations into syngeneic mice. Citation Format: Fredrik I. Thege, Bhargavi B. Barathi, Dhwani N. Rupani, Sonja M. Woermann, Andrew D. Rhim. Mapping the pharmacotranscriptomic landscape of pancreatic circulating tumor cell organoids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C55.