SUMMARY Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo , we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo -relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

In pancreatic ductal adenocarcinoma (PDAC), the basal-like and classical transcriptional subtypes are associated with differential chemotherapy sensitivity and patient survival. These phenotypes have been defined using bulk transcriptional profiling, which can mask underlying cellular heterogeneity and the biologic mechanisms that distinguish these subtypes. Furthermore, few studies have interrogated metastases, which are the cause of mortality in most patients with this highly lethal disease. Using single-cell RNA-sequencing of metastatic needle biopsies and matched organoid models, we demonstrate intra-tumoral subtype heterogeneity at the single-cell level and define a continuum for the basal-like and classical phenotypes that includes hybrid cells that co-express features of both states. Basal-like tumors show enrichment of mesenchymal and stem-like programs, and demonstrate immune exclusion and tumor cell crosstalk with specific macrophage subsets. Conversely, classical tumors harbor greater immune infiltration and a relatively pro-angiogenic microenvironment. Matched organoid models exhibit a strong bias against the growth of basal-like cells in standard organoid media, but modification of culture conditions can rescue the basal-like phenotype. This study reframes the transcriptional taxonomy of PDAC, demonstrates how divergent transcriptional subtypes associate with unique tumor microenvironments, and highlights the importance of evaluating both genotype and transcriptional phenotype to establish high-fidelity patient-derived cancer models.

Metastatic pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal malignancy with few therapeutic options. Tumor transcriptional state is a strong predictor of clinical outcome in PDAC, with two primary cell states, basal-like and classical, identified by bulk transcriptional profiling. Basal-like tumors carry a worse prognosis, but the mechanisms underlying this survival difference, the degree of cellular heterogeneity within a given tumor, and the subtype-specific contributions from the local immune microenvironment are not well understood. In addition, there are ongoing efforts to use patient-derived organoid models as functional surrogates for an individual patient’s disease, but the degree to which patient transcriptional phenotypes are preserved in their matched organoid models remains unclear. Here, we describe a pipeline that enables both direct characterization of the liver metastatic niche via single-cell RNA-sequencing and functional assessment of PDAC tumor biology in patient-matched organoid models. Starting from core needle biopsies of metastatic PDAC lesions, we applied this approach to profile 22 patient samples and their matched organoid models using single-cell RNA-sequencing with Seq-Well. We demonstrate significant heterogeneity at the single-cell level across the basal-like to classical transcriptional spectrum. Basal-like cells expressed more mesenchymal and stem-like features, while classical cells expressed features of epithelial and pancreatic progenitor transcriptional programs. A population of “hybrid” malignant cells co-expressed markers of both basal-like and classical states, suggesting that these phenotypes lie on a continuum rather than as discrete entities. Microenvironmental composition also differed by subtype across T/NK and macrophage populations. Specifically, basal-like tumors exhibited tumor cell crosstalk with specific macrophage subsets, while classical tumors harbored greater immune infiltration and a relatively pro-angiogenic microenvironment, raising important considerations for subtype-specific microenvironmental directed therapy. Finally, we found that matched organoids exhibited transcriptional drift along the basal-like to classical axis relative to their parent tumors, with evidence for selection against basal-like phenotypes in vitro. However, tumor cells in organoid culture exhibited remarkable plasticity and could recover in vivo basal-like phenotypes in response to changes in their growth conditions. Taken together, our work provides a framework for the analysis of human cancers and their matched models using single-cell methods to dissect tumor-intrinsic and extrinsic contributions, and reveals novel insights into the transcriptional heterogeneity and plasticity of PDAC. Citation Format: Srivatsan Raghavan, Peter S. Winter, Andrew W. Navia, Hannah L. Williams, Alan DenAdel, Radha L. Kalekar, Jennyfer Galvez-Reyes, Kristen E. Lowder, Nolawit Mulugeta, Manisha S. Raghavan, Ashir A. Borah, Sara A. Vayrynen, Andressa Dias Costa, Junning Wang, Emma Reilly, Dorisanne Y. Ragon, Lauren K. Brais, Alex M. Jaeger, James M. Cleary, Lorin Crawford, Jonathan A. Nowak, Brian M. Wolpin, William C. Hahn, Andrew J. Aguirre, Alex K. Shalek. Transcriptional subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-058.