Despite extensive efforts to characterize the transcriptional landscape of pancreatic ductal adenocarcinoma (PDA), reproducible assessment of subtypes with actionable dependencies remains challenging. Systematic, network-based analysis of regulatory protein activity stratified PDA tumours into novel functional subtypes that were highly conserved across multiple cohorts, including at the single cell level and in laser capture microdissected (LCM) samples. Identified subtypes were characterized by activation of master regulator proteins representing either gastrointestinal lineage markers or transcriptional effectors of morphogen pathways. Single cell analysis confirmed the existence of Lineage and Morphogenic states but also revealed a dominant population of more differentiated Oncogenic Precursor (OP) cells , present in all sampled patients, yet not apparent from bulk tumor analysis. Master regulators were validated by pooled, CRISPR/Cas9 screens, demonstrating both subtype-specific and universal dependencies. Conversely, ectopic expression of Lineage MRs, such as OVOL2, was sufficient to reprogram Morphogenic cells, thus providing a roadmap for the future targeting of patient-specific dependencies in PDA.
Despite extensive efforts, reproducible assessment of pancreatic ductal adenocarcinoma (PDA) heterogeneity and plasticity at the single cell level remains elusive. Systematic, network-based analysis of single cell RNA-seq profiles showed that most PDA tumors comprise three coexisting lineages whose aberrant transcriptional state is mechanistically controlled by distinct regulatory programs. These lineages were characterized by the aberrant activation of either gastrointestinal lineage markers (GLS), transcriptional effectors of morphogen pathways (MOS) and acinar to ductal metaplasia markers (ALS). Each lineage was characterized by cells in two different cell states determined by the differential activation of MEK signaling (M+/M-) and high cellular plasticity. These states were confirmed in multiple cohorts, cell lines, PDX models and harmonized with bulk profile analyses. Master regulators (MRs) of GLS and MOS state were predictive of patient’s survival in bulk profiles. Cross-state plasticity was confirmed by lineage tracing assays, while pooled CRISPR/Cas9 assays confirmed the essentiality of identified MR proteins. Finally, mechanistic MR-mediated cell state control was confirmed by MR expression-mediated reprogramming of MOS cells to a GLS state. Our work provided a mechanistic model of pancreatic cancer heterogeneity and testable hypothesis to target cell state-specific pancreatic cancer dependencies. Citation Format: Pasquale Laise, Mikko Turunen, Hans Carlo Maurer, Alvaro Curiel Garcia, Ela Elyada, Bernhard Schmierer, Lorenzo Tomassoni, Jeremy Worley, Mariano J. Alvarez, Jordan Kesner, Xiangtian Tan, Somnath Tagore, Ester Calvo Fernandez, Kelly Wong, Alexander L. E. Wang, Sabrina Ge, Alina C. Iuga, Aaron T. Griffin, Winston Wong, Gulam A. Manji, Faiyaz Notta, David A. Tuveson, Kenneth P. P. Olive, Andrea Califano. Pancreatic cancer comprises co-existing transcriptional states regulated by distinct master regulator programs [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A007.
Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an extremely heterogeneous tumor microenvironment (TME) paired with a sparse and compromised vascular network. These features of the TME shape drug responses and contribute to PDAC’s distinctive chemotherapeutic resistance. Prior work demonstrated that inhibition of the Hedgehog (Hh) signaling pathway using Smoothened (Smo) inhibitors led to increased angiogenesis. However, the precise mechanisms through which the Hh pathway regulates angiogenesis has remained elusive and subsequent studies with different agents and timepoints yielded conflicting data. Frustratingly, the phenotype could not be recreated in simple co-culture models. In order to maintain the cellular heterogeneity and spatial organization that characterizes PDAC, we developed a novel tumor explant system for short-term cultures of both murine and human PDAC. Briefly, we cultured intact 300 μm slices of PDAC tissue on a modified gelatin platform system with a novel culture medium informed by the metabolic composition of PDAC tumor interstitial fluid. This system replicates some of the nutrient/waste gradient of PDAC and enables tumors to be cultured up to one week maintaining the representation of multiple cell types. Studies in explants were complemented by work in the Kras-LSL.G12D/+, p53-LSL.R172H/+, Pdx1-Cre (KPC) genetically engineered model of PDAC. Comparison of short versus long-term Smo inhibition in KPC mouse pancreatic tumors revealed a robust increase in vessel density in short-term treatment that is lost at later timepoints. Studies in murine and human PDAC explants employing different Smo inhibitors demonstrated increased endothelial tip cell formation upon two days of treatment, resulting in increased vessel density after four days. Computational analyses of human PDAC single cell RNAseq data identified candidate cellular subsets potentially involved in the signaling cascade. RNAseq of pre- and on-treatment tumor samples from KPC mice identified Wif1 as a candidate Hh pathway target gene that effected these programs. Mechanistic studies in tumor explants reveal a complex, multi-step mechanism involving several cell types that led to suppression of angiogenesis in PDAC. These studies both provide a candidate mechanism for Hh-mediated angiosuppression and also demonstrated how the tumor explant platform can elucidate complex intercellular signaling cascades in both murine and human PDAC. Citation Format: Marie C. Hasselluhn, Amanda R. Decker, Alvaro Curiel Garcia, Carlo Maurer, Dafydd H. Thomas, Kenneth P. Olive. Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3648.
Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an extremely heterogeneous tumor microenvironment (TME) paired with a sparse and compromised vascular network. These features of the TME shape drug responses and contributes to PDAC’s distinctive chemotherapeutic resistance. Prior work demonstrated that inhibition of the Hedgehog (Hh) signaling using Smoothened (Smo) inhibitors relieved some of the local angiogenic suppression. However, the precise mechanisms through which the Hh pathway regulates angiogenesis has remained elusive. Subsequent studies evaluating different timepoints yielded conflicting data on the role of Hedgehog signaling in angiogenesis. In order to replicate the heterogeneity and spatial patterning of cells in pancreatic tumors, we developed a novel tumor explant system for short-term cultures of both murine and human PDAC. Briefly, we cultured intact 300 μm slices of PDAC tissues in a modified gelatin platform system with a novel culture medium informed by the metabolic composition of PDAC tumor interstitial fluid. This system replicates some of the nutrient/waste gradient of PDAC and enables tumors to be culture up to one week with good representation of multiple cell types. Studies in explants were complemented by work in the KPC genetically engineered model of PDAC. Comparison of short versus long-term Smo inhibition in KPC mouse pancreatic tumors revealed a robust increase in vessel density in short-term treatment, which is lost at later timepoints. Studies in murine and human PDAC explants employing different Smo inhibitors demonstrated increased endothelial tip cell formation upon two days of treatment, resulting in increased vessel density after four days. Computational analyses of human PDAC single cell RNAseq data identified candidate cellular subsets potentially involved in the signaling cascade. RNAseq of pre- and on-treatment tumor samples from KPC mice identified Wif1 as a candidate Hh target gene that effected these programs. Mechanistic studies in tumor explants reveal a complex, multi-step mechanism involving multiple cell types that led to suppression of angiogenesis in PDAC. These studies demonstrate how the tumor explant platform can elucidate complex intercellular signaling cascades and provide a candidate mechanism for the Hh-mediated suppression of angiogenesis in PDAC. Citation Format: Marie C. Hasselluhn, Amanda R. Decker, Alvaro Curiel Garcia, Carlo Maurer, Dafydd Thomas, Kenneth P. Olive. Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-014.
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