Experimental microdissection enables functional harmonisation of pancreatic cancer subtypes

© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ. Objective Pancreatic ductal adenocarcinoma (PDA) has among the highest stromal fractions of any cancer and this has complicated attempts at expression-based molecular classification. The goal of this work is to profile purified samples of human PDA epithelium and stroma and examine their respective contributions to gene expression in bulk PDA samples. Design We used laser capture microdissection (LCM) and RNA sequencing to profile the expression of 60 matched pairs of human PDA malignant epithelium and stroma samples. We then used these data to train a computational model that allowed us to infer tissue composition and generate virtual compartment-specific expression profiles from bulk gene expression cohorts. Results Our analysis found significant variation in the tissue composition of pancreatic tumours from different public cohorts. Computational removal of stromal gene expression resulted in the reclassification of some tumours, reconciling functional differences between different cohorts. Furthermore, we established a novel classification signature from a total of 110 purified human PDA stroma samples, finding two groups that differ in the extracellular matrix-associated and immune-associated processes. Lastly, a systematic evaluation of cross-compartment subtypes spanning four patient cohorts indicated partial dependence between epithelial and stromal molecular subtypes. Conclusion Our findings add clarity to the nature and number of molecular subtypes in PDA, expand our understanding of global transcriptional programmes in the stroma and harmonise the results of molecular subtyping efforts across independent cohorts

Abstract A007: Pancreatic cancer comprises co-existing transcriptional states regulated by distinct master regulator programs

Abstract 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.</jats:p

Erratum: \u3b22 Adrenergic-Neurotrophin Feedforward Loop Promotes Pancreatic Cancer (Cancer Cell (2018) 33(1) (75\u201390.e7), (S153561081730510X) (10.1016/j.ccell.2017.11.007))

(Cancer Cell 33, 75\u201390.e1\u2013e7; January 8, 2018) In the originally published version of this article, the representative image of pancreatic H&E slides from LSL-Kras+/G12D;Pdx1-Cre (KC) mice at 20 weeks (Figure 1D) and the representative image of pancreatic H&E slides from cerulein-injected KC mice at 20 weeks on PLX-7486-containing diet (Cer PLX) (Figure 5E, right) were the same image. This happened because, during the first revision of our paper, we inadvertently labeled Figure 1D incorrectly and put the same image in both panels. We have gone back and reviewed pathology, and the images are consistent with what we described. We provide here a revised Figure 5, which includes a correct representative image of pancreatic H&E slides from cerulein-injected KC mice at 20 weeks on PLX-7486-containing diet in Figure 5E. We also identified two additional errors in Figures 3B and 3C. In the second bar from the right, respectively, Isoproterenol and Propanolol treatment was indicated, which is incorrect. These bars depict sphere number and sphere diameter of Propranolol only treated LSL-Kras+/G12D pancreatic spheres treated with an Adeno-Cre virus after 7 days in culture, respectively. Both Figure 3 and Figure 5 have now been corrected here and in the online version of the paper. The authors apologize for any confusion these errors may have caused. [Figure presented

\uce\ub22 Adrenergic-Neurotrophin Feedforward Loop Promotes Pancreatic Cancer

Catecholamines stimulate epithelial proliferation, but the role of sympathetic nerve signaling in pancreatic ductal adenocarcinoma (PDAC) is poorly understood. Catecholamines promoted ADRB2-dependent PDAC development, nerve growth factor (NGF) secretion, and pancreatic nerve density. Pancreatic Ngf overexpression accelerated tumor development in LSL-Kras+/G12D;Pdx1-Cre (KC) mice. ADRB2 blockade together with gemcitabine reduced NGF expression and nerve density, and increased survival of LSL-Kras+/G12D;LSL-Trp53+/R172H;Pdx1-Cre (KPC) mice. Therapy with a Trk inhibitor together with gemcitabine also increased survival of KPC mice. Analysis of PDAC patient cohorts revealed a correlation between brain-derived neurotrophic factor (BDNF) expression, nerve density, and increased survival of patients on nonselective \uce\ub2-blockers. These findings suggest that catecholamines drive a feedforward loop, whereby upregulation of neurotrophins increases sympathetic innervation and local norepinephrine accumulation. Renz et al. show that catecholamines promote ADRB2-dependent pancreatic ductal adenocarcinoma development and secretion of neurotrophins (NT), which in turn promote tumor innervation leading to increased NE and tumor growth. Blockade of ADRB2 or NT receptors improves gemcitabine's therapeutic effect