Spatial Transcriptomics of Intraductal Papillary Mucinous Neoplasms of The Pancreas Identifies NKX6-2 as a Driver of Gastric Differentiation and Indolent Biological Potential

Abstract

Intraductal Papillary Mucinous Neoplasms (IPMNs) of the pancreas are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). The most common subtype of IPMNs harbor a gastric foveolar type lining epithelium, and these low-grade mucinous neoplasms are harbingers of IPMNs with high-grade dysplasia and cancer. The molecular underpinning of gastric differentiation in IPMNs is unknown, although identifying drivers of this indolent phenotype might enable opportunities for intercepting progression to high-grade IPMN and cancer. We conducted spatial transcriptomics on a cohort of patient IPMNs, followed by orthogonal and cross species validation studies, which established the transcription factor NKX6-2 as a key determinant of gastric cell identity in low-grade IPMNs. Loss of NKX6-2 expression is a consistent feature of IPMN progression, while restitution of NKX6-2 in murine IPMN lines recapitulates the aforementioned gastric transcriptional program. Our study identifies NKX6-2 as a previously unknown transcription factor driving indolent gastric differentiation in IPMN pathogenesis.

Integrated spatial transcriptomics and lipidomics of precursor lesions of pancreatic cancer identifies enrichment of long chain sulfatide biosynthesis as an early metabolic alteration

“…Specifically, Matrix Assisted Laster Desorption/Ionization (MALDI) mass spectrometry-based imaging was performed on 23 surgically resected human IPMN and IPMN-associated PDAC cases, which led to the discovery of distinct enrichment of long-chain hydroxylated sulfatides in neoplastic epithelium. Leveraging our recent spatial transcriptomic (ST) dataset in IPMNs obtained via the 10x Genomics Visium platform 17 for serial sections derived from the same tissues evaluated via MALDI-MS imaging, we demonstrated elevated transcripts encoding for enzymes involved in sulfatide biosynthesis to co-localize with enrichment of long-chain hydroxylated sulfatides found in epithelium lining the IPMN. These findings were validated in a genetically engineered mouse model of IPMN, co-expressing mutant Kras;Gnas 18 .…”

“…Tissues from 9 additional PDAC cases without a concurrent IPMN were also analyzed. Samples were cryo-sectioned onto Visium Spatial Gene Expression slides for ST analysis, as previously published 19 . For the murine samples, pancreata were collected from p48-Cre; LSL-KrasG12D; Rosa26RLSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice) at 25 weeks on doxycycline diet.…”

“…After MALDI-MS imaging, the same and an adjacent tissue section were stained with hematoxylin and eosin (H&E) for pathological annotation. MALDI-MS imaging was performed on serial sections from a subset of the same tissue samples used for spatial transcriptomics using the Visium technology in our recently study 17 , thereby allowing us to directly compare the localization of the RNA transcripts and the sulfatides observed on the same tissue sample.…”

“…Spatial transcriptomic data (Visium 10X platform) was derived from our recent foundational publication 17 . Single-cell data integration method to match and compare (scMC) pipeline was used for initial processing and clustering of the human IPMN ST dataset; to analyze spatially-resolved RNA-seq data, we used the Seurat package (version 3.2) implemented in R statistical software (version 4.3) (https://www.r-project.org/).…”

Generative interpolation and restoration of images using deep learning for improved 3D tissue mapping

“…This resolution is further limited during the common practice of intermixing stains (hematoxylin and eosin [H&E], immunohistochemistry [IHC], spatial transcriptomics) at regular intervals. 6,[8][9][10][11][12] Second, the axial resolution of the sample is diminished due to physical artifacts of sectioning, where tissue splitting, folding, and warping can dramatically limit the user's ability to reconstruct continuous structures. 4,13,14 In contrast, intact imaging approaches such as magnetic resonance imaging (MRI), computed tomography (CT), and tissue clearing enable 3D views of continuous structures.…”