Novel Approach for Pancreas Transcriptomics Reveals the Cellular Landscape in Homeostasis and Acute Pancreatitis.

BACKGROUND & AIMS: Acinar cells produce digestive enzymes that impede transcriptomic characterization of the exocrine pancreas. Thus, single-cell RNA-sequencing studies of the pancreas underrepresent acinar cells relative to histological expectations, and a robust approach to capture pancreatic cell responses in disease states is needed. We sought to innovate a method that overcomes these challenges to accelerate study of the pancreas in health and disease. METHODS: We leverage FixNCut, a single-cell RNA-sequencing approach in which tissue is reversibly fixed with dithiobis(succinimidyl propionate) before dissociation and single-cell preparation. We apply FixNCut to an established mouse model of acute pancreatitis, validate findings using GeoMx whole transcriptome atlas profiling, and integrate our data with prior studies to compare our method in both mouse and human pancreas datasets. RESULTS: FixNCut achieves unprecedented definition of challenging pancreatic cells, including acinar and immune populations in homeostasis and acute pancreatitis, and identifies changes in all major cell types during injury and recovery. We define the acinar transcriptome during homeostasis and acinar-to-ductal metaplasia and establish a unique gene set to measure deviation from normal acinar identity. We characterize pancreatic immune cells, and analysis of T-cell subsets reveals a polarization of the homeostatic pancreas toward type-2 immunity. We report immune responses during acute pancreatitis and recovery, including early neutrophil infiltration, expansion of dendritic cell subsets, and a substantial shift in the transcriptome of macrophages due to both resident macrophage activation and monocyte infiltration. CONCLUSIONS: FixNCut preserves pancreatic transcriptomes to uncover novel cell states during homeostasis and following pancreatitis, establishing a broadly applicable approach and reference atlas for study of pancreas biology and disease.

More than acinar identity? A novel cystic phenotype suggests broader roles for NR5A2 in pancreatic cancer†.

The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains dismal. Multiple genome-wide association studies (GWAS) have implicated the nuclear receptor NR5A2 in modulating PDAC risk, but mechanisms for this association are not understood. NR5A2 is a transcription factor that maintains acinar cell identity, and heterozygous loss of Nr5a2 in mice accelerates oncogenic Kras-driven formation of pancreatic intraepithelial neoplasia (PanIN), a PDAC precursor derived from acinar cells. In a recent issue of The Journal of Pathology, Cobo et al characterize a novel mouse model that uses Ptf1a:Cre to drive oncogenic Kras as well as heterozygous Nr5a2 inactivation. In addition to the expected PanIN lesions, these mice exhibited a surprising phenotype: large pancreatic cystic lesions which have not been previously reported. Comparing expression of oncogenic Kras and heterozygous Nr5a2 in various mouse models reveals several possible explanations for these cystic lesions. Importantly, these differences across mouse models suggest that NR5A2 may contribute to PDAC precursors in ways beyond its previously characterized acinar cell-autonomous role. These observations highlight that pathways implicated by GWAS may have roles in unexpected cell types, and an understanding of these roles will be critical to guide new preventive and treatment strategies for PDAC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chronic metabolic stress drives developmental programs and loss of tissue functions in non-transformed liver that mirror tumor states and stratify survival.

Under chronic stress, cells must balance competing demands between cellular survival and tissue function. In metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD/NASH), hepatocytes cooperate with structural and immune cells to perform crucial metabolic, synthetic, and detoxification functions despite nutrient imbalances. While prior work has emphasized stress-induced drivers of cell death, the dynamic adaptations of surviving cells and their functional repercussions remain unclear. Namely, we do not know which pathways and programs define cellular responses, what regulatory factors mediate (mal)adaptations, and how this aberrant activity connects to tissue-scale dysfunction and long-term disease outcomes. Here, by applying longitudinal single-cell multi -omics to a mouse model of chronic metabolic stress and extending to human cohorts, we show that stress drives survival-linked tradeoffs and metabolic rewiring, manifesting as shifts towards development-associated states in non-transformed hepatocytes with accompanying decreases in their professional functionality. Diet-induced adaptations occur significantly prior to tumorigenesis but parallel tumorigenesis-induced phenotypes and predict worsened human cancer survival. Through the development of a multi -omic computational gene regulatory inference framework and human in vitro and mouse in vivo genetic perturbations, we validate transcriptional (RELB, SOX4) and metabolic (HMGCS2) mediators that co-regulate and couple the balance between developmental state and hepatocyte functional identity programming. Our work defines cellular features of liver adaptation to chronic stress as well as their links to long-term disease outcomes and cancer hallmarks, unifying diverse axes of cellular dysfunction around core causal mechanisms.

CaMad2 Promotes Multiple Aspects of Genome Stability Beyond Its Direct Function in Chromosome Segregation.

Mad2 is a central component of the spindle assembly checkpoint required for accurate chromosome segregation. Additionally, in some organisms, Mad2 has roles in preventing mutations and recombination through the DNA damage response. In the fungal pathogen Candida albicans, CaMad2 has previously been shown to be required for accurate chromosome segregation, survival in high levels of hydrogen peroxide, and virulence in a mouse model of infection. In this work, we showed that CaMad2 promotes genome stability through its well-characterized role in promoting accurate chromosome segregation and through reducing smaller scale chromosome changes due to recombination and DNA damage repair. Deletion of MAD2 decreased cell growth, increased marker loss rates, increased sensitivity to microtubule-destabilizing drugs, and increased sensitivity to DNA damage inducing treatments. CaMad2-GFP localized to dots, consistent with a role in kinetochore binding, and to the nuclear periphery, consistent with an additional role in DNA damage. Furthermore, deletion of MAD2 increases growth on fluconazole, and fluconazole treatment elevates whole chromosome loss rates in the mad2∆/∆ strain, suggesting that CaMad2 may be important for preventing fluconazole resistance via aneuploidy.

Mitotic DNA Synthesis Is Differentially Regulated between Cancer and Noncancerous Cells.

Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress. This unusual form of DNA synthesis occurs in the absence of RAD51 or BRCA2, which led to the identification of RAD52 as a key player in this process. Notably, mitotic DNA synthesis is predominantly observed at chromosome loci that colocalize with FANCD2 foci. However, the role of this protein in mitotic DNA synthesis remains largely unknown. In this study, we investigated the role of FANCD2 and its interplay with RAD52 in mitotic DNA synthesis using aphidicolin as a universal inducer of this process. After examining eight human cell lines, we provide evidence for FANCD2 rather than RAD52 as a fundamental supporter of mitotic DNA synthesis. In cancer cell lines, FANCD2 exerts this role independently of RAD52. Surprisingly, RAD52 is dispensable for mitotic DNA synthesis in noncancerous cell lines, but these cells strongly depend on FANCD2 for this process. Therefore, RAD52 functions selectively in cancer cells as a secondary regulator in addition to FANCD2 to facilitate mitotic DNA synthesis. As an alternative to aphidicolin, we found partial inhibition of origin licensing as an effective way to induce mitotic DNA synthesis preferentially in cancer cells. Importantly, cancer cells still perform mitotic DNA synthesis by dual regulation of FANCD2 and RAD52 under such conditions. IMPLICATIONS: These key differences in mitotic DNA synthesis between cancer and noncancerous cells advance our understanding of this mechanism and can be exploited for cancer therapies.