A novel single-cell RNA-sequencing method pushes the boundaries of research and clinical care in pancreatic cancer

From novel laboratory research that enables scientists to define pancreatic cancer cells better to innovative clinical approaches that help patients recover from surgery more quickly, Dana-Farber Brigham Cancer Center (DFBCC) is steadily advancing the diagnosis and management of pancreatic cancers.

Reversible Fixation Overcomes Challenges of Single-Cell Profiling

Sahar Nissim, MD, PhD, of the Brigham’s Division of Genetics and Division of Gastroenterology, Hepatology and Endoscopy is an award-winning physician-scientist. His team studies the earliest events that cause a normal pancreas cell to progress toward cancer to devise “interception,” a strategy to block those events and prevent cancer from forming altogether.

Researchers in Dr. Nissim’s laboratory leverage developmental biology, genetic discovery in hereditary pancreatic cancer families, integrative transcriptomic and epigenomic approaches, and pre-clinical experiments in mouse models and human tissue to study determinants of pancreas cell identity and cancer initiation.

In a recent study, Dr. Nissim and first co-authors Woo-Jeong Jeong and Katherine Jean Aney presented a single-cell method to define the transcriptomes of pancreas cells, providing a vital new tool for future investigation of the pancreas in health and disease. The study, A Novel Approach for Pancreas Transcriptomics Reveals the Cellular Landscape in Homeostasis and Acute Pancreatitis, was published in Gastroenterology. It marked the first-time reversible fixation was applied to single-cell analysis of the pancreas.

Single-cell RNA-sequencing studies have advanced the understanding of signaling pathways and cell diversity in many organs. However, digestive enzymes produced in acinar cells hamper such investigation of the pancreas. FixNCut, the novel method described by Dr. Nissim and his co-authors, uses reversible fixation to achieve unprecedented single-cell transcriptomic definition of challenging pancreas cells, including acinar and immune populations in homeostasis and following acute pancreatitis.

This method overcomes the endogenous digestive enzymes that have historically challenged single-cell profiling of the pancreas, Dr. Nissim explains.

“We know that certain stressors can cause pancreatic cells to evolve and become precursor lesions to pancreatic cancer,” he says. “Studying these cells and cancer precursors will give us critical insights into why cancer forms and how we might intercept that process. However, the pancreas environment is hostile to traditional single-cell assays. Moreover, a permanent fixative approach to address this challenge precludes the ability to conduct subsequent research that requires generation and understanding of the genes expressed.”

FixNCut is a single-cell RNA sequencing approach where tissue is reversibly fixed with dithiobis (succinimidyl propionate) (DSP) prior to dissociation and single-cell preparation. The approach completely shuts off RNase, DNase, and protease activity—the activity of the digestive enzymes that traditionally impede this type of work—and then allows that fixation to be reversed for subsequent analyses.

“DSP is a cross-linker that has been considered for use as a reversible fixative,” Dr. Nissim says. “However, this is the first time anyone has harnessed this reagent for single-cell analysis of pancreas cells. This method is a transformative advance in our ability to understand the pancreas at homeostasis and disease states. These insights, in turn, will inform new strategies for prevention, early detection, and treatment.”

Aney adds that while this initial FixNCut study was conducted in mouse models, a future goal is to apply the approach to human pancreatic tissue.

“Due in part to issues around access to human pancreatic tissue, the causes of pancreatic cancer—cell types and states as well as risk factors—are more easily studied in mouse models than in humans,” she says. “FixNCut gives us the ability to better understand cell types and how they evolve so that we might intercept pancreatic cancer in different contexts.”

“This approach,” Jeong adds, “may also be applied in other organs that pose challenges for single-cell RNA sequencing and other omics pipelines as well.”

SourceBrigham and Women’s Hospital

Aney KJ, Jeong WJ, Vallejo AF, Burdziak C, Chen E, Wang A, Koak P, Wise K, Jensen K, Pe’er D, Dougan SK, Martelotto L, Nissim S. (2024) Novel Approach for Pancreas Transcriptomics Reveals the Cellular Landscape in Homeostasis and Acute Pancreatitis. Gastroenterology [Epub ahead of print]. [abstract]

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