ABOVE: Histological image of pancreatic cancer, with cancerous cells in red National Cancer Institute / Fox Chase Cancer Center
Researchers have long known that there is a link between pancreatic injury and cancer, but the underlying mechanisms behind that connection have proven elusive. Now, a study published October 22 in Gastroenterology reveals key mutations that explain how the very cells that aid in healing can give rise to cancer, finally shedding light on a process of tumor formation and progression that’s long left researchers confused.
The discovery is likely “going to be of lasting importance to the field,” says University of Utah geneticist and pancreatic cancer researcher Charles Murtaugh, who didn’t work on the study.
The pancreas plays a key role in digesting food and regulating blood sugar. When the pancreas is inflamed, either from physical injuries or inflammatory diseases like pancreatitis, the organ’s secretory acinar cells lose their defining characteristics and revert to a stem cell-like state via a process called acinar-to-ductal metaplasia (ADM). From there, the cells can redifferentiate to take on new forms to promote healing. Typically, this process goes off without a hitch—but sometimes, the newly-formed cells promote cancer growth, and the specific pathways of that process have remained unknown.
To gain insight into this mystery, a research team led by Vanderbilt School of Medicine cell biologist Kathy DelGiorno tagged the acinar cells of mice bred to be cancer-prone with a fluorescent protein and then induced pancreatitis for either two or four weeks straight. The researchers removed the pancreases at least one month after they finished inducing pancreatitis and performed RNA sequencing on individual fluorescent cells. “We took an agnostic approach where we labeled the pancreas, injured it, and then did single cell sequencing to see what we could capture,” DelGiorno tells The Scientist. This not only identified all of the cell types the acinar cells transitioned into, it also allowed the researchers to infer the trajectory that cells followed from one intermediate form to the next.
The team confirmed the presence of each cell type identified through both electron microscopy and immunostaining, and then compared their findings to existing data sets and tissue samples as an extra layer of validation. That revealed that a surprising diversity of cells—including transient intermediates that had never been spotted in this context—are formed in the aftermath of pancreatic injury.
“This study represents a tremendous amount of work,” cancer researcher and Pancreatic Cancer Action Network’s Associate Director of Scientific Communications Allison Rosenzweig, who didn’t work on the study, writes in an email to The Scientist. “Utilizing leading-edge technology to conduct single-cell DNA sequencing of more than 13,000 cells is a tour de force,” she adds, saying that the findings offer a “deeper understanding” of how acinar cells within the pancreas transition into tumor precursors.
Prior to this study, the possible pathways to cancer were almost endless. “One possible explanation had been that things are just so deranged that you’re getting random differentiation happening,” says Murtaugh. “Their results suggest that it’s not a random process.”
Instead, as the former acinar cells redifferentiate, they may pick up a mutation in the oncogene KRAS that’s known to be a one of the major drivers of pancreatic cancer. In order to determine how the mutation interacts with the redifferentiating cells, the researchers triggered the activating mutation specifically in metaplastic acinar cells after they’d already damaged the pancreas. Only with the KRAS mutation did tumor precursors arise, confirming that KRAS activity in metaplastic cells is an important driver of oncogenesis. Further in vitro experiments revealed this mutation turns off the cells’ healing, anti-inflammatory effects, allowing tumor progenitors to form. It does this by causing the cells to enter developmentally unstable hybrid cell states (DUHCS), which promote a constant state of inflammation that leads to tumorigenesis, study coauthor and Salk Institute for Biological Studies professor Geoffrey Wahl explains.
This “is a tough experiment to do,” Murtaugh tells The Scientist of the team’s demonstration that KRAS mutations specifically in metaplastic cells formed tumor precursors. “Nobody’s ever shown that before.”
Strangely enough, the team also found that when acinar cells reach an intermediate form in the metaplasia process, they can take an alternate path. Whether or not they had the KRAS mutation, the cells sometimes became enteroendocrine cells—a type of cell typically found elsewhere in the gastrointestinal tract that secretes hormones to aid digestion and nutrition intake—making their emergence in the pancreas all the more surprising. “You’d never think that you’d find these cells here because they’re so well-defined, structurally,” says DelGiorno, adding that the pancreas has “completely different cell types than you’d find in the gut.”
“We don’t know exactly what those cells are doing there,” says study coauthor Zhibo Ma, a postdoc in Wahl’s lab.
In addition to secreting hormones, enteroendocrine cells have a sensory role, Ma explains, and he suspects the cells play a role in detecting injury and regulating the injury response. “But that’s just speculation,” he adds. This paper only scratched the surface of what functions these metaplastic and transient cell networks perform, Ma says, so “very interesting direction moving forward would be understanding how these cells are contributing [to] or regulating the disease expression.”
The authors say they suspect that the same ADM process occurs in humans. Indeed, they compared their findings to screens of tissue samples from human pancreatitis patients and identified many of the same cell types they’d found in mice, indicating that a similar metaplasia pathway takes place.
Pancreatic cancer is particularly deadly. The American Cancer Society estimates that the disease will kill more than 48,000 Americans in 2021, and only ten percent of patients live to see the fifth anniversary of their diagnosis. The challenge, DelGiorno explains, is that diseases like pancreatitis and pancreatic cancer don’t cause noticeable symptoms until they’ve progressed into later, untreatable stages. Some of the symptoms that do occur, like the onset of diabetes and weight gain, are ambiguous and make diagnosis difficult, Wahl says.
“Pancreatitis is a really debilitating disease,” Murtaugh says. “If one knew what these [enteroendocrine] cells are doing and whether they’re enhancing or inhibiting its activity, you might be able to get pancreatitis to resolve or at least be less intense.”
Furthermore, DelGiorno points out that if scientists manage to identify the specific hormone signals that these cells secrete and determine which are important to tumorigenesis, they may be able to develop a diagnostic tool that identifies pancreatitis or pancreatic cancer when it’s still early enough for treatment or removal. “We have cell types that are forming in really early-stage disease and we have some indication of what changes in early and late-stage disease because of this study,” she says, suggesting that the work could lead to a blood or urine test that screens for early-stage pancreatic cancer.
It might also be possible, Machado writes in her email, to develop drugs for patients with pancreatic injury that target the metaplasia pathway to prevent pancreatic diseases from taking hold in the first place.
That, Wahl says, is the goal at the forefront of his work to understand cancer. “Wouldn’t it be great to stop cancer,” he adds, “to stop these cells at the very beginning?”