The glycan CA19-9 promotes pancreatitis and pancreatic cancer in mice.

We designed a Dox pulse/chase approach to determine if CA19–9 expression is required to maintain pancreatitis. CA19–9-mediated pancreatitis was completely reversed in C;R LSL ;F mice following a 3-day Dox pulse and 4-day recovery period ( fig. S22 , A to C ). Partial resolution of chronic pancreatitis in the C;R LSL ;F model (28-day Dox treatment) was also observed after a 14-day recovery period ( fig. S22 , D and E ). In a preventive setting of acute pancreatitis, two antibodies directed against CA19–9 both reduced immune infiltration, ductal metaplasia, and fibrosis in vivo ( Fig. 5A , and fig. S22 , F and G ). In addition, decreased release of amylase and lipase into the circulation, and reduced hyperactivation of EGFR was observed in vivo ( Fig. 5 , B and C , and fig. S22 , H and I ). In an intervention setting of existing acute pancreatitis, we found that two forms of 5B1 significantly reduced secretion of amylase into the circulation with modest normalization of the pancreatic histology ( fig. S22 , J to L ). CA19–9 antibody treatment of existing acute pancreatitis also reduced the levels of phosphorylated EGFR in both the ductal and acinar compartments and decreased recruitment of macrophages ( fig. S22 , M and N ). These data suggest that CA19–9 plays a role in disease pathogenesis and maintenance and that CA19–9 targeted therapy may warrant further therapeutic exploration. Interestingly, inhibition of EGFR in vivo using Erlotinib was not as effective as CA19–9 antibody blockade to mitigate pancreatitis induction in mice ( fig. S23 , A to F ). Indeed, Erlotinib treatment of CA19–9 expressing mice caused severe weight loss, necessitating euthanasia. These effects were unrelated to Erlotinib toxicity in control mice. Although serum levels of amylase and lipase decreased in Erlotinib-treated animals, pancreatic atrophy and acinar cell vacuolization was observed, suggesting that the weight loss was due to increased pancreatitis severity and resulting exocrine insufficiency ( fig. S23 , B and C ). Erlotinib treatment also incompletely blocked phospho-EGFR levels in vivo ( fig. S22D ). Acinar-ductal metaplasia (ADM) can be detected by Sox9 IHC ( 36 ) and occurred after 7 days of Dox treatment in the C;R LSL ;F model ( fig. S22E ). Although ADM was less apparent in the Erlotinib treated C;R LSL ;F mice ( fig. S22F ), lymphocyte infiltration and fibrosis remained unaffected. CA19–9 sequestration is unlikely to interfere with ADM survival mechanisms in the acinar compartment given that these cells are largely CA19–9 negative ( 46 ). Therefore, EGFR kinase inhibition alone cannot substitute for CA19–9 blockade, and may be a harmful therapy in the setting of pancreatitis.

CA19–9 is expressed at low levels in the normal ducts of the pancreas, but becomes elevated in benign reactive, metaplastic, and malignant ducts in humans. It is conceivable that the degree of CA19–9 elevation could impart a way to control the degree of fibroinflammatory response in both pancreatitis and PDAC. Interestingly, mice retain the ability to respond to elevations in CA19–9 despite lacking the ability to express this glycan. The ability of mice to respond to CA19–9 elevation may be due to the existence of similar regulatory mechanisms for related Lewis antigens (e.g. sialyl Lewis x) that are involved in similar processes in other organs and have also been shown to substitute for CA19–9 in individuals lacking this glycan ( 49 ). Patients with pancreatitis have an elevated risk of 2.7 to 16.5-fold for developing PDAC while individuals with hereditary pancreatitis have a 40–55% lifetime risk of developing pancreatic cancer ( 1 , 5 ). Therapeutic options for pancreatitis patients are currently focused on treating the symptoms and little can be done to facilitate the resolution of idiopathic pancreatitis or to prevent its recurrence, highlighting the pressing need for new treatments. Prophylactic intervention could also be beneficial in the setting of recurrent or hereditary pancreatitis, and following certain routine procedures for which pancreatitis is a common outcome. For example, 3.5% (1–16% range) of the >700,000 patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) each year in the United States will develop pancreatitis ( 50 , 51 ) and several risk factors can be used to identify patients with elevated risk for ERCP-associated pancreatitis (40%) ( 52 ). Fully human CA19–9 antibodies have passed phase 1A clinical trials PET-imaging of pancreatic cancer ( 53 ), facilitating rapid translation of CA19–9 targeted therapy to the clinic for the treatment of pancreatitis patients. Therefore, not only would a new treatment strategy for pancreatitis itself serve an unmet need, there is also an intriguing possibility that effective pancreatitis treatment could also lead to prevention approaches for PDAC. Furthermore, such an approach may also reduce the severity of PDAC due to the fibroinflammatory and EGFR-activating properties of CA19–9 modified proteins, including Fibulin 3.

To determine if CA19–9 expression promoted PDAC, we intercrossed the C;R LSL ;F alleles with the conditional Kras LSL-G12D allele (K;C;R LSL ;F) ( fig. S24A ) ( 47 , 48 ). CA19–9 expression significantly accelerated pancreatic cancer lethality relative to untreated mice and control littermates ( Fig. 6A ). When treated with Dox, K;C;R LSL ;F mice rapidly succumbed to primary and metastatic pancreatic cancer with a median survival of 202 days relative to 460 days in the K;C control cohort and 420 days in the untreated K;C;R LSL ;F cohort. The primary tumors were anaplastic with glandular features ( Fig. 6B ). Widespread metastases were observed in the peritoneum, diaphragm, liver, and lung in multiple Dox treated K;C;R LSL ;F mice. To better understand the role of CA19–9 in PDAC initiation, we examined the effect of short-term CA19–9 expression on pancreatic transformation. Whereas littermate controls exhibited the expected low burden of mPanIN-1A lesions, CA19–9 expressing animals harbored a high penetrance of cystic and fibroinflammatory disease with abundant mPanIN-1B and occasional mPanIN-2 lesions after 2 weeks of Dox ( Fig. 6C ) and increased macrophage infiltration relative to the K;C control cohort ( fig. S24 , B and C ). After 4 weeks of Dox, cystic papillary neoplasia and invasive carcinoma could be detected ( Fig. 6C ). CA19–9 expression was elevated in normal, benign reactive, and metaplastic ducts as well as in mPanIN and PDAC lesions in K;C;R LSL ;F mice, similar to the human expression pattern ( Fig. 6D and fig. S12 ). Equivalent levels CA19–9 were also observed in human PDA and mouse K;C;R LSL ;F organoids ( fig. S24D ). Phosphorylated EGFR ( Fig. 6E ) was detected at high levels in Dox-treated K;C;R LSL ;F mice whereas CA19–9 negative K;C mice exhibited low to negative levels of phospho-EGFR.

Pancreatitis, or inflammation of the pancreas, is a painful, recurrent and occasionally lethal medical disorder with limited treatment options. Pancreatitis is a common disease, with 33.74 acute and 9.62 chronic pancreatitis cases per 100,000 people worldwide ( 1 ). Pancreatitis accounts for more than 275,000 hospitalizations in the United States per year and the number of hospital admissions has increased by 20% over the past decade ( 2 ). The causes of pancreatitis include blockage of the pancreatic duct by gallstones, alcohol and certain drugs that cause acinar cell damage, medical procedures or trauma that damage pancreatic tissue, and autoimmune diseases ( 2 ). In approximately one third of cases, the underlying etiology of the pancreatitis is unknown (idiopathic) ( 3 , 4 ). Most acute pancreatitis cases will resolve with supportive care, however up to 20% of patients will develop severe tissue damage and will either succumb to multi-organ system failure or suffer from bouts of recurrent disease with markedly diminished quality of life ( 2 – 4 ). Individuals with hereditary acute pancreatitis progress to chronic pancreatitis with a much higher penetrance and furthermore have a 40 to 55% lifetime risk of developing pancreatic cancer ( 1 , 5 ). Indeed, chronic pancreatitis promotes mutant Kras-mediated development of pancreatic cancer in mice ( 6 ). The glycan CA19–9 is found in the serum of 10–30% of pancreatitis patients, 75% of pancreatic cancer patients, as well as in patients with other gastrointestinal diseases ( 7 – 16 ). CA19–9 elevation is also detected in Pancreatic Intra-epithelial Neoplasms (PanINs), which are precursors to pancreatic ductal adenocarcinoma (PDAC) ( 17 ). CA19–9 (sialyl-Lewis a , sLe a ) is generated by the stepwise addition of sugar moieties to Type 1 precursor chains present on proteins and other molecules, culminating in the α1,4 linkage of fucose to N-Acetylglucosamine (GlcNAc) ( fig. S1A ). The FUT3 fucosyltransferase is the only enzyme with the ability to add fucose moieties through an α1,4 linkage and generate CA19–9. Mice lack this enzyme because Fut3 is a pseudogene in rodents ( 18 , 19 ). To facilitate the discovery of PDAC biomarker candidates, we sought to create a mouse model of PDAC that recapitulated the elevation of CA19–9 observed in human patients. This model would enable prioritization of biomarkers that outperform CA19–9. Furthermore, changes to glycosylation often result in functional consequences. Here, we investigate the role of CA19–9 elevation in mouse and organoid models of pancreatic disease.

To express CA19–9 in mouse cells, we transduced mouse PDAC cells with human FUT3 . However, expression of FUT3 alone was insufficient for CA19–9 production, but did lead to increased levels of Lewis x antigens following removal of terminal galactose moieties present in rodents, but not humans ( Fig. 1A ). The generation of the related Lewis x epitopes suggested that reprogramming of the precursor substrates would be necessary for the production of CA19–9 in pancreatic ductal cells. β3GALT5 is required for the production of Type I chain precursors ( 20 ), which serve as the precursors for the Lewis a modification ( fig. S1A ). Accordingly, expression of both FUT3 and β3GALT5 in mouse PDAC cells led to the cell surface expression of CA19–9 at levels equivalent to those observed in human cancer cell lines (Colo205, Suit2) ( Fig. 1B and fig. S1B ). Comparable CA19–9 levels were observed in the blood of mice following orthotopic transplantation of the CA19–9 expressing mouse and human cells ( fig. S1C ). To determine whether the murine PDAC cell proteins harboring CA19–9 modification are similar to those in human PDAC cells, CA19–9 protein carriers were immunoprecipitated (IP) and identified by mass spectrometry (MS) ( fig. S2A and table S1 ). These analyses identified known CA19–9 protein carriers in the FUT3 and β3GALT5 expressing mouse cells (n=3, FC1199, FC1242, FC1245), including CD44, Lgals3bp, Muc1and Muc5ac ( 21 – 24 ). The human PDAC cell line, MiaPaCa-2, is CA19–9 negative and therefore we used it as a control to identify human CA19–9 core proteins in the CA19–9 positive cell lines, Capan2, Suit2, and hM1–2D ( fig. S2B and table S2 ). We compared mouse and human CA19–9 protein carriers and found that an average of 72.3% (95% CI 60.3–84.3%, n=3) of the CA19–9 modified proteins identified in all three human PDAC cell lines were also found in the engineered murine PDAC cell lines. Thus, expression of the human FUT3 and β3GALT5 genes in mouse cells largely recapitulates the human CA19–9 carrier profile ( Fig. 1C ).