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Cancers of the biliary tract system affect the network of ducts that connect the liver, gallbladder, and small intestine, and are aggressive, killing 9 out of 10 patients within just a few years of diagnosis. New research from the ӳ����ý and Massachusetts General Hospital has uncovered new subtypes of the rare cancer and possible molecular targets for new treatments, and generated much needed cell models that can drive more research.

In a new study published in , researchers generated 30 new cell lines derived from cells from patients with various biliary tract cancers (BTCs), nearly doubling the number available for study. The team conducted a multi-omic analysis of the entire set of more than 60 cell lines, providing the deepest look yet at the molecular and functional diversity of BTCs. The team has integrated their BTC cell atlas into the ӳ����ý’s , which has identified genetic vulnerabilities in more than 1,000 cancer cell lines. The scientists have made their data and cell lines available to other researchers to spur more research.

“In this study, by integrating new cell lines, expression and functional data, and validation in clinical specimens, we were able to define meaningful cell state differences, paving the way to new treatment approaches that could one day benefit more patients,” said co-senior author and ӳ����ý associate member Nabeel Bardeesy, who is also John R. Gallagher III and Katherine A. Gallagher Endowed Chair in Gastrointestinal Cancer Research at MGH and professor of medicine at Harvard Medical School.

Bardeesy led the work along with co-first authors Vindhya Vijay and Negin Karisani and co-senior authors Francisca Vazquez, director of DepMap, and Gad Getz, a ӳ����ý core institute member, professor of pathology at Harvard Medical School, and faculty member and director of bioinformatics in the Department of Pathology and Krantz Family Center for Cancer Research at MGH, where he is also the Paul C. Zamecnik Chair in Oncology.

An understudied cancer

BTCs include intrahepatic and extrahepatic cholangiocarcinoma (bile duct cancer within and outside of the liver), gallbladder carcinoma, and ampullary carcinoma. Together, these cancers are rare, comprising only 3 percent of all gastrointestinal cancers, but cases increase each year. Even among tumors from the same anatomical site, BTC cells can carry a variety of unique genetic mutations and features, making this a highly diverse type of cancer. Yet most patients receive the same chemotherapy and immunotherapy treatment with limited benefit, regardless of where in the network of bile ducts their cancer occurs or which molecular changes it carries.

Bardeesy and his collaborators knew they needed more cell models of BTCs to better study this cancer in the lab. They worked with surgeons and clinicians for years to generate the new patient-derived BTC cell lines. They also gathered cell models produced by other researchers across the globe, bringing the number in their collection to 63. The researchers performed transcriptomic, proteomic, DNA sequencing, and copy number analyses of the cell lines as well as genome-wide CRISPR screening. These data were integrated into the ӳ����ý’s DepMap, which comprises such “multi-omics” profiling of more than 1,000 cell lines from other cancers.

The team used the data to define “dependencies” — pathways that the tumor cells need to survive — that could be targeted by new and better drugs in specific subtypes of BTC.

“We knew that to make our cell collection more impactful, we needed to work with DepMap to make sense of all the data and generate a coherent picture that might lead to new treatment hypotheses,” said Bardeesy.

A patient partnership

Their work revealed marked differences among BTC cell lines based on molecular characteristics, gene dependencies, and drug sensitivity profiles. Within cholangiocarcinomas, they identified two clear subtypes each with their own dependencies and transcriptional patterns: one “ductal” type with similarities to bile duct cells, and one “bi-lineage” type that has features of both duct cells and liver cells and is associated with better prognosis in patients. These lineage states are governed by master transcription factors, such as HNF1B in bi-lineage cells and GRHL2 and KLF5 in ductal cells. The subtypes help explain the heterogeneity of biliary tract cancer, in addition to helping shed light on why some patients fare better than others on different therapies.

Their analysis uncovered dependencies unique to each subtype that could one day be targets for new treatments. They also found that most biliary tract cancers broadly rely on the EGFR cell signaling pathway, raising the possibility of treating patients with combination therapy that targets both the EGFR pathway and a mutation-related dependency to thwart drug resistance.

In future work, the researchers plan to use single-cell RNA sequencing and spatial transcriptomic methods to further evaluate these lineage-based subtypes in human patient samples and learn how the dependencies promote survival of BTC cells. They are also exploring how AI-based methods might be able to classify a patient’s cells without molecular testing.

Bardeesy credits for its support of this research. The foundation was created by a patient with bile duct cancer, Paul Poth, in 2009 shortly before he died of the disease. He wanted to help fund research into rare cancers, including efforts to generate new cell lines for studying BTC.

“With generous support from TargetCancer, we’ve created an invaluable resource for the research community that may one day help improve the lives of patients with this aggressive illness,” said Bardeesy.

"After 15 years of partnership, I am tremendously proud and grateful to see Dr. Bardeesy’s groundbreaking work and dedication to cholangiocarcinoma research reach this pivotal point," said Kristen Palma, President of TargetCancer Foundation. "My late husband Paul Poth founded TargetCancer Foundation with the idea that cell lines were the key to understanding this rare cancer. We are truly honored to have provided funding that led to the development of the cell line atlas — a critical step in bringing life-saving treatment options to patients while honoring Paul’s legacy."

Bardeesy also expressed gratitude to the Cholangiocarcinoma Foundation for their generous and sustained support through multiple research fellowships and the C2T2 Think Tank initiative, which helped advance key aspects of this work.