March 4, 2026 | The vast number and diversity of chemicals that exist in the natural world represent both the cause of and solution to all the world’s problems, according to Amit Rai, Ph.D., assistant professor in the department of crop sciences at the University of Illinois College of Agricultural, Consumer and Environmental Sciences. As such, they are foundational, high-stakes agents irrespective of the biological system—be they comprised of plants, microorganisms, or human beings.
Plant biologists are particularly well suited to the study of human metabolomics because the human body harbors somewhere between 5,000 and 10,000 chemicals while plants produce upwards of 100,000 to 1 million different metabolites (Plant Physiology, DOI: 10.1093/plphys/kiaf408), “so it’s like nothing,” he says. The relative ease of the chemical discovery exercise is how he became a key player in the quest to identify the distinct metabolic signatures in gallbladder cancer patients with, and without, accompanying gallstone disease (Journal of Proteome Research, DOI: 10.1021/acs.jproteome.5c00403).
Gallstones is a major risk factor for gallbladder cancer but is also a highly prevalent condition worldwide that rarely turns cancerous. This has created a dilemma for doctors who invariably see these cancers in advanced stages when they may no longer be treatable, Rai says. Gallbladder cancer currently does not have any sort of standard, routine screening test for the general population.
Treating physicians have long sought reliable diagnostic biomarkers appearing post-gallstones that would give them an early alert when patients are at heightened cancer risk, so they get regular checkups with a specialist and proactively manage their gallbladder health by reducing obesity, quitting tobacco, and eating a diet rich in fruits and vegetables. But detection of these biomarkers needs to be approached with caution, since they are known biological indicators of multiple cancer types, Rai says.
“We have not discovered one biomarker that has never been linked to cancer before,” he points out. “But we were able to detect the very specific ones linking to the gallbladder stone converting into cancer.”
Before coming to the University of Illinois, Rai was working as a research scientist within the Metabolomics Research Group at the RIKEN Center for Sustainable Resource Science in Japan. His focus was on discovering the pharmacological properties of chemicals produced by medicinal plants that could potentially be useful for human medicines, as well as the chemicals shaping the rhizosphere community around plant roots.
His lab is also creating a “social network of chemicals” of plants, ranked based on their similarity to one another. “We want to use chemicals to discover new chemicals,” says Rai, noting that this new resource is expected to be published sometime in the next few months.
In 2019, he met a visiting researcher from India, Sheelendra P. Singh from the CSIR-Indian Institute of Toxicology Research (CSIR-IITR), and they became fast friends. Five years later, when Rai relocated to the U.S., they began talking about the difficulty in distinguishing the different gallbladder cancer types and how they might collaboratively decipher the chemical clues when gallstones are likely to convert into the highly deadly and aggressive cancer.
The next step was to generate the dataset, annotate it, and then analyze all those annotations, says Rai. “Chemicals are chemicals, irrespective of whether they come from crops or blood,” and the discovery work lies in examining them from an overall biochemical pathways' perspective.
Although the odds of someone with gallstones developing cancer is very low, the conversion is disproportionately common in parts of India, including Assam, he says. The availability of 30 samples from his collaborators in the region made the search for the metabolic signature possible, although many times more than that number are needed to confirm and refine the findings as well as gain a mechanistic understanding of why those biomarkers mark the progression from stones to cancer.
The research team will now try to link gallstone-associated gallbladder cancer with other cancer types, says Rai, adding that he is confident there is a common “control center” connecting several of these abnormalities. “That’s when we’ll probably be in a better position to discover the unique biomarker that is only for gallstone [-related] gallbladder cancer.”
Mass spectrometry for the initial pilot study was performed at the CSIR-IITR, since the data generation was done in India, says Rai. That required a computational metabolomics expert to collaborate with an analytical chemist to ensure that the data output was correct and would enable biological interpretation.
The first phase of the project involved annotating as many biochemicals as possible from the blood for profiling using high-resolution mass spectrometry, Rai says. But less than 1,000 of the 19,000-plus molecular fragments could be annotated. “What that shows is we are still missing so many compounds ... that might be more relevant.”
Each of those chemicals couldn’t be extracted equally because extraction methods are specific to chemical families. “It’s impossible to use all possible extraction means because of ... [limitations on] how many compounds [in a sample] you can analyze,” he says.
Considerable manual effort is required for the one-by-one “forensic discovery” of the different chemical fingerprints, points out Rai. The hundreds of chemicals that the research team could confidently identify after many months of work were sorted as being specific to one of the three phenotypes.
One missing element was an imaging mass spectrometrist who could have mapped the metabolites on patient tissue samples that were at the research team’s disposal. Rai says he is now attempting to see if he can get prepared tissue samples sent to his lab for analysis with his mass spectrometry system, called desorption electrospray ionization (DESI), for the pixel-by-pixel discovery of chemicals localized in different areas.
Exploring the Differences
Bile acid metabolism seems to be one of the core control centers linked to the biochemical pathway of gallbladder cancer, whether there is an association with gallstone disease or not. Yet, surprisingly, their metabolic signatures look quite different, says Rai.
In the gallstone-free cancer cases, researchers detected 180 altered metabolites, but in the gallstone-associated cases the number jumped to 225, with 138 shared by both. Subsequent analysis identified 12 gallbladder-cancer-specific metabolic signatures with high diagnostic efficiency, 20 for the cancer plus stones cases, and 30 shared between the two groups.
The news created a media stir in India, where the team has secured a grant permitting the larger study to proceed. It will look at different stages of gallbladder cancer, including cases with and without associated gallstones, and group patients based on their gender and age. Researchers also hope to see how genetics links with the biochemistry to learn if there is an inheritability signature, he says, provided the team in India can find a way to sequence so many samples.
As is already known, the frequency and probability of different cancers vary significantly across global population groups on earth, continues Rai, each of which carries a unique genomic signature. In addition to genomics, the forthcoming study will include proteomics to aid mechanistic understanding of how this gallbladder cancer evolves, which will be done using readily available resources in Rai’s lab.