Molecular maps lead the way to better medicine
By Bill Snyder
Researchers at Vanderbilt University and Vanderbilt University Medical Center are creating high-resolution, single-cell “atlases” of human tissues that promise to lead to more precise ways to prevent and treat everything from diabetes-related blindness to colorectal cancer.
Human cell atlases are complete and multi-dimensional catalogs that encompass information on cell location, function, gene expression, and more. But they’re not mere lists of biological components. They’re more akin to the interactive maps that can call up an address with a tap in a cellphone application.
Thanks to cutting-edge tissue-mapping technologies, researchers can generate images of specific cells, proteins, lipid molecules, and genetic material that shimmer across tissues and organs like constellations of stars in the night sky.
While cellular mapping projects are underway around the world, Vanderbilt scientists are uniquely positioned to make their mark in the field. They have pioneered technologies such as imaging mass spectrometry and multiplex immunofluorescence that can bring individual proteins and other biomolecules into sharp focus.
VU and VUMC also have a long tradition of collaboration and cross-fertilization between the basic and clinical sciences. Biochemists and cell biologists routinely team up with specialists in cancer, digestive diseases, diabetes, lung disease, and blindness. Out of that synergy have come some astonishing discoveries.
Major mapping projects currently underway at Vanderbilt include:
- Creating atlases of the kidney, pancreas, and eye
- Tracking lung development and revealing why COVID-19 is much worse in people with chronic lung disease
- Searching for clues to the chronic intestinal inflammatory disorder known as Crohn’s disease
- Identifying the key first steps to colorectal cancer
The Human Biomolecular Atlas Program
One of the key technologies that enables human cell mapping at Vanderbilt is imaging mass spectrometry.
Developed in the late 1990s by Richard Caprioli, the Stanford Moore Professor of Biochemistry and director of the Mass Spectrometry Research Center, IMS is essentially a “molecular microscope” that can visualize the distribution, spatial rearrangement, and alterations in expression levels of proteins, lipids, and other biomolecules in cells and tissues.
In 2015, Caprioli and his colleagues achieved the first “image fusion” of mass spectrometry and microscopy—a technological tour de force that revealed the molecular makeup of tissues in high resolution.
Three years later they received a four-year, $5.5-million grant from the Common Fund of the National Institutes of Health to establish the Vanderbilt Biomolecular Multimodal Imaging Center, or BIOMIC—one of 18 tissue-mapping centers in the NIH Human Biomolecular Atlas Program consortium.
Jeffrey Spraggins, assistant professor of cell and developmental biology, and Caprioli are the principal investigators of BIOMIC, which initially was charged with creating an atlas of the human kidney. Its mission has since been expanded with an additional $4.4 million in HuBMAP consortium funding to include atlases of the pancreas and eye.
“It’s early days, but I’m quite excited about it,” said Ken Lau, an associate professor of cell and developmental biology who is a member of the BIOMIC team and two other Vanderbilt mapping projects.
By applying computational and systems biology tools to single-cell analysis, Lau investigates interacting networks of cells, metabolites, and gene expression patterns. “We now can look at human tissues with a different lens, a finer resolution,” he said.
Another team member, Bennett Landman, develops applications for integrating image processing, artificial intelligence, and informatics to combine data across scales from microscopic tissue studies with RNA and protein expression data to whole-body imaging.
He develops three-dimensional averages of anatomical images from hundreds of clinical computerized tomography and magnetic resonance scans that the entire HuBMAP consortium is using as a common framework to help with atlas construction.
Landman is a professor of electrical and computer engineering and started the Center for Computational Imaging in the Vanderbilt University Institute of Imaging Science. He now chairs the Department of Electrical and Computer Engineering.
“The ability to generate and integrate imaging data across broad spatial and molecular scales is what makes BIOMIC so unique,” Spraggins said.
Leading efforts to create atlases of the pancreas and eye, respectively, are Dr. Alvin Powers, the Joe C. Davis Professor of Medical Science and director of the Vanderbilt Diabetes Research Center, and Kevin Schey, professor of biochemistry and chemistry.
Caprioli noted that a multidisciplinary approach is crucial for meeting such a complex challenge. “It’s an absolutely great melding together of clinicians and basic scientists that is done so well at Vanderbilt,” he said.
Other HuBMAP centers are mapping the heart, bone, spleen, lymph nodes, intestines, urinary tract, and the female reproductive system. The hope is that atlases of normal, healthy tissues will lead the way to understanding how abnormalities—diseases—occur.
One such disease is diabetes, the leading cause of end-stage renal disease and a leading cause of blindness. High blood glucose levels, the hallmark of type 1 diabetes, can damage the glomeruli, the kidney’s filtration units, as well as the retina in the back of the eye.
HuBMAP’s rationale is that to decipher the molecular basis for disease, researchers first must know what each cell type normally does in its tissue environment. Only then can we fully appreciate changes that, for example, block insulin production by pancreatic beta cells and that consequently damage the eye and kidney.
Ultimately this knowledge may lead to treatments precisely tailored to individual patients—the ultimate “precision medicine,” Spraggins said.
Single-cell RNA-sequencing, or scRNA-seq, is one of the most important tools for building human cell atlases. By measuring RNA, a marker of the expression of specific genes, the technique can distinguish between different populations of cells.
Using scRNA-seq, Dr. Jennifer Sucre and her colleagues earlier this year reported the creation of a single-cell atlas that tracked the lineages of major cell types during the later stages of lung development.
Dr. Jonathan Kropski and colleagues who participate in the international Human Cell Atlas Lung Biological Network are using scRNA-seq to study the molecular mechanisms and gene regulatory architecture underlying lung diseases like idiopathic pulmonary fibrosis.
Pulmonary fibrosis is a condition in which the lungs become progressively scarred over time. Idiopathic pulmonary fibrosis, a subset of the disease with no known cause, kills up to 40,000 Americans each year.
Sucre, an assistant professor of pediatrics, and Kropski, an assistant professor of medicine, both have secondary appointments in the Department of Cell and Developmental Biology.
Last year, Sucre and Kropski teamed up with Dr. Bryce Schuler, a resident physician in pediatrics and genetics at VUMC, to deter-mine why SARS-CoV-2, the virus that causes COVID-19, causes such severe illness in older patients and those with chronic lung diseases.
Using the scRNA-seq technology, they discovered that older patients have increased expression of a cellular enzyme called TMPRSS2. The enzyme enables SARS-CoV-2 to enter certain lung cells, where it hijacks their genetic machinery to copy itself.
In patients with chronic lung diseases, the researchers also found an increased expression of inflammatory and immuneresponse genes, which may increase the risk for severe reactions to COVID-19.
Although vaccination against SARS-CoV-2 has proven to lessen the severity of or even prevent COVID-19 illness in most people, it may not protect patients whose immune systems are suppressed, such as those undergoing cancer treatment.
In this vulnerable population, targeting TMPRSS2 and turning down the inflammatory response potentially could help avoid severe illness and adverse outcomes from COVID-19, the researchers concluded.
Fire in the gut
Inflammation also plays a prominent role in Crohn’s disease, a debilitating chronic disorder of the upper and lower intestines. The challenge to improving treatment is that the disease is highly variable and presents with a wide range of phenotypes and clinical characteristics, including patient history, symptoms, and response to medications.
Thus the need for the Gut Cell Atlas: an initiative of the New York City-based Leona M. and Harry B. Helmsley Charitable Trust.
VUMC is among several institutions participating in the Gut Cell Atlas, which is part of a larger international effort called the Human Cell Atlas. In turn, the Human Cell Atlas is collaborating with the NIH HuBMAP consortium to map all of the cells in the human body.
The Vanderbilt project, led by Dr. Keith Wilson, the Thomas F. Frist Sr. Professor of Medicine, and Dr. Lori Coburn, assistant professor of medicine, is funded for more than $3.6 million and applies scRNA-seq and bioinformatics analyses to cells isolated from biopsies and surgical resections obtained from patients seen in the Vanderbilt Inflammatory Bowel Disease Center.
By comparing samples from patients at different stages of disease, as well as from healthy controls, the researchers hope to identify key cell types and patterns of gene and protein expression that will enable them to correlate inflammation at the cellular level with clinical presentations of the disease.
“Each patient is different—that’s what makes it difficult,” Coburn said. “Determining what’s going on at the cellular level will aid the search for new therapies and help physicians choose treatments that are most likely to work in their patients.”
Gut Cell Atlas team members include Ken Lau and Bennett Landman, as well as Gregor Neuert, assistant professor of molecular physiology and biophysics and pharmacology, and Qi Liu, associate professor of biostatistics and biomedical informatics.
Liu and her colleagues have developed techniques for analyzing the scRNA-seq data, while Neuert is an expert in RNA-FISH, a method for detecting and localizing RNA in cells. When combined with multiplex immunofluorescence, which allows up to 40 proteins to be examined on a single tissue section, this technology can quantify RNA and protein expression at the single-cell level.
One of Vanderbilt’s most ambitious mapping projects, funded by a five-year, $11-million Cancer Moonshot Grant, is to build a single-cell-resolution atlas that tracks the routes that polyps in the colon take to progress to colorectal cancer, the second leading cause of cancer death in the United States.
Leading the Colon Molecular Atlas Project are Dr. Robert Coffey, Ingram Professor of Cancer Research and director of the Epithelial Biology Center at VUMC; Martha Shrubsole, research professor of medicine in the Division of Epidemiology; and Ken Lau. Qi Liu is also contributing to this project.
Vanderbilt, one of five institutions to be designated as a Pre-Cancer Atlas Research Center (and grant recipient) by the National Cancer Institute in 2018, has been a powerhouse of colorectal cancer research for decades. In 2002 it gained NCI recognition as a Gastrointestinal Specialized Program of Research Excellence.
Coffey and his colleagues in the Epithelial Biology Center have pioneered single-cell technologies such as multiplex immunofluorescence.
Complementing the protein analysis, Lau’s lab uses a microfluidic approach—handling liquids in channels that are less than a sixteenth of an inch wide—to isolate thousands of cells encapsulated in an oil-liquid emulsion and analyze their gene expression patterns.
“We know that within a tumor there is a highly heterogeneous ecosystem,” Lau said. “You have tumor cells, you have immune cells, you have microbes—all of them coexisting.” Single-cell technologies are revealing how “behavior of the ecosystem” may drive the development of cancer, he said.
Most colorectal cancers arise from adenomas, a type of polyp or small clump of cells that forms in the epithelium, the lining of the colon.
Neoplastic adenomas, those that progress to cancer, are characterized by loss of the tumor suppressor gene APC, which normally keeps a tight rein on the Wnt signaling pathway that regulates cell growth and proliferation in the epithelium. Without the APC “brake,” Wnt signaling accelerates in an out-of-control manner.
About a third of colorectal cancers arise from another type of polyp, called a sessile serrated lesion or SSL, which is characterized by its flattened (sessile) appearance. Surprisingly, these lesions, at least in their early stages, don’t exhibit a loss of APC or over-activation of the Wnt pathway.
Rather, the Vanderbilt researchers have found SSLs in a setting of metaplasia, a precancerous transformation of the epithelium that is suggestive of inflammation and potentially a microbial insult. In much the same way, infection by the bacterium Helicobacter pylori triggers metaplastic changes that lead to stomach cancer.
“It’s a very new perspective on the origin of a not-uncommon type of colon cancer,” Coffey said. “To me that is fascinating and offers perhaps new diagnostic and therapeutic opportunities.”
“We will have a couple more years of work to go,” Shrubsole added, “but we’re very hopeful that we’ll have some potential targets that can then move onto the next phase of testing.”
Over at the Gut Cell Atlas, researchers are collecting as many tissue samples from patients with Crohn’s disease as they can. The deeper they get into their investigations, the more questions about the biology of the disease arise.
“This is a really hard project,” Wilson said. Given the disruptions caused by the COVID-19 pandemic, “it’s pretty miraculous that we are where we are.”
Other contributors to Vanderbilt’s single-cell atlas projects
Biomolecular Multimodal Imaging Center
- Dr. Mark de Caestecker, professor of medicine in the Division of Nephrology and professor of cell and developmental biology
- Dr. Agnes Fogo, the John L. Shapiro Professor of Pathology and director of the Division of Renal Pathology in the Department of Pathology, Microbiology and Immunology
- Dr. Raymond Harris Jr., the Ann and Roscoe R. Robinson Professor of Nephrology in the Department of Medicine and professor of molecular physiology and biophysics
- From the Mass Spectrometry Research Center: Danielle Gutierrez, research assistant professor of biochemistry, Heath Patterson, research instructor in biochemistry, Elizabeth Neumann and Angela Kruse, postdoctoral fellows, and David Anderson, staff scientist
- From outside of Vanderbilt: former postdoctoral fellow Raf Van de Plas, now at Delft University of Technology in the Netherlands (Kidney Atlas), and Christine Curcio at the University of Alabama at Birmingham (Eye Atlas)
Gut Cell Atlas and Colon MAP
- Dr. Kay Washington, professor of pathology, microbiology and immunology
- Yu Shyr, the Harold L. Moses Professor of Cancer Research and chair of the Department of Biostatistics
- Dr. Timothy Geiger, associate professor of surgery (Colon MAP)
- Dr. Reid Ness, associate professor of medicine (Colon MAP)
- From outside of Vanderbilt: Dr. Cindy Sears, Johns Hopkins University School of Medicine (Colon MAP)