The method, described Jan. 2 in a paper in Nature Biotechnology, records gene activity patterns and the presence of key proteins in cells across tissue samples, while retaining information about the cells’ precise locations. This enables the creation of complex, data-rich “maps” of organs, including diseased organs and tumors, which could be widely useful in basic and clinical research.

“This technology is exciting because it allows us to map the spatial organization of tissues, including cell types, cell activities and cell-to-cell interactions, as never before,” said study co-senior author Dr. Dan Landau, an associate professor of medicine in the Division of Hematology and Medical Oncology and a member of the Sandra and Edward Meyer Cancer Center at Weill Cornell Medicine and a core faculty member at the New York Genome Center.

The other co-senior author was Dr. Marlon Stoeckius of 10x Genomics, a California-based biotechnology company that makes laboratory equipment for the profiling of cells within tissue samples. The three co-first authors were Dr. Nir Ben-Chetrit, Xiang Niu, and Ariel Swett, respectively a postdoctoral researcher, graduate student, and research technician in the Landau laboratory during the study.

The new method is part of a broad effort by scientists and engineers to develop better ways of “seeing” at micro scale how organs and tissues work. Researchers in recent years have made big advances particularly in techniques for profiling gene activity and other layers of information in individual cells or small groups of cells. However, these techniques typically require the dissolution of tissues and the separation of cells from their neighbors, so that information about profiled cells’ original locations within the tissues is lost. The new method captures that spatial information as well, and at high resolution.

The method, called Spatial PrOtein and Transcriptome Sequencing (SPOTS), is based in part on existing 10x Genomics technology. It uses glass slides that are suitable for imaging tissue samples with ordinary microscope-based pathology methods, but are also coated with thousands of special probe molecules. Each of the probe molecules contains a molecular “barcode” denoting its two-dimensional position on the slide. When a thinly sliced tissue sample is placed on the slide and its cells are made permeable, the probe molecules on the slide grab adjacent cells’ messenger RNAs (mRNAs), which are essentially the transcripts of active genes. The method includes the use of designer antibodies that bind to proteins of interest in the tissue — and also bind to the special probe molecules. With swift, automated techniques, researchers can identify the captured mRNAs and selected proteins, and map them precisely to their original locations across the tissue sample. The resulting maps can be considered alone, or compared to standard pathology imaging of the sample.

The team demonstrated SPOTS on tissue from a normal mouse spleen, revealing the complex functional architecture of this organ including clusters of different cell types, their functional states, and how those states varied with the cells’ locations.

Highlighting SPOTS’ potential in cancer research, the investigators also used it to map the cellular organization of a mouse breast tumor. The resulting map depicted immune cells called macrophages in two distinct states as denoted by protein markers — one state active and tumor-fighting, the other immune-suppressive and forming a barrier to protect the tumor.

“We could see that these two macrophage subsets are found in different areas of the tumor and interact with different cells — and that difference in microenvironment is likely driving their distinct activity states,” said Dr. Landau, who is also an oncologist at NewYork-Presbyterian/Weill Cornell Medical Center.

Such details of the tumor immune environment — details that often can’t be resolved due to immune cells’ sparseness within tumors — might help explain why some patients respond to immune-boosting therapy and some don’t, and thus could inform the design of future immunotherapies, he added.

This initial version of SPOTS has a spatial resolution such that each “pixel” of the resulting dataset sums gene activity information for at least several cells. However, the researchers hope soon to narrow this resolution to single cells, while adding other layers of key cellular information, Dr. Landau said.

Many Weill Cornell Medicine physicians and scientists maintain relationships and collaborate with external organizations to foster scientific innovation and provide expert guidance. The institution makes these disclosures public to ensure transparency. For this information, see profile for Dr. Landau.

Cells programmed to produce these chains continuously add building blocks that encode particular cellular events. Later, the ordered protein chains can be labeled with fluorescent molecules and read under a microscope, allowing researchers to reconstruct the timing of the events.

This technique could help shed light on the steps that underlie processes such as memory formation, response to drug treatment, and gene expression.

“There are a lot of changes that happen at organ or body scale, over hours to weeks, which cannot be tracked over time,” says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology, a professor of biological engineering and brain and cognitive sciences at MIT, a Howard Hughes Medical Institute investigator, and a member of MIT’s McGovern Institute for Brain Research and Koch Institute for Integrative Cancer Research.

If the technique could be extended to work over longer time periods, it could also be used to study processes such as aging and disease progression, the researchers say.

Boyden is the senior author of the study, which appears today in Nature Biotechnology. Changyang Linghu, a former J. Douglas Tan Postdoctoral Fellow at the McGovern Institute, who is now an assistant professor at the University of Michigan, is the lead author of the paper.

Cellular history

Biological systems such as organs contain many different kinds of cells, all of which have distinctive functions. One way to study these functions is to image proteins, RNA, or other molecules inside the cells, which provide hints to what the cells are doing. However, most methods for doing this offer only a glimpse of a single moment in time, or don’t work well with very large populations of cells.

“Biological systems are often composed of a large number of different types of cells. For example, the human brain has 86 billion cells,” Linghu says. “To understand those kinds of biological systems, we need to observe physiological events over time in these large cell populations.”

To achieve that, the research team came up with the idea of recording cellular events as a series of protein subunits that are continuously added to a chain. To create their chains, the researchers used engineered protein subunits, not normally found in living cells, that can self-assemble into long filaments.

The researchers designed a genetically encoded system in which one of these subunits is continuously produced inside cells, while the other is generated only when a specific event occurs. Each subunit also contains a very short peptide called an epitope tag — in this case, the researchers chose tags called HA and V5. Each of these tags can bind to a different fluorescent antibody, making it easy to visualize the tags later on and determine the sequence of the protein subunits.

For this study, the researchers made production of the V5-containing subunit contingent on the activation of a gene called c-fos, which is involved in encoding new memories. HA-tagged subunits make up most of the chain, but whenever the V5 tag shows up in the chain, that means that c-fos was activated during that time.

“We’re hoping to use this kind of protein self-assembly to record activity in every single cell,” Linghu says. “It’s not only a snapshot in time, but also records past history, just like how tree rings can permanently store information over time as the wood grows.”

Recording events

In this study, the researchers first used their system to record activation of c-fos in neurons growing in a lab dish. The c-fos gene was activated by chemically induced activation of the neurons, which caused the V5 subunit to be added to the protein chain.

To explore whether this approach could work in the brains of animals, the researchers programmed brain cells of mice to generate protein chains that would reveal when the animals were exposed to a particular drug. Later, the researchers were able to detect that exposure by preserving the tissue and analyzing it with a light microscope.

The researchers designed their system to be modular, so that different epitope tags can be swapped in, or different types of cellular events can be detected, including, in principle, cell division or activation of enzymes called protein kinases, which help control many cellular pathways.

The researchers also hope to extend the recording period that they can achieve. In this study, they recorded events for several days before imaging the tissue. There is a tradeoff between the amount of time that can be recorded and the time resolution, or frequency of event recording, because the length of the protein chain is limited by the size of the cell.

“The total amount of information it could store is fixed, but we could in principle slow down or increase the speed of the growth of the chain,” Linghu says. “If we want to record for a longer time, we could slow down the synthesis so that it will reach the size of the cell within, let’s say two weeks. In that way we could record longer, but with less time resolution.”

The researchers are also working on engineering the system so that it can record multiple types of events in the same chain, by increasing the number of different subunits that can be incorporated.

The research was funded by the Hock E. Tan and K. Lisa Yang Center for Autism Research, John Doerr, the National Institutes of Health, the National Science Foundation, the U.S. Army Research Office, and the Howard Hughes Medical Institute.

Using health data gathered from 11,255 adults over a 30-year period, researchers analyzed links between serum sodium levels — which go up when fluid intake goes down — and various indicators of health. They found that adults with serum sodium levels at the higher end of a normal range were more likely to develop chronic conditions and show signs of advanced biological aging than those with serum sodium levels in the medium ranges. Adults with higher levels were also more likely to die at a younger age.

“The results suggest that proper hydration may slow down aging and prolong a disease-free life,” said Natalia Dmitrieva, Ph.D., a study author and researcher in the Laboratory of Cardiovascular Regenerative Medicine at the National Heart, Lung, and Blood Institute (NHLBI), part of NIH.

The study expands on research the scientists published in March 2022, which found links between higher ranges of normal serum sodium levels and increased risks for heart failure. Both findings came from the Atherosclerosis Risk in Communities (ARIC) study, which includes sub-studies involving thousands of Black and white adults from throughout the United States. The first ARIC sub-study started in 1987 and has helped researchers better understand risk factors for heart disease, while shaping clinical guidelines for its treatment and prevention.

For this latest analysis, researchers assessed information study participants shared during five medical visits — the first two when they were in their 50s, and the last when they were between ages 70-90. To allow for a fair comparison between how hydration correlated with health outcomes, researchers excluded adults who had high levels of serum sodium at baseline check-ins or with underlying conditions, like obesity, that could affect serum sodium levels.

They then evaluated how serum sodium levels correlated with biological aging, which was assessed through 15 health markers. This included factors, such as systolic blood pressure, cholesterol, and blood sugar, which provided insight about how well each person’s cardiovascular, respiratory, metabolic, renal, and immune system was functioning. They also adjusted for factors, like age, race, biological sex, smoking status, and hypertension.

They found that adults with higher levels of normal serum sodium — with normal ranges falling between 135-146 milliequivalents per liter (mEq/L) — were more likely to show signs of faster biological aging. This was based on indictors like metabolic and cardiovascular health, lung function, and inflammation. For example, adults with serum sodium levels above 142 mEq/L had a 10-15% associated increased odds of being biologically older than their chronological age compared to ranges between 137-142 mEq/L, while levels above 144 mEq/L correlated with a 50% increase. Likewise, levels of 144.5-146 mEq/L were associated with a 21% increased risk of premature death compared to ranges between 137-142 mEq/L.

Similarly, adults with serum sodium levels above 142 mEq/L had up to a 64% increased associated risk for developing chronic diseases like heart failure, stroke, atrial fibrillation and peripheral artery disease, as well as chronic lung disease, diabetes, and dementia. Conversely, adults with serum sodium levels between 138-140 mEq/L had the lowest risk of developing chronic disease.

The findings don’t prove a causal effect, the researchers noted. Randomized, controlled trials are necessary to determine if optimal hydration can promote healthy aging, prevent disease, and lead to a longer life. However, the associations can still inform clinical practice and guide personal health behavior.

“People whose serum sodium is 142 mEq/L or higher would benefit from evaluation of their fluid intake,” Dmitrieva said. She noted that most people can safely increase their fluid intake to meet recommended levels, which can be done with water as well as other fluids, like juices, or vegetables and fruits with a high water content. The National Academies of Medicine, for example, suggest that most women consume around 6-9 cups (1.5-2.2 liters) of fluids daily and for men, 8-12 cups (2-3 liters).

Others may need medical guidance due to underlying health conditions. “The goal is to ensure patients are taking in enough fluids, while assessing factors, like medications, that may lead to fluid loss,” said Manfred Boehm, M.D., a study author and director of the Laboratory of Cardiovascular Regenerative Medicine. “Doctors may also need to defer to a patient’s current treatment plan, such as limiting fluid intake for heart failure.”

The authors also cited research that finds about half of people worldwide don’t meet recommendations for daily total water intake, which often starts at 6 cups (1.5 liters).

“On the global level, this can have a big impact,” Dmitrieva said. “Decreased body water content is the most common factor that increases serum sodium, which is why the results suggest that staying well hydrated may slow down the aging process and prevent or delay chronic disease.”

This research was supported by the Division of Intramural Research at NHLBI. The ARIC study has been supported by research contracts from NHLBI, NIH, and the Department of Health and Human Services.