Like people, bacteria have their preferences when it comes to relationships. Some are totally independent, while others prefer company. Salmonella and many other kinds of bacteria are of the social type: They can live and even thrive inside a host cell. But unlike us, these bacteria do not spend a long time wooing the cell in the hope that it will welcome them in. Instead, they inject proteins that take control of the host cell’s systems.

In recent years, thanks in part to studies conducted by Prof. Roi Avraham’s team at the Weizmann Institute of Science, researchers have identified differences among the proteins that various bacterial subspecies inject into their hosts, which could explain why some of these subspecies are more virulent than others. For example, there are more than 2,500 subspecies of salmonella, but only a handful of them cause life-threatening disease. In a paper published in the Proceedings of the National Academy of Sciences (PNAS), scientists from Avraham’s team presented a new research method that shines fresh light on the relationship between bacteria and their host cells – and reveals what makes some bacteria particularly virulent.

Over the past decade, scientists have gained the ability to examine molecular processes at an unprecedented resolution, revolutionizing research in the life sciences. New methods of sequencing genetic material on the single-cell level have contributed to this revolution. But existing tools cannot be used to apply single-cell RNA sequencing to examine all the molecular relationships between thousands of bacterial subspecies and the no-less impressive array of hosts. To map the differences in virulence and disease-causing ability between the various subspecies of salmonella, for example, researchers need to sequence the DNA of the bacterial cells on the single-cell level, do the same for the DNA of the specific host cells that were infected and match up the findings of guest and host, which amounts to a daunting task.

“We discovered how bacteria manipulate host cells and how cells respond, which may help treat antibiotic-resistant infections”

In their study, the researchers applied their new method to 25 mutant salmonella species that infected immune system cells called macrophages. The method allowed them to study the relationship between each species of bacteria and its host cell and to identify a species that causes an exceptionally powerful immune response in the host. The species in question lacks a protein that the other bacteria express and successfully inject into the host. The researchers inferred that this protein is essential for repressing the host’s immune system. “We discovered a new role of a familiar protein and showed that it sabotages the host’s defense mechanisms,” Avraham explains. “In fact, bacteria inject many proteins into their hosts, and we still have not figured out which roles most of them play. Our method, which is already used by researchers around the world, will make it possible to continue systematically revealing these roles. Moreover, it can be applied to any kind of bacteria, including friendly bacteria that are vital to many of our body’s systems.”

Beyond advancing basic research, the new method might help develop ways of battling bacterial resistance to antibiotics, defined by the World Health Organization as one of the main threats to human health and food security. “There are two new potential defense strategies, both still in the early stages of development,” Avraham says. “One is reducing the virulence of disease-causing bacteria and the other is bolstering the immune response of the host cells. Our method makes it possible to study both: simultaneously understanding how the bacterium launches its attack and how the host cell defends itself.”

The cells that make up cancerous brain tumors are extremely varied and sometimes create unique three-dimensional shapes. As far back as 1932, American neurosurgeon Percival Bailey attempted to label these cells and discovered that they can be divided into several families of cells with similar properties. More than ninety years later, we still know precious little about the identities of cell groups that make up different kinds of brain tumors, these groups’ organization and how they affect the course of the disease and the outcome of treatment. This is why the success rate for treatment of most brain cancers is typically not high.

Over the past decade, genetic sequencing technology that works at the single-cell level has made it possible to examine, in minute detail and in one fell swoop, thousands of cells in the same tissue, to understand which genes they express and to then categorize them and study the role of each group. Scientists in Dr. Itay Tirosh’s research group in the Weizmann Institute of Science’s Molecular Cell Biology Department, in collaboration with Prof. Mario L. Suvà’s lab at Massachusetts General Hospital, harnessed this technology in order to reexamine some of the unanswered questions in the field of brain tumors.

The most common type of primary brain tumor is the glioma, which originates from the support cells that assist our nerve cells. There are two main types of glioma tumors: those that are usually less aggressive and have a mutation in the gene encoding an enzyme called IDH, and those without this mutation, which are highly aggressive and known in medical terminology as glioblastoma. In the past few years, researchers from Tirosh’s lab have been using single-cell RNA sequencing to analyze the cellular composition of both kinds of tumors. They revealed that the tumor cells are divided into groups, each of which expresses a unique genetic program that determines the biological “state” of the cancer cells in this group. Among other findings, the researchers discovered groups of cells that use their unique genetic programs to mimic normal brain cells.

In a new study published recently in Cell, researchers from Tirosh’s lab – led by Dr. Alissa Greenwald, Noam Galili Darnell and Dr. Rouven Hoefflin – harnessed technologies that make it possible to not only sequence the RNA on the single-cell level but also spatially map its expression. This allowed them, for the first time, to identify which genes are uniquely expressed in each of the thousands of areas within a brain tumor. As a result, they were able to precisely map how glioblastoma and glioma tumors are organized. To conduct the study, they took biopsies from 13 patients with glioblastomas and from six patients with gliomas that had the IDH mutation.

The driving force behind the tumor’s layered structure is the lack of oxygen, which is exacerbated as the disease progresses and the tumor develops

The researchers’ first discovery was that the groups of various cells within a glioma tumor are not distributed evenly across the tumor; rather, they are concentrated in various environments inside the growth. These microenvironments are not entirely homogenous: Cells from other groups were always found in proximity to other types of cells. In the next stage of the study, the researchers checked whether there were groups of tumor cells that usually exist in proximity to each other. They discovered that the cells not only had preferred neighbors but also that these good-neighbor couplings were consistent in different patients.

Certain neighboring pairs imitated the natural behavior of brain tissue. For example, cells that imitate the parent cells of the oligodendrocyte support cell were found close to endothelial cells, which line the walls of blood vessels. This coupling also occurs in healthy tissue, since endothelial cells release substances that are vital for the survival and proliferation of oligodendrocyte precursor cells. Similarly, cells that imitate neuron progenitor cells were found in the parts of the tumor that penetrated healthy brain tissue, just as progenitor cells in healthy tissue migrate when the tissue is regenerated.

Taking an overview to gain a fuller understanding of these couplings, the researchers realized that the cells created five distinct layers by organizing themselves into separate environments within the tumor. The innermost layer – the core of the tumor – is made up of necrotic cells, which do not receive enough oxygen to survive. In the layer surrounding the necrotic core, the researchers found cells similar to embryonic connective tissue, as well as additional cells, including immune system cells responsible for causing inflammation. The third layer was primarily made up of blood vessels, endothelial cells forming blood vessel walls and additional immune system cells.

Cells in the two outer layers of the tumor don’t suffer from a lack of oxygen. This enables groups of tumor cells that mimic healthy brain tissue – progenitors of neurons and support cells – to develop in the fourth layer. The fifth, outermost layer contains healthy brain tissue, into which the tumor penetrates. These findings about the different layers of a tumor indicate that the driving force behind the tumor’s layered structure is the lack of oxygen, which is exacerbated as the disease progresses and the tumor develops.

Based on these findings, the researchers noticed a much more chaotic structure in less aggressive tumors – which are also usually smaller – and in areas of the tumor with a plentiful supply of oxygen. In most glioma tumors with the IDH mutation, for example, there was usually no necrotic tissue, and the structure of the tumor was disorganized; in the rare cases when there was necrotic tissue, the biopsies also showed a relatively well-ordered structure.

“We discovered that an organized spatial structure is characteristic of the more aggressive tumors,” Tirosh explains. “The lack of oxygen in the tumor cells’ environment influences the gene program that they express and therefore affects their state. As the tumor grows, distinct layers are formed, some of which may be less accessible to drugs and to cells from the immune system, and these could make the tumor more resilient.”

The changing status of cancer cells

Researchers from Tirosh’s lab used the information they collected on the cellular composition of glioma tumors to work out how a new, promising drug helped some of the patients with this type of cancer. To do so, they used biopsies from tumors of three patients who had participated in a clinical trial of the new drug and who had responded to the treatment, as well as biopsies from six patients who had not undergone any treatment. To complete the picture, they also used data from biopsies taken from an additional 23 patients who had taken the drug and 134 patients who had not.

The research team, led by Dr. Avishay Spitzer, found that the drug, which works by inhibiting the mutant IDH enzyme, caused the cells to alter the gene program that they expressed. In fact the treatment encourages the cancerous stem cells to differentiate into mature cells, thereby undermining their ability to divide rapidly, blocking the disease’s progress.

The researchers postulated that if the drug works by causing cancerous cells to differentiate into mature cells, the mutation attacking the gene that is critical to the differentiation process could explain those cases in which the drug does not work. In the biopsies taken from patients who did not receive the drug, they identified a certain gene that is linked to low levels of mature cancerous cells. When they silenced that gene in a mouse model of cancer, they found, as expected, that the drug did not work. “This indicates that the gene mutation we identified could be a biological marker allowing us to determine in advance which patients will benefit from the treatment and which will not,” Tirosh explains. These new findings could also help find a course of treatment that combines IDH inhibitors with another drug that encourages the differentiation process and increases the treatment’s impact on the tumor.

“Our two most recent studies revealed the forces that shape the character of cancerous cells in a tumor, be that in their untouched environment or in one resulting from a therapy that alters the cells’ genetic program,” Tirosh says. “These findings pave the way for a new approach to cancer treatment, since once we are familiar with the cell groups that populate every area of the tumor and we know how a cell can move from one state to another, we might be able to develop new targeted treatments that will alter the course of the disease. The understanding that both the composition of cells within the tumor and its three-dimensional structure are linked to the level of the tumor’s aggressiveness could also lead to new diagnostic methods that do not rely solely on the volume of the tumor and the mutations it contains.”

Heart attacks happen when the supply of blood to part of the heart muscle is cut off. Unless this supply is renewed quickly, the heart muscle cells start to die. Unlike skeletal muscle and other tissues that can recover from injury unscarred, heart muscle cells do not divide and do not replace dead cells with a new muscle. Instead, the heart’s fibroblasts (that is, fiber cells) divide rapidly in the damaged area and create a network of protein fibers that replace the damaged cells with scar tissue. This tissue ensures the integrity of the heart but reduces its ability to contract and pump blood. In the long term, therefore, a heart attack increases the chances of heart failure, a chronic condition in which the heart is incapable of meeting all the body’s needs, initially during physical exertion and later even while at rest. Heart failure affects around 64 million people around the world.

“Because the patent for Copaxone has expired, we are finding it hard to find partners in the pharmaceutical industry for continuing this research”

Over the past decade, the immune system’s response to heart damage has been shown to be directly involved in cardiac recovery and rehabilitation. But when the inflammation triggered by this response fails to subside and becomes chronic, the damage grows even worse and can lead to heart failure. Since it was already established that Copaxone alters the composition of cells in the immune system and the proteins they release, thereby suppressing inflammation, Sarig wondered whether it would be possible to use the drug to examine how the immune system influences recovery from a heart attack.

In the new study – led by Sarig and two research students from Tzahor’s lab, physician Dr. Gal Aviel and Jacob Elkahal – researchers treated mice that had suffered heart attacks with a daily abdominal injection of Copaxone. Echocardiograms revealed that the drug improved the functioning of the damaged mouse hearts and that their heart chambers were sending more blood to the large arteries with each heartbeat, which in turn supplied more of the vital blood to other organs. The scar area in the treated mice was relatively small; moreover, large scars covering at least 30 percent of the left chamber were observed only in mice that were not treated. People who suffer heart attacks do not always go to the emergency room right away, but the researchers discovered that Copaxone was effective in mice even when treatment began 24 to 48 hours after the heart attack.

The next stage of the study was to test the treatment in a rat model, but this time the researchers started treatment almost a month after the heart attack, when the rats already had chronic heart failure. By the end of the two-month treatment, the percentage of blood that was pumped out with each heartbeat climbed by an average 30 percent, and cardiac contractility – the ability of the ventricles to contract – improved by almost 60 percent. One month after the end of the treatment, the pumping of blood continued to improve and improvements in cardiac contractility persisted. So, while seeking to answer a fundamental scientific question – the extent to which the immune system affects heart rehabilitation – the scientists discovered a promising new possibility for treating a common heart disease.

To their surprise, the team also discovered that the drug works by not only influencing the composition of the immune system cells in the damaged area of the heart but also, apparently, by directly protecting the cardiac muscle cells themselves: Copaxone was found to protect heart muscle cells in tissue cultures that contained no immune cells. At a later stage, the treatment also halted the division of the fiber cells that form scar tissue and boosted the production of new blood vessels.

“Treatment with Copaxone does not cause heart muscle cells to divide,” Sarig explains. “It helps the existing cells to survive and contract effectively, enhances the production of blood vessels that supply them and delays the creation of scar tissue.” In light of their promising laboratory results, the Weizmann scientists, along with Aviel and other clinicians, joined forces with Prof. Offer Amir and Prof. Rabea Asleh from the Hadassah Medical Center in Jerusalem to conduct a phase 2a clinical trial examining the effectiveness of subcutaneous injections of Copaxone in patients with heart failure.

The results of this trial are yet to be published, but they are expected to show a rapid improvement in markers of both inflammation and heart damage. “Because the patent for Copaxone has expired, we are finding it hard to find partners in the pharmaceutical industry for continuing this research,” Tzahor says. “Still, repurposing an existing drug for a new use is quick and inexpensive compared to developing a new drug, and I hope that some donor or organization will pick up the gauntlet.”

It follows that a balance between dividing and mature cells is a precondition for the existence of any multicellular organism, but how is it maintained? In a new study published recently in Cell, researchers from the Weizmann Institute of Science used single-celled organisms to better understand how multicellular organisms maintain this equilibrium and protect themselves from cancer.

Cell differentiation is a biological “specialization training,” in which a stem cell divides into two daughter cells, one of which assumes a defined role and acquires the characteristics needed to fulfill it. When cells undergo differentiation, their new specialty is useful to the multicellular organism of which they are a part, but they pay a heavy individual toll: The further they get along this specialization pathway, the more their ability to replicate decreases, until they are no longer able to divide at all. This slow division of differentiated cells makes them vulnerable to populations of cells that divide and grow at a faster rate and can therefore take over the tissue and its resources. In some types of blood cancer, for example, stem cells in the bone marrow undergo a mutation that slows their differentiation and allows them to produce more daughter stem cells. These mutant cells take advantage of the natural weak point in the differentiation process, overcoming the population of healthy cells in a process known as mutant takeover.

“To determine which differentiation rate works best, we held a competition between 11 strains of E. coli, each of which cuts out DNA segments – that is, differentiates – at a different rate”

Even though one mutation, on average, occurs in every cell division in our bodies, most of us enjoy decades of good health, through countless cell divisions, without experiencing mutant takeover. This suggests that there are effective mechanisms for dealing with this threat, even if they are hard to identify in complex organisms. Scientists in Prof. Uri Alon’s research group at Weizmann’s Molecular Cell Biology Department decided to engineer E. coli bacteria, which do not usually differentiate, so as to make them undergo an artificial differentiation process, allowing researchers to study how a cell population deals with mutant takeover.

“There are a number of clear advantages to the E. coli model,” explains Dr. David Glass, who led the study in Alon’s lab. “One of them is a short generation time, which allowed us to study the development of mutants over hundreds of generations in the lab.” In order to produce E. coli bacteria capable of differentiating, researchers took inspiration from cyanobacteria called Anabaena, which differentiate – by cutting out certain segments of their DNA – in response to a shortage of nitrogen in their environment. Although the differentiated bacteria lose the ability to divide, they gain an important survival edge: the ability to supply themselves and the entire colony with nitrogen.

To mimic the differentiation process in the E. coli model, the scientists grew the bacteria in an environment that included antibiotics but lacked an essential amino acid. Using genetic engineering, they inserted into each bacterium several copies of a gene for resistance to antibiotics and several copies of a gene that produced the missing amino acid. Before the process of artificial differentiation began – that is, when the bacteria were in a state equivalent to that of stem cells – the antibiotic-resistance genes were active, so the bacteria were able to divide and differentiate at a high rate despite the presence of the antibiotic. When the differentiation process started by means of cutting out the antibiotic resistance genes, the bacteria gradually lost their ability to divide and differentiate, but they gained a survival advantage: The cuts in the DNA gradually activated the genes that produced the essential amino acid.

“To determine which differentiation rate works best, we held a competition between 11 strains of E. coli, each of which cuts out DNA segments – that is, differentiates – at a different rate,” Glass explains. “We mixed equal quantities of the bacteria, grew them over the course of a few days and then checked to see which had survived. We discovered a very strong selection in favor of bacteria that differentiated at a moderate rate and found that strains of bacteria with a moderate rate of differentiation maintained the optimal balance of cell types in their population. At any given moment, only a minority of the cells were ‘pure stem cells’ or ‘fully differentiated cells,’ and a majority were found in intermediate states of the process.”

This optimal, moderate differentiation rate is shared by various systems in the human body, in which a quantitative balance is maintained among stem cells, progenitor cells at different stages of differentiation and differentiated cells that occasionally die and are replaced by new ones.

To keep the population size steady, it is important to maintain that equilibrium even when environmental conditions change. To find out whether the bacteria in their model indeed maintained this equilibrium even under changed conditions, the researchers grew them in 36 different combinations of antibiotic and amino acid concentrations in the culture medium. “We saw that in every situation – apart from the most extreme ones, such as a total absence of antibiotics – the cells’ optimal differentiation rate remained in the moderate range and the equilibrium was maintained,” Glass explains. “This means that the population equilibrium characterizing the differentiation model we developed is, to a large extent, immune to environmental changes and threats.” But is a population of bacteria that is differentiating at an optimal rate also immune to mutant takeover, like the systems in multicellular organisms?

To test the ability of these bacteria to withstand mutant takeover, the researchers grew them over many generations and checked whether random mutations appeared during the long growth period, creating bacteria that do not differentiate at all and divide uncontrollably. In other words, do mutant bacteria bring about mutant takeover, or are they suppressed at an early stage? The first time they conducted the experiment, the researchers were disappointed to find mutant takeovers in half of the cases. “We found that when a genetic change breaks the connection between differentiation slowdown and getting that survival advantage, mutants that do not differentiate can take over,” Glass adds.

“Many diseases are unique to multicellular organisms. When we genetically engineer more and more characteristics of multicellular systems in single-celled organisms, we can uncover the weak points”

Next the researchers repeated the experiment with a new bacterial strain that was genetically engineered to be immune to the identified mutation. “We managed to grow around 270 generations of differentiating bacteria, and no mutant takeover occurred. Unfortunately, the invasion of Israel on October 7 cut the experiment short, and the bacteria may well be even more resilient,” Glass says. “We showed that a system in which differentiating E. coli cells stop dividing but gain a survival advantage can maintain an optimal population balance and avert mutant takeover. Many diseases, such as cancer and autoimmune disorders, are unique to multicellular organisms. When we genetically engineer more and more characteristics of multicellular systems in single-celled organisms, we can uncover the weak points and look for them in human tissue too.”

“Beyond basic science, these new findings could also have an impact on the use of bacteria in industry,” Glass adds. “Genetically engineered bacteria are currently used in the large-scale production of insulin, enzymes and other substances used by humans. Creating a population of differentiating bacteria that maintains its equilibrium, renews itself and even prevents mutant takeover could be very useful in these production processes.”

Study participants included Dr. Anat Bren, Elizabeth Vaisbourd and Dr. Avi Mayo from Weizmann’s Molecular Cell Biology Department.