Berlin Institute of Health

Multiple myeloma is one of the most common forms of cancer of the immune cells in the bone marrow. It is considered incurable. Even when patients respond to treatment at first, the cancer comes back. To be able to intervene faster and on a more targeted basis, researchers completed a comprehensive study of this disease at the molecular level. Multiple myeloma is one of the most common forms of cancer of the immune cells in the bone marrow. It is considered incurable. Even when patients respond to treatment at first, the cancer comes back. To be able to intervene faster and on a more targeted basis, researchers at Charité -- Universitätsmedizin Berlin, the Berlin Institute of Health at Charité (BIH), and the Max Delbrück Center teamed up with other partners for a comprehensive study of this disease at the molecular level. The team now describes how highly aggressive types of tumors can be detected early on in an article published in the journal Nature Cancer.* They show how changes in genetic material affect the protein pro the tumor cells, and thus the mechanisms involved in the disease. Multiple myeloma is a form of cancer in which the immune cells in the bone marrow, known as plasma cells, mutate and become cancerous. Plasma cells are responsible for producing antibodies. All humans have many different kinds of plasma cells that form large numbers of different antibodies. This allows the body to recognize and fight various pathogens. In multiple myeloma, a single plasma cell mutates into a tumor cell. That cell reproduces unchecked, forming a monoclonal cell population. This means many cells are formed, all of them exactly the same and genetically identical at first. The mutated cells often also produce large volumes of antibodies or fragments of them -- but they do not function properly. Over the course of the disease, most patients develop tumors at various locations in the bone marrow, hence the "multiple" in the disease's name. Immunodeficiency, kidney failure, bone loss, and bone fractures are just some of the consequences of this uncontrolled cell growth. Despite advances in treatment and the introduction of new gene and cell therapies, there is no cure for multiple myeloma at present. With this issue in mind, a team of researchers led by Jan Krönke from the Department of Hematology, Oncology and Cancer Immunology at Charité and Dr. Philipp Mertins, head of the Proteomics technology platform of the Max Delbrück Center and BIH, set out in search of new approaches to diagnosis and treatment. What path does the tumor take? No two cases of cancer are alike, and multiple myeloma is no exception. Tumors develop differently in different individuals, including at different rates. This makes it more difficult to predict how the disease will progress and choose the optimum treatment. While the mutated plasma cells do not spread much in some cases, in others they are extremely aggressive, leading to a poor prognosis. But what causes so much divergence in the course of multiple myeloma? In cooperation with protein analysis experts from the Max Delbrück Center and BIH, the researchers conducted a detailed study of genetic and molecular changes occurring in the tumor cells in a group of more than one hundred patients. The study included data from patients in the German Multiple Myeloma Study Group (DSMM), which is coordinated by the University Hospital of Würzburg. This allowed the researchers to include clinical data on patients who had received standardized treatment over a period of eight years or more following initial diagnosis. Systems medicine and big data While changes in the genome and their effects on the proteome are already well described for other types of cancer, this is the first detailed proteo-genomic study of multiple myeloma. "Genetic data alone is insufficient to explain the mechanisms involved in this disease," Mertins says. "We wanted to know the consequences of genetic changes at the protein level and compare this molecular biology data against the actual course of the disease in patients." The team was supported in collecting and analyzing the large volumes of data by experts at Charité, BIH, and the German Cancer Consortium (DKTK). Cutting-edge mass spectrometry methods made it possible to map the protein pro mutated plasma cells and compare it against that of healthy plasma cells in people without the disease. The researchers found that both genetic changes and changes in signaling pathways lead to uncontrolled activation of cancer cells. Regulatory processes at the protein level had the stronger influence. The researchers identified a protein constellation that suggests the disease will take a particularly aggressive course, regardless of other known risk factors. Unlocking new therapies "Our findings will help subcategorize patients more effectively going forward, personalizing their treatment," Krönke concludes. "We've identified key proteins and signaling pathways that can serve as the basis for even more effective, better tolerated treatments for multiple myeloma, for example for immune therapies such as CAR T-cell therapy." In further steps, the researchers plan to study which of the target structures they have identified are in fact good candidates for new therapeutic approaches. The study is a crucial resource for research and applied development, says Dr. Evelyn Ramberger, first author of the study: "To make the complex data set manageable, we programmed an interactive, freely available online tool." This has given cancer researchers easy access to the results, so they can use the information to develop new therapies and tests to help guide treatment. For example, it may be possible to treat patients with an especially aggressive form of multiple myeloma with more intensive therapies right at the outset. Mass spectrometry Mass spectrometry is a technique for analyzing the mass of molecules and atoms. The substance to be analyzed is ionized and converted to a gas phase. The ions formed are sharply accelerated using an electric field and then sorted by mass-to-charge ratio in the mass spectrometer's analytical unit. The mass spectrum of a substance provides information on its molecular composition. Mass spectrometry is therefore useful for identifying, characterizing, and quantifying a large number of biomolecules, such as proteins, metabolites, sugars, and fats, which may behave differently depending on the disease and the individual organism.

Collaborative work has developed a cell culture system that differentiates human pluripotent stem cells to amniotic ectoderm and surface ectoderm based on cell density. Research conducted by Dr Shota Nakanoh in a collaboration between Dr Teresa Rayon's lab at the Institute and Professor Ludovic Vallier's lab at the Wellcome-MRC Cambridge Stem Cell Institute has determined that the degree of cell crowding in the early human embryo influences whether cells develop as extra-embryonic cells or become a part of the embryo and eventually give rise to skin, hair and nails. The research is published in the current issue of Science Advances. Combining the new culture method with three-dimensional culture and single cell RNA sequencing analysis, Dr Nakanoh, the lead researcher on the project, used human pluripotent stem cells (hPSC) to learn more about how amniotic and surface ectoderms arise in human embryos at around gastrulation (around two weeks after fertilisation). The mystery of similar but different tissues -- amniotic and surface ectoderms Amniotic ectoderm is a single cell layer that forms the membrane surrounding the developing embryo. It also provides essential signals for human embryo development, however how the amniotic membrane comes about is still not fully known in humans. Surface ectoderm is a dense sheet of epidermal progenitors, which gives rise to the surface covering of the body, such as skin, and the associated features of teeth, hair and fingernails. Its artificial production is of medical interest if technologies to generate it could be improved. Amniotic and surface ectoderm cells are specialised at an early stage of development and largely share biological features, such as gene expression patterns and signal requirements. It has been a mystery why these distinct tissues are so similar and how they differentiate during development. Cell crowding affects cell identity Dr Nakanoh customised culture conditions for hPSCs and found that the supplements Activin A, BMP4, a GSK3-beta inhibitor, and a MEK inhibitor promoted their differentiation into amniotic ectoderm. Using a co-culture technique to form cellular aggregates, he was able to confirm the ability of the generated amniotic ectoderm cells to initiate gastrulation. Also, single cell RNA sequencing analysis suggested the differentiation pathway for amniotic ectoderm, where surface ectoderm genes are expressed before the activation of the amniotic ectoderm gene programme. Comparison of the gene expression data against public data from primate embryos at comparable developmental stages, performed with the expertise of Dr Irina Mohorianu, Head of Bioinformatics at the CSCI, validated the cell types generated in the culture system, demonstrating their similarity to the corresponding embryonic tissues. Moreover, the developmental pathway through surface ectoderm state to amniotic ectoderm state was also observed in primate embryos. Vitally, Dr Nakanoh identified that the distinction between the two cell types depends on cell density; where cells become amniotic ectoderm in sparse conditions. In contrast, high density culturing led to cells that expressed markers for surface ectoderm but not amniotic ectoderm. These cells were also able to differentiate into a downstream epidermal cell type, keratinocytes, which represent a key cell type in the skin. The researchers tested variations in the culture media but cell density was the only factor that influenced the cell fate choice between amniotic ectoderm and surface ectoderm. The research team also found that the new culture system produces cells like extraembryonic mesoderm, which is not yet well characterised in human embryos. Dr Shota Nakanoh, a postdoctoral researcher in the Rayon lab, said: "Amnion protects the embryo and provides key developmental cues while surface ectoderm contributes to a substantial part of adult body. Both cell types are of high clinical interest and are important elements for successful in vitro models for human embryos. Although there are protocols to differentiate cells to either amniotic or surface ectoderms, researchers were not able to make a clear distinction between these cell types. Our findings about cellular density as a key regulator fills this knowledge gap and thus facilitate us to be more certain about generating the cell types of interest." Applying this cell crowding effect back to the developing human embryo, the researchers propose how these similar tissues are differentiated in the pre-gastrulation embryo, where amniotic ectoderm arises as a loose sheet with relatively small numbers of cells, while surface ectoderm is formed as a continuous dense sheet of cells. Enabling better stem cell embryo models A method to correctly specify amnion formation is important to be able to recapitulate human embryo development as closely as possible using stem cell-based embryo models. These models are increasingly being used to explore human development beyond the technical and regulatory limits that govern human embryo research. Dr Teresa Rayon, tenure-track group leader in the Institute's Epigenetics research programme, said: "We have only recently begun to explore the generation of amnion during human development. These findings advance our understanding on how to generate extra-embryonic cells in the lab in vitro, and sheds light on the mechanisms that drive the formation of cell types at the stages that correspond to the 'black box' of human development. Given the growing interest in using stem cell embryo models as proxies of human embryos, this work provides more knowledge for the generation of successful integrated models." Prof. Vallier, now Professor of Stem Cells in Regenerative Therapies at the Berlin Institute of Health at Charité (BIH), said: "Our culture system also generates extra-embryonic mesoderm, another tissue not studied well in human embryos. It will provide better understanding of human development and could improve our knowledge about diseases affecting first step of foetal life. This work also opens the door for new studies regarding the role of cellular density in cell fate decision."

New findings reveal that the body undergoes significant, systematic changes across multiple organs during prolonged periods of fasting. The results demonstrate evidence of health benefits beyond weight loss, but also show that any potentially health-altering changes appear to occur only after three days without food. New findings reveal that the body undergoes significant, systematic changes across multiple organs during prolonged periods of fasting. The results demonstrate evidence of health benefits beyond weight loss, but also show that any potentially health-altering changes appear to occur only after three days without food. The study, published today in Nature Metabolism, advances our understanding of what's happening across the body after prolonged periods without food. By identifying the potential health benefits from fasting and their underlying molecular basis, researchers from Queen Mary University of London's Precision Healthcare University Research Institute (PHURI) and the Norwegian School of Sports Sciences provide a road map for future research that could lead to therapeutic interventions -- including for people that may benefit from fasting but cannot undergo prolonged fasting or fasting-mimicking, such as ketogenic, diets. Over millennia, humans have developed the ability to survive without food for prolonged periods of time. Fasting is practiced by millions of people throughout the world for different medical and cultural purposes, including health benefits and weight loss. Since ancient times, it has been used to treat diseases such as epilepsy and rheumatoid arthritis. During fasting, the body changes its source and type of energy, switching from consumed calories to using its own fat stores. However, beyond this change in fuel sources, little is known about how the body responds to prolonged periods without food and any health impacts -- beneficial or adverse -- this may have. New techniques allowing researchers to measure thousands of proteins circulating in our blood provide the opportunity to systematically study molecular adaptions to fasting in humans in great detail. Researchers followed 12 healthy volunteers taking part in a seven-day water-only fast. The volunteers were monitored closely on a daily basis to record changes in the levels of around 3,000 proteins in their blood before, during, and after the fast. By identifying which proteins are involved in the body's response, the researchers could then predict potential health outcomes of prolonged fasting by integrating genetic information from large-scale studies. As expected, the researchers observed the body switching energy sources -- from glucose to fat stored in the body -- within the first two or three days of fasting. The volunteers lost an average of 5.7 kg of both fat mass and lean mass. After three days of eating after fasting, the weight stayed off -- the loss of lean was almost completely reversed, but the fat mass stayed off. For the first time, the researchers observed the body undergoing distinct changes in protein levels after about three days of fasting -- indicating a whole-body response to complete calorie restriction. Overall, one in three of the proteins measured changed significantly during fasting across all major organs. These changes were consistent across the volunteers, but there were signatures distinctive to fasting that went beyond weight loss, such as changes in proteins that make up the supportive structure for neurons in the brain. Claudia Langenberg, Director of Queen Mary's Precision Health University Research Institute (PHURI), said: "For the first time, we're able to see what's happening on a molecular level across the body when we fast. Fasting, when done safely, is an effective weight loss intervention. Popular diets that incorporate fasting -- such as intermittent fasting -- claim to have health benefits beyond weight loss. Our results provide evidence for the health benefits of fasting beyond weight loss, but these were only visible after three days of total caloric restriction -- later than we previously thought." Maik Pietzner, Health Data Chair of PHURI and co-lead of the Computational Medicine Group at Berlin Institute of Health at Charité, said: "Our findings have provided a basis for some age-old knowledge as to why fasting is used for certain conditions. While fasting may be beneficial for treating some conditions, often times, fasting won't be an option to patients suffering from ill health. We hope that these findings can provide information about why fasting is beneficial in certain cases, which can then be used to develop treatments that patients are able to do."