In patients with familial adenomatous polyposis, a genetic disease predisposing to colon cancer, mutations of the APC gene induce the formation of intestinal polyps, but also reduce immune system activity. In a new study, researchers from the Institut Pasteur, INSERM⁽¹⁾ and Université Paris Cité describe the mechanisms that modify the structure of T lymphocytes and hinder their migration towards the tumors to be destroyed. This discovery, published in the journal Science Advances on April 13, 2022, provides new perspectives on the migration of immune cells, a key process in antitumor immune defense.

As its name suggests, familial adenomatous polyposis is transmitted from generation to generation. The cause: mutations of the tumor suppressor gene APC (adenomatous polyposis coli). People who inherit these mutations develop hundreds, possibly thousands, of polyps in their colon from adolescence, then colorectal cancer⁽²⁾ in adulthood if the polyps are not surgically removed. “As it’s a hereditary disease, all of the body’s cells carry the mutation and can be affected in different ways”, explains Andrés Alcover, Head of the Lymphocyte Cell Biology Unit at the Institut Pasteur and joint senior author of the study. “Today we know that these mutations disrupt the functioning of colon cells but also cells of the immune system”.

In previous studies, the team of researchers from the Institut Pasteur, CNRS and Inserm – funded by the French Cancer League since 2018⁽³⁾ – demonstrated the dual impact of APC mutations.

Not only do these mutations prevent intestinal epithelial cells from differentiating correctly and cause them to form tissue growths (polyps), they also adversely affect the functioning of immune cells, thereby preventing them from effectively combating polyps and tumors. Two mechanisms that together promote the growth of tumors.

In order to better understand what prevents immune cells from fulfilling their role, the researchers this time decided to take a closer look at the T lymphocytes whose mission is to detect and destroy tumors by infiltrating them. To this end, biologists and clinical research physicians of the Institut Pasteur’s ICAReB platform, Dr. Hélène Laude and Dr. Marie-Noëlle Ungeheuer, approached the patient association POLYPOSES FAMILIALES France. A new clinical research project involving the association recruited patient volunteers for the collection of blood samples. “Thanks to the association, we met patients and also clinicians specialized in polyposis. We learned a lot about this complex pathological condition, the experience of patients and families, and the different levels of disease severity. We recognize the valuable role of the patients, who were highly motivated to take part in the study, and the input of specialists”, pointed out Andrés Alcover.

The naturally mutated T lymphocytes present in the blood of these patients were cultured then subjected to several in vitro experiments. Using several microdevices – filters, channels, protein substrates and layers of vascular endothelial cells – the researchers could compare the behavior of diseased lymphocytes with that of lymphocytes from healthy volunteers.

“In order to move along blood vessel walls, cross them and reach the tumor to be infiltrated, healthy lymphocytes change their morphology. Something akin to a large adhesive foot, supported by the lymphocyte’s cytoskeleton, grows longer in the direction of migration. This polarization is essential for movement in the right direction,” explains Marta Mastrogiovanni, researcher in the Institut Pasteur’s Lymphocyte Cell Biology Unit and lead author of the study. In mutated lymphocytes, the microtubules making up the cytoskeleton are disorganized and there are fewer adhesion proteins. The cells lose their polarity and their ‘muscles'”.

Although the mutated T lymphocytes are not necessarily moving more slowly than healthy lymphocytes, they adhere less well to the walls and have more difficulty moving in a given direction and passing through the walls. In short, this research showed their migration to be less effective. “This discovery is important because the motility of immune cells is a key process in antitumor immune defense. “We know that the immune system is very important in combating pathogens but we sometimes forget that it also contributes to combating cancer cells”, concludes Vincenzo Di Bartolo, researcher in the Institut Pasteur’s Lymphocyte Cell Biology Unit and joint senior author of the study.

(1) Collaborative project: Institut Pasteur, Department of Immunology and  and Center for Translational Science (CRT, ICAReB), and Institut Pasteur, Institut Cochin, Institut Curie, and Institut Pierre-Gilles de Gennes.

(2) Familial adenomatous polyposis accounts for 1% of all colorectal cancers. 

(3) Funding via the French Cancer League (La Ligue Contre Le Cancer), 2018-2022 “Équipe Labellisée” program, the Institut Pasteur and Inserm. Marta Mastrogiovanni was funded by the Pasteur-Paris University International Doctoral Program and the European Union Horizon 2020 Research and Innovation Programme under Marie Sklodowska-Curie grant agreement 665807 and La Ligue Contre Le Cancer, doctoral grant 4th year of PhD.

Brain microglia (green) initiating expression of cell division marker (red), but unable divide due to co-expression of a senescence marker (blue), due to the chemotherapy treatment (busulfan). © K. Sailor/ PM Lledo, Institut Pasteur.

Annually over 50,000 bone marrow transplantations occur worldwide as a therapy for multiple cancerous and non-cancerous diseases. Yet, how this procedure gives rise to bone marrow-derived cells that engraft the brain, despite being absent in the normal brain, remains unknown. In the present study, scientists from the Institut Pasteur, the CNRS and the Paris Brain Institute (Inserm) discovered how the host’s microglia, the brain’s innate immune cells, are replaced by bone marrow-derived macrophages. The key discovery was that transplantation chemotherapy eliminated the microglia’s regenerative capacity, gradually causing the engraftment of macrophages to replace microglia, providing a potential mechanism for future cell-based therapies to treat central nervous system diseases. This finding will be published in Nature Medicine on February 21, 2022.

Progressive demyelination of the central nervous system leads to devastating neurologic symptoms and premature death. As the number of cases diagnosed through pregnancy and newborn screening is increasing, more efficient therapeutic strategies are required. Recently, the successful use of gene therapy to correct disease-causing gene mutations in bone marrow stem cells, and their subsequent autologous transplantation into patients, has been clinically used for treating white matter diseases, while eliminating the need for finding matching donors. Clinical studies showed that pre-transplantation ablation of host bone marrow stem cells using the chemotherapy agent busulfan allows for efficient engraftment and tolerance to gene-modified cells, yet the mechanism involved was largely known.

Using an animal model, neuroscientists from the Institut Pasteur (CNRS) and the Paris Brain Institute (Inserm) have demonstrated the broad impact of busulfan chemotherapy used for bone marrow transplant therapy on various cell populations of the host brain.

Microglia are tissue-resident brain immune cells that are instrumental for maintaining healthy brain physiology in normal and diseased states. These cells exhibit a robust self-renewal capacity and gradually turnover throughout life. However, in this study, Sailor and colleagues show that following pre-transplantation busulfan chemotherapy, microglia completely lose their regenerative capacity, becoming senescent, which thereby allowed the engraftment of bone marrow-derived macrophages into the brain. They also show these macrophages to become resident with similar surveillant dynamics as the microglia they replaced.

They demonstrate that busulfan administration temporarily depleted oligodendrocyte precursor cells and, importantly, caused the complete and permanent ablation of all adult neurogenesis, which may explain cognitive deficits in patients after busulfan chemotherapy, i.e. the “chemo brain”. Microglia that were eliminated because of busulfan chemotherapy, coupled with their loss of regenerative capacity, leave empty niches in the brain which are replaced by engrafted bone marrow-derived macrophages. These macrophages assumed the morphology and cellular process dynamics like normal microglia.

This study is highly relevant since for the first time it shows a mechanism dependent on microglia senescence, with the loss of their regenerative capacity, to explain how macrophages are permissively engrafted into the brain after bone marrow transplantation.

“Microglial cells play an essential role in brain function and in the pathophysiology of many severe neurological diseases, both genetic and complex, such as multiple sclerosis and Alzheimer’s disease. Understanding the fate of these cells after the transplant process is essential both to clarify the consequences of chemotherapy and to develop new therapeutic strategies for serious neurodegenerative diseases,” said Nathalie Cartier, research director at Inserm and member of the NeuroGenCell team at the Brain Institute (ICM), and last co-author of the study.

“This study sheds light for the first time on a mechanism explaining how stem cell-derived macrophages enter the brain after bone marrow cell transplantation. This better understanding is essential to develop new strategies for gene and cell therapy applied to diseases of the central nervous system,” said Pierre-Marie Lledo, research director at the CNRS and head of the Perception and Memory Unit at the Institut Pasteur, and last co-author of the study.

“We show bone marrow transplantation chemotherapy to cause microglia, the brain’s resident immune cells, to lose their regenerative capacity. The microglia were unable to sustain their population which allowed bone marrow derived cells to replace them. This demonstrates how bone marrow transplantation is an effective therapy for certain neurological diseases and provides a strategy for cellular gene therapy in the central nervous system,” said Kurt Sailor, research fellow at the Institut Pasteur Unit Perception and Memory in Paris, and first author of the study.

Microscopic visualization of lymphocytes (in green) infiltrating a tumor HEV (in red) during combination anti-PD-1 plus anti-CTLA-4 immunotherapy. The white arrow indicates a lymphocyte that is leaving the bloodstream and entering the tumor (in black). © Elisabeth Bellard and Jean-Philippe Girard – IPBS (CNRS/UT3 Paul Sabatier)

Immunotherapy, a therapeutic strategy aimed at increasing the activity of the immune system in order to recognize and destroy cancer cells, has revolutionized cancer treatment over the past decade. A better understanding of how this therapeutic approach works, and more particularly how killer lymphocytes access tumors during immunotherapy, could improve the efficacy of the treatments. The team of Jean-Philippe Girard, Inserm Research Director at the Institute of Pharmacology and Structural Biology (French National Center for Scientific Research [CNRS]/Université Toulouse III – Paul Sabatier), in collaboration with Gustave Roussy, has recently discovered the essential role played in this process by specific blood vessels known as tumor-associated HEVs. For the first time, the scientists were able to film the lymphocytes infiltrating the walls of the HEV vessels to enter the tumors. What is more, the researchers have shown in animal models that increasing the proportion of HEV vessels in a tumor improves the efficacy of the immunotherapy and leads to the eradication of the tumors. Finally, they found that the likelihood of recovery of patients with metastatic melanoma (skin cancer) and treated with immunotherapy is increased when a large number of HEV vessels are present in tumors. The findings of this study have been published in the February 3, 2022 issue of Cancer Cell[1].   

Immunotherapy with therapeutic antibodies represents a real revolution in cancer treatment. In particular, it is used to cure certain patients with metastatic melanoma (skin cancer), who would previously not have survived. Unfortunately, immunotherapy is not effective in all patients or on all cancers. A better understanding of the mechanism of action of the treatment could improve it and make it effective in a larger number of patients. 

Killer lymphocytes – white cells present in the blood – are capable of eradicating cancer cells. It is essential that many of these killer cells have access to tumors in order to defend the body against cancer. The Toulouse team has lifted the veil on the mechanisms that allow killer lymphocytes to penetrate tumors in order to destroy them – either spontaneously or following immunotherapy with anti-PD-1 plus anti-CTLA-4 antibodies.

The scientists have discovered that the HEVs, very specific blood vessels known as high endothelial venules, constitute the major gateway of lymphocytes to tumors. Using sophisticated microscopy techniques, the researchers were able to film the passage of lymphocytes from the blood to the tumor in animal models. For the first time, they were able to visualize, directly and in real time, the lymphocytes in the process of infiltrating the walls of the HEV vessels in order to access the cancer cells in the tumor. “We thought that HEV vessels played an important role in the entry of lymphocytes into the tumor, but we were surprised to see that they were virtually the only gateway,” says Jean-Philippe Girard, Inserm Research Director and last author of the study.

The researchers then observed in their models that the presence of a large number of killer lymphocytes in tumors is associated with the presence of a large number of HEV vessels. What is more, they provided proof of concept that increasing the proportion of HEV vessels in a tumor improves the efficacy of combination anti-PD-1 plus anti-CTLA-4 immunotherapy and leads to tumor eradication.

Labeling of HEV vessels (brown) on a tumor section from a patient with metastatic melanoma and treated with immunotherapy. © Jean-Philippe Girard – IPBS (CNRS/UT3 Paul Sabatier)

Finally, in collaboration with Caroline Robert’s team at Gustave Roussy[2], the scientists studied patients with metastatic melanoma. They discovered that the presence of a large number of HEV vessels in tumors is associated with a better response to combination anti-PD-1 plus anti-CTLA-4 immunotherapy.

The next step for the researchers will be to develop treatments to increase the proportion of HEV vessels in tumors, in order to improve the efficacy of immunotherapy, allowing massive recruitment of killer lymphocytes to eradicate cancer cells.

[1] This study was funded by Fondation ARC, the Foundation for Medical Research (FRM), the French National Cancer Institute (INCa), the French National Research Agency (ANR), and Labex TOUCAN

[2] And also team leader at Inserm U981

The patients who best respond to the dual therapy in this study have a “Mitoscore signature” associated with strong mitochondrial activity. © Adobe Stock

While the care and treatment of acute myeloid leukemia (AML) have greatly improved in recent years, overall survival remains low. Resistance to the various treatments continues to present a major clinical challenge. Using animal models, and also by working with patients themselves, scientists from Inserm, CNRS and Université Toulouse III – Paul Sabatier at the Cancer Research Center of Toulouse have identified a new biomarker predictive of response to dual therapy (chemotherapy plus targeted therapy) used in AML, as well as resistance mechanisms behind relapses. The findings of this research have been published in Nature Cancer.

Leukemia groups several types of blood cancer that affect nearly 10,000 people each year in France. These include acute myeloid leukemia (AML), which affects the hematopoietic cells[1] in the bone marrow.

For a long time, intensive chemotherapy has been the treatment of choice for patients. Although the majority respond favorably and go into remission, overall survival in the longer term remains low, with certain resistant cancer cells persisting in the body following chemotherapy and leading to relapse.

In recent years, the development of targeted therapies has improved the treatment and response of patients, prolonging survival a little – particularly in elderly people ineligible for chemotherapy. However, even with these therapies, relapse remains a major issue. Understanding the mechanisms underlying resistance to leukemia treatments and finding a way to resolve them are a central focus of the work of Inserm researcher Jean-Emmanuel Sarry and his team at the Cancer Research Center of Toulouse (Inserm/CNRS/Université de Toulouse III – Paul Sabatier).

While most scientists working on the subject are more interested in the genetic mechanisms associated with resistance, Sarry’s team is studying the non-genetic mechanisms in order to understand why some patients are more likely to relapse.

Identification of a “Mitoscore signature”

In their new study, the researchers looked at a recently approved dual therapy (conventional chemotherapy combined with a new targeted therapy), which is increasingly used in the treatment of AML.

Using patient transcriptomes (i.e. all messenger RNA derived from genome expression), they show that people who respond best to the dual therapy and who have prolonged survival have a specific biomarker – a “Mitoscore signature” – that is associated with a high level of mitochondrial activity[2]. “In other words, this strong Mitoscore signature, which reflects a high level of mitochondrial activity, is predictive of an improved response to these treatments,” explains Sarry.

Finally, thanks to single-cell sequencing[3]of residual disease[4] following this dual therapy, the researchers observed a particular remodeling of mitochondrial function allowing cancer cells to adapt to therapies and induce the patient’s relapse. In mice, the team also showed that treatment based on a molecule that inhibits the action of the mitochondria makes it possible to block this mitochondrial function remodeling, prevent relapse, and prolong the animals’ survival.

“The objective is now to test this Mitoscore signature on very large cohorts in order to validate its utility. Ultimately, the idea would be to use this biomarker to improve patient follow-up and offer more personalized therapies – by giving dual therapy, possibly also with the mitochondrial inhibitor, for those likely to benefit from it. This research could therefore have a real clinical impact in the years to come,” explains Sarry.

1 Hematopoietic stem cells are made by the bone marrow and develop into the various blood cells: red cells, white cells, and platelets. Source INCa

2 Mitochondria are intracellular organelles whose role is to provide the cells with the energy they need. They therefore play a central role in cellular energy metabolism.

3 Single-cell sequencing is a set of molecular biology techniques used to analyze genetic information at single-cell level, using next generation sequencing technologies.

4 Residual disease is the persistence in tissue of malignant cells below the detection limit of conventional techniques.