Primary tumors are currently treated by a combination of therapies including, in most cases, surgery, local radiotherapy, and adjuvant chemotherapy. Even when the tumor has apparently been defeated, micrometastases of dormant tumor cells frequently lead to tumor relapse and final therapeutic failure. Accumulating evidence indicates that the innate and adaptive immune systems can make a crucial contribution to the antitumor effects of conventional chemotherapy-based cancer treatments. However, the tumor-mediated immunosuppression of the host often limits the initiation of an effective anticancer immune response. Therefore, to defeat cancer, it is essential to relieve host immunosuppression to unleash an immune response that eradicates residual tumor cells. We have identified that 5-Fluorouracil (5-FU), a pyrimidine analog that is mainly used in the treatment of breast and colon cancer, has a superior and selective ability to deplete Myeloid derived suppressor cells (MDSC) in vivo. The elimination of MDSC by 5-FU partly restored anticancer responses and improved 5-FU therapeutic effect. However, our preliminary data indicate that an additional molecular pathway activated by the 5-FU-mediated cell death of MDSC limits the efficacy of 5-FU and contributes to immune evasion. Specifically, we hypothesize that 5-FU triggers an inflammatory, caspase-1-dependent cell death of MDSC, which supports the IL-1ß-mediated polarization of intratumor Th17 cells that impair the development of anticancer responses. Thus, by identifying the molecular mechanisms by which 5-FU affects MDSC, this project ultimately aims at unraveling new molecular targets that could be modulated to achieve optimal anticancer responses.

Atherosclerosis is a chronic systemic inflammatory disease affecting the large and medium-sized arteries. The development of atherosclerosis which can progress silently during decades is characterized by the thickening and loss of elasticity of the arteries owing to the formation of atherosclerotic plaques in lesion-prone areas. These plaques are built from the accumulation of fatty materials within the vessel walls and by the modification of the connective tissue of the vessel walls. As a result, the luminal narrowing (stenosis) of the modified arteries occurs and limits therefore the blood flow which can lead to tissue ischaemia. However the most severe complications arise from the rupture of the atherosclerotic plaques which accounts for 70% of heart attacks. Since atherosclerosis is involved in most cardiovascular diseases which are the leading cause of morbidity and death in the world, the identification of vulnerable plaque (i.e. rupture-prone plaques) constitute an urgent need which would result in health benefits. Among the numerous imaging modalities, the combination of magnetic resonance imaging (MRI) and positron emission tomography (PET) constitutes a promising strategy because it allies the high resolution of MRI to the exceptional sensitivity of PET imaging. Such a combination should be a significant breakthrough in the early detection of vulnerable atherosclerotic plaques. If the development of imaging device which integrates both MRI and PET is in itself a major challenge, the elaboration of nanoprobes for exploiting this promising hybrid technology for medical imaging represents a crucial step for the early detection of vulnerable atherosclerotic plaques. Facing the real and urgent need, the CARGOLD project aims at developing nanoprobes for early detection of vulnerable atherosclerotic plaques and therapy by magnetic hyperthermia from multifunctional nanostructures whose physical and chemical properties render possible the specific targeting of these plaques, their follow-up by integrated MRI/PET device and also by computed tomography (CT) after intravenous injection, a therapeutic activity and their removal by bio-degradation and renal clearance. Such attractive characteristics should be obtained by assembling in a controlled manner bio-resorbable maghemite nanoflowers and multifunctional gold nanoparticles. The gold cores which can generate the contrast enhancement of the images acquired by CT will be coated by bio-targeting groups (peptides) and by gadolinium and positron emitter chelates for a simultaneous follow-up by MRI and PET. Although these multifunctional gold nanoparticles exhibit the potential for a targeted imaging, they are handicapped by a too rapid renal clearance which should impede a sufficient accumulation in the vulnerable plaques. Their grafting onto bio-resorbable nanocarriers (iron oxide nanoflowers (~30 nm)) is expected to enhance their circulation time after intravenous injection by postponing their renal clearance which remains a pre-requisite for the in vivo application of gold nanoparticles. Besides a greater circulation time, the accumulation of these golden nanoflowers designed for multimodal imaging (MRI/PET and CT) will be ensured by the avidity of macrophages for nanoparticles which are present in a large amount in the vulnerable plaques and by the specific interaction between the peptides coated to the golden nanoflowers and cell adhesion molecules (VCAM-1). This strategy carries the promise to significantly improve the detection of vulnerable plaques and therefore to prevent from the dramatic issue of their rupture. For achieving this ambitious goal, the CARGOLD project gathers six partners (4 academic partners, 1 medical imaging platform and an industrial specialized in the production of customized particles) which are recognized for their expertise in the complementary fields explored in CARGOLD.

Atherosclerosis is a leading cause of death worldwide. This complex disease is the subject of intensive research to improve detection/diagnosis and consequently therapeutic approaches. The exploration of atherosclerotic disease requires different imaging techniques such as echography, X rays, magnetic resonance and scintigraphy. However, while some techniques lack specificity, some other are dedicated to only one aspect of the diagnosis and only give specific information on either structural, metabolic or cellular characteristics of the aortic sections of interest. Therefore, gathering enough information in order to choose the most adequate therapeutic strategy often requires the use of multiple imaging techniques. A key feature of atherosclerosis is altered metabolism of LDL (Low Density Lipoproteins), natural particles that carry cholesterol in the bloodstream. Indeed, high LDL plasma levels are a critical risk factor for atherosclerosis, and abnormal LDL uptake occurs in atherosclerotic plaques. The use of LDL as imaging tracers should therefore constitute a new versatile tool to complement the former imaging techniques in order to detect, diagnose and increase our understanding of the mechanisms involved in this silent disease. The DLLICATE project aims at generating bimodal (radioactive and fluorescent) imaging probes that will be bound to LDL in order to determine the fate of these particles by analyzing simultaneously whole body distribution (non-invasive scintigraphy), and tissue and cell distribution (in situ imaging and fluorescence microscopy). Two generations of dually-labelled LDL or oxidized LDL will be synthesized and characterized in order to define a labelling protocol that does not alter their natural biological features. The imaging efficacy of the most promising conjugates will be tested in models of atherosclerosis with different levels of progression and inflammatory status. On the short term, this project will establish the preclinical proof of concept of the approach and validate our labelling techniques for the production of dual-labelled LDL. Efficient bimodal labelling of LDL will enable to determine the metabolic fate of LDL in vivo by the visualization of arterial sections and cell types showing high LDL uptake in pathological conditions, such as immune cells in atheromatous plaques. Whole body imaging (radioactive mode) will allow exhaustive localization of atherosclerotic plaques. Then, the use of a near-infrared fluorescent probe will allow direct visualization the LDL uptake pattern in arterial sections exposed on the surgical field. Finally, the analysis of plaque biopsies obtained from our pre-clinical models will help to establish the possible link between localization, type and inflammatory status of immune cell subtypes which take up LDL as markers of plaque stability. In this way, our technique will help to provide new risk markers of plaque rupture and infarction. On the long term, our concept based on the use of endogenous, well-tolerated particles as imaging agents will allow a possible translation in the context of human atherosclerosis. Our LDL conjugates could constitute new clinical diagnostic tools for the exhaustive detection and precise localization of atheroma plaques in subjects at high cardiovascular risk. In addition, the use of NIR fluorescent probes might also be helpful for intravascular imaging and fluorescence-assisted surgery. Finally, ex vivo fluorescence microscopy on tissues biopsies will allow a better understanding of the disease (plaque stability, immune cell profiling) and thus will help in selecting the best therapies to be implemented.

Esophageal Atresia (EA) is a rare developmental defect of the foregut that presents with or without a Tracheo-Esophageal Fistula (TEF). The prevalence of EA/TEF over time and around the world has been relatively stable (> 165 new EA/year on average in France). EA/TEF is manifested in a broad spectrum of anomalies: in some patients it manifests as an isolated atresia, but in more than 60% of the cases it affects several organ systems. While the associated malformations are often those of the VACTERL spectrum (Vertebral, Anorectal, Cardiac, Tracheo-Esophageal, Renal and Limb), many patients are affected by other malformations, such as anomalies of the genitourinary, respiratory and gastrointestinal systems. Though EA/TEF is a genetically heterogeneous condition, recurrent genes and loci are sometimes affected. Trachea-Esophageal (TE) defects are in fact a variable feature in several known single gene disorders and in patients with specific recurrent Copy Number Variations and structural chromosomal aberrations. At present, a causal genetic aberration can be identified in less than 10% of patients. In most, EA/TEF is a sporadic finding; the familial recurrence rate is low (1%). As this suggests that epigenetic and environmental factors also contribute to the disease, non-syndromic EA/TEF is generally believed to be a multifactorial condition. Several population-based studies and case reports describe a wide range of associated risks, including age, diabetes, drug use, herbicides, smoking and fetal alcohol exposure. The phenotypical and genetic heterogeneity seen in EA/TEF patients indicates not one underlying cause, but several. In this project we will combine the French register of EA and multiomic studies in order to elucidate new causes or mechanisms in the etiology within specific sub-populations. Improved knowledge of predictive factors and molecular mechanisms may improve prediction and parental counseling and prevent co-morbidity. In this context, state of the art multi-omics will be performed from esophageal biopsies. Systemic/integrative biology will be then undertaken to establish predictive networks. Validation of EA pathways will be investigated using cross link coupled to mass spectrometry (XL-MS) and with BioID in order to evaluate the protein-protein partner involved in networks to better understand physiopathological mechanisms occurring in EA etiology. Integration of all the data using robust bioinformatics and biostatistics will give hypotheses for EA etiology. Based on these fundamental knowledges acquired on the EA pathology, a translational research step will be launched. Multi-omics analyses will be then performed on extracellular vesicles (EVs) issued from amniotic liquid. EVs have raised interest as a potential source of biomarker discovery because of the resemblance of their molecular content to that of the releasing cells. EVs in amniotic liquid will be the mirror of the EA pathology occurring in course of fetus development. Thus, multi-omic analyses of these EVs will be the first steps to improve the prenatal EA/TEF diagnostic.

Borna disease virus (BoDV), a single-stranded RNA virus of negative polarity, has puzzled researchers for decades, because of its neurotropism and non-cytolytic multiplication strategy. Its recent recognition as a zoonotic agent causing severe encephalitis and brain dysfunction has provided further impetus for increasing our knowledge on this enigmatic pathogen. To date, very little is known regarding the BoDV replication modalities. It is the only animal Mononegavirales to replicate in the nucleus, where it assembles viral factories that, strikingly, are physically bound to the neuronal chromatin. The only available structural information is the X-ray structure of the RNA-free nucleoprotein and nothing is known regarding its recognition of the genomic RNA or the organization of its nucleocapsid. Intriguingly, sequence analysis predicts that the BoDV polymerase is radically different from that of other Mononegavirales, being 20 % shorter and not containing the canonical RNA-capping motifs. In particular, the methyltransferase domain seems absent, strongly suggesting the recruitment of specialized host factors at viral factories -such as the capping machinery- to complement this activity which is essential for the maturation of viral mRNAs. Finally, the functional consequences of the binding of viral factories onto the chromatin, notably its impact of neuronal epigenetics and neuronal communication, are also completely unexplored. In this context, the Bavarian project aims at providing an integrative and multiscale vision of the BoDV replication complex. Our multi-pronged proposal will describe the structural organization and explore the BoDV replication complex modus operandi within infected cells. To tackle these questions, our three teams will combine their expertise, implementing cutting-edge structural biology, biochemistry, virology and neuronal functional assays. Our complementary approaches will allow a fine characterization of: (i) the architecture of the BoDV replicative machinery, (ii) its viral-host polypeptides interplays and (iii) the impact of altering the viral replication on viral fitness, neuronal epigenetics and function. Our project will provide an unmatched deciphering of BoDV pathogenic mechanisms and, in a broader view, will represent a breakthrough in the field of RNA virus family evolutionary mechanisms. Indeed, Bavarian will focus on the only non-retroviral RNA virus known so far that needs to interact with the host DNA genome to persist and proliferate. To achieve its goals, our project groups three teams with complementary expertise. The team of the coordinator (IBS, Grenoble) is composed of biochemists and structural virologists, with recognized experience in viral replication. Partner-2 (Infinity, Toulouse) has a long-standing expertise in the analysis of the mechanisms and consequences of viral persistence, in particular BoDV, in the central nervous system. Partner-3 (Prism, Lille) has a strong background in proximal interactomics (notably BioID) and systems biology, illustrated by his recent description of whole proteome proximal interactomes of emerging viruses such as Zika and SARS-CoV-2. These recent months, the three teams have established a very efficient collaboration, which has led to a very convincing set of preliminary data that strongly ensures the chances of success of the proposed program. The recent SARS-CoV-2 pandemic is a vivid reminder that no pathogen should be under-estimated. Indeed, deciphering the interaction of viruses with target cells is essential to gather clues for therapeutic intervention. Notably, increasing our knowledge on the organization of viral replication complexes is instrumental, because it often allows to identify strategies to block not only one, but often several viruses. Obtaining original data on the unexplored BoDV system could therefore open the road for finding new antivirals and/or therapeutic strategies, which may be applicable to many pathogens.