Research

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PRISM is based on its two pillars. The Technological innovation pillar developed molecular tools based on omics used by the Therapeutic innovation pillar to understand the cellular communication and the mechanisms occurring in non-classical endocrine cells such as macrophages or cancer cells to give an appropriate therapeutic response. This response is based on biotherapy or specific drugs developments in Therapeutic innovation pillar and new surgical tools such as Spidermass or Irosmass in Technological innovation pillar. The Organomic platform reinforce the technical skills needed to answer to our main questions. The fundamental research supports the translational one in which O’DREAMS support the pre-clinical studies before addressing the clinical. Thus, the two main axes during converge to the pre-clinical then clinical studies which are valorized through publications, patents, start-up, and societal benefit for the patient.

Technological innovations Pillar

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The Technological Innovation axis – (Coordinated by Pr. I. Fournier) arose from the MALDI Imaging Team (MIT) created in 2004 after Prof. I. Fournier obtained a French National Starting Grant (ACI young researchers) based on the research that was conducted since 2002 on MALDI MS Imaging (MALDI MSI) ^{1, 2}. Since 2004, this research was organized around i) technological developments and ii) clinical applications transversally to the second axis of the unit. However, the developments towards clinical applications were strengthened over the past years. Technological Innovation is focused since its beginnings in the laboratory on the development of MALDI MSI first with improvements in i) tissue preparation for both Fresh frozen ^3-5 and Formalin–Fixed Paraffin-Embedded (FFPE) samples ^{6, 7} and ii) biomolecules identification with preserved spatial localization ^8-10. This was pursed through the development of novel MALDI matrices (ionic matrices) ^{5, 11}, of matrix deposition methods (spraying devices, automatic spotting with piezoelectric head) ^12-14, on-tissue microdigestion and on-tissue tryptic peptides derivatization, unlocking the use of FFPE tissue ^{8, 9, 15, 16}, bioinformatics (novel imaging software, MITICS, Principal-Component Analysis-Symbolic Discriminant Analysis method (PCA-SDA)) ^16-19, novel pre-spotted MALDI plates with ionic matrices ²⁰, as well as targeted MALDI-MSI based on Tag-Mass ^21-25. The Tag-mass technology allow to perform multiplex images and is now sell by Ambergen and was previously sell by fluidigm as the hyperion system. Following these developments, the ones we named the spatial proteomic guided by MSI has open the door to revisit the clinical proteomics. Since we can localize some regions of interest defined by MALDI MSI based on molecular differences, the spatial proteomic was developed using liquid junction extraction technology after on tissue micro-digestion using a micro-spotter. From 600µm to 250µm tissues extraction diameter, it was possible to analyse the tissue micro-environment which is lack using complete tissue extraction or single cell analyses ^{8, 9, 26, 27}. We thus be able to determine the whole of the actors in interactions in a tissue region and associate protein clusters to patients’ classification, overall survival, and prognosis ^{8, 24, 28-34}. This was the early stages of the integration of multiomics and in same time the evidence of existence of alternative proteins identified in MS spectra but not associated to human genome. These alternative proteins, we named the Ghost proteome, were neglected during the last ten years but our discovery with the ones of other groups has pushed this field to grow up very fast now ^35-51. Moreover, the discovery of their functions and interactions with partners has consolidated the fact that these proteins issued from the non-coding region of mRNA and from ncRNA are not considered as noise. We started to organize the field with several groups in 2023. Tissue spatially resolved proteomic has shifted to spatially resolved interactomic with the use of cross-link associated to MS ^{35, 41}. We developed in same time the spatially resolved top-down proteomic also guided by MSI ^{46, 52}. Finally, we integrate in our workflow the glycoproteomic associated to MSI and surfaceome proteomic ^{53, 54}. These are the major improvements achieved by MIT over the last 20 years on tissue omics and MSI. However, all these technologies are performed ex vivo or in vitro on tissues sections. Two new instruments (SpiderMass and Snoop-I), we created give a new dimension of the mass spectrometry of biomolecules since they offer the possibility to get in vivo in real time in a non-invasive way the possibly to handle the dynamic of the biomolecules in cellulo. SpiderMass is based on a Water assisted laser desorption/ionisation process (WALDI-MS) can be performed in vivo, in real time with a low invasives ^55-63. SpiderMass is associated with deep and transfer learning ⁶⁴. Altogether, this new technology offers the possibility to detect in real time metabolites and lipids but also proteins in certain conditions. This instrument is devoted to help surgeons for margin detection and decision making. It has also been developed for performing imaging and molecular topography imaging. The Snoop-I is an instrument for detecting volatile organic compounds (VOCs) based on low temperature plasma (LTP) directly coupled to MS instrument⁶⁵. Volatilome is known to be produce as communication messenger from cells and microbiota. Its detection can be used as an early diagnosis element. We thus developed SNOOP-I in this optic for breast cancer diagnosis based on patch led on breast skin for a couple of hours before LTP-MS analysis and real tile diagnosis. Taken together, based on the different grants obtained, the technological innovations axis is now structured in two WPs i.e. WP1 Omics tools for microenvironnement study and WP2: consist of the next generation surgery (therapy) through developments of novel instruments for real-time in vivo analyses. The contents of these 2 WPs will be detailed in the "Research" section of this document. Clinicians from the COL including oncologist, pathologist, and surgeons (8 clinicians in total) have joined this axis to develop a project devoted to precision medicine based on tumor heterogeneity analysis, medialization and interpolation with clinical data.

Early Diagnosis

Therapy

Therapeutic innovations Pillar

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The Therapeutic Innovation pillar (Coordinated by Pr. M.Salzet) is organized in 2 integrated work packages which are translational from basic science to clinics is issued from neuroimmune research developed by Prof. M. Salzet and its team since 2000 on neuroendocrine factors expression in non-classical endocrine cells such as macrophages and their role in the immune response ^66-71. These results have led to reconsider the term neuroendocrine, not only as cells that secrete their products in a regulated manner, in response to a specific stimulus but to also include the notion that activation of specific genetic switches can lead to the expression of a partial or full neuroendocrine phenotype in a variety of cell types, including immune cells or neuroendocrine cancer cells. Thus, besides functional similarities, there is potentially a particular developmental link that bridges the neuroendocrine and immune system which can lead the discovery of early markers of the neuroendocrine switch and thus provide potential therapeutic targets, which constitutes the aim of WP1 i.e. investigation through basic sciences and translational research, this concept of neuroendocrine factors expression in non-endocrine cells such as neuroendocine cancers (Merkel, GBM, Gynaecological cancers) or immune cells like macrophages using tools developed by the technological innovation pillar.

In this context, we demonstrated the ability to switch macrophages in a pro-inflammatory phenotype by knocking out the proprohormone convertase PC1/3 ⁷² or knocking down the NR8282 rat macrophages cell line ⁷³. When PC1/3 is inhibited a pro-inflammatory cytokine storm occurs leading a drop in blood pressure and a least death ⁷⁴. We established that PC1/3 is involved as a chaperone through UNC93B-GramD4-NOGOB modulation on the Toll-like receptor (TLR) Myd88 dependant signalling pathway ^75-77. Since PC1/3 inhibition leads pro-inflammatory cytokines release, we investigate the anti-tumoral potential of such macrophages ^78-81. We established that PC1/3 inhibition in macrophages switch them into anti-tumoral profile unsensible to epigenetic modifications from the tumours ⁸² and orienting the immune response to Th1 ⁷⁹. Moreover, these modified macrophages produce extracellular vesicles with the ability to reactivate tumour infiltrated macrophages (TAM) in anti-tumoral profile by increasing phagocytosis and cytotoxic factors production. ⁸⁰. In this context, we investigate the development of therapeutic strategy based on proprotein hormones invalidation since we demonstrate that besides PC1/3, furin inhibition in macrophages leads anti-viral immune response ⁷⁹. Similarly, we investigate presence of such enzyme in tumours. PACE4 is present in prostate and ovarian cancers ^{83, 84}. PACE4 inhibition decrease the tumour proliferation in xenografted mice. We thus deceived to use a proprotein convertase inhibitor that can interact with both the cancer cells and TAMs ^{78, 80}. Results obtained confirmed the beneficial effect of such inhibitor for GBM therapy. In this context, we will use modified macrophages as cargo of this inhibitor. using spatially-resolved proteomic guided by MSI and link to patients survival, we were able the ability to classify for GBM (clinical trial) the patients in 3 survival prognosis groups linked to macrophages infiltration, neuroendocrine factors or oncoviral factors expression ^{28, 85}. Infiltration of macrophages are clearly very bad prognosis for GBM and several biomarkers linked to this bad prognosis were identified e.g. MAOB, IgHM, ANXA6 and ANX11. Development of inhibitors in WP2 towards these markers is under process. It must be noticed that MAOB is linked to depression but can also be considered as an immune check point. In same time, we investigate the expression of tumour surface proteins though surfaceome proteomic In GBM ⁵⁴. We discovered new biomarkers e.g. mutated proteins such as RELL1, CYBA, EGFR, and MHC I proteins and ghost proteins. We then performed similar experiments in breast cancers and identified several non-classical HLA. Besides HLA-G, HLA-E and HLA-F, we identified two novel non-classical HLA issued from pseudogenes (HLA-J and HLA-H). These non-classical HLA were found specifically by breast cancers subtypes. These new targets have allowed to start chimeric antigen receptor modified Macrophages (CAR-MAC) directed against these specific targets as novel therapeutic strategy and will constitute the WP2.