Etude du rôle d'un donneur de monoxyde d'azote (Glycéryl Trinitrate) dans la mort cellulaire immunogène induite par des chimiothérapies (FOX) et/ou radiothérapie dans le cancer colique

Le cancer colorectal se situe au 3ème rang mondial des cancers les plus fréquents derrière les cancers du sein et du poumon. Dans le cas de cancer colorectal métastatique, la première ligne thérapeutique repose sur l’utilisation d’une association de chimiothérapies : le FOLFOX (acide FOLinique, 5-Fluorouracile et OXaliplatine). Malgré une forte efficacité anti-tumorale de cette combinaison, de nombreuses rechutent apparaissent nécessitant l’utilisation de nouvelles stratégies basées sur l’association du FOLFOX à d’autres agents anti-cancéreux ou stimulant le système immunitaire. Cette étude a eu comme objectif d’étudier l’effet anti-tumoral del’association du FOX (5-Fluorouracile et OXaliplatine) à un donneur de monoxyde d’azote (NO) tel que le Glycéryl Trinitrate (GTN) combiné ou non à de la radiothérapie dans le cancer colique. En clinique, l’utilisation du GTN, un agent couramment utilisé dans le traitement des angines de poitrine, a montré un bénéfice réel chez des patients porteurs de cancer du poumon non à petites cellules et traités par chimiothérapies associées ou non à de la radiothérapie. Dans nos modèles précliniques de cancer du côlon, nous avons montré que l’association du GTN au FOX potentialisait l’effet anti-tumoral de ces chimiothérapies. L’utilisation de modèles murinsimmunodéficients nous a permis de montrer l’importance du système immunitaire dans l’activité anti-tumorale de cette combinaison. Nous avons alors recherché par quel mécanisme le GTN pouvait induire l’activation du système immunitaire et avons alors montré que le GTN induisait l’externalisation de la calréticuline, un des éléments nécessaire à l’induction de la mort cellulaire immunogène (ICD). Cette mort immunogène est caractérisée par la libération de facteurs immunogènes (HMGB1 et ATP) et par l’externalisation de la calréticuline. Cette ICD représente un des mécanismes par lequel la radiothérapie mais également certaines chimiothérapies peuvent activer le système immunitaire et ainsi potentialiser l’efficacité de ces thérapies. Ainsi et pour la première fois, nous avons montré le rôle primordial du NO dans le mécanisme d’externalisation de la calréticuline induit par l’ajout d’un donneur de NO, ou par ajout de chimiothérapies pouvant induire la production de NO. Tous ces travaux montrent l’importance du NO dans l’efficacité des thérapies conventionnelles (chimiothérapies, radiothérapie) et mettent en exergue l’utilisation de GTN en association à des thérapies non immunogènes afin de potentialiser leur efficacité anti-tumorale

HSPB5 Inhibition by NCI-41356 Reduces Experimental Lung Fibrosis by Blocking TGF-β1 Signaling

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-β gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-β1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-β1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-β1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-β1 signaling. Further investigations are needed to determine whether these results can be transposed to humans

Lipoproteins LDL versus HDL as nanocarriers to target either cancer cells or macrophages

International audienceIn this work, we have explored natural unmodified low- and high-density lipoproteins (LDL and HDL) as selective delivery vectors in colorectal cancer therapy. We show in vitro in cultured cells and in vivo (NanoSPECT/CT) in the CT-26 mice colorectal cancer model that LDLs are mainly taken up by cancer cells, while HDLs are preferentially taken up by macrophages. We loaded LDLs with cisplatin and HDLs with the heat shock protein-70 inhibitor AC1LINNC, turning them into a pair of “Trojan horses” delivering drugs selectively to their target cells as demonstrated in vitro in human colorectal cancer cells and macrophages, and in vivo. Coupling of the drugs to lipoproteins and stability was assessed by mass and raman spectrometry analysis. Cisplatin vectorized in LDLs led to better tumor growth suppression with strongly reduced adverse effects such as a renal or liver toxicity. AC1LINNC vectorized into HDLs induced a strong oxidative burst in macrophages and innate anti-cancer immune response. Cumulative anti-tumor effect was observed for both drug-loaded lipoproteins. Altogether, our data show that lipoproteins from patient’s blood can be used as natural nanocarriers allowing cell specific targeting, paving the way toward more efficient, safer and personalized use of chemo-and immunotherapeutic drugs in cance

Coumarin-Phosphine-Based Smart Probes for Tracking Biologically Relevant Metal Complexes: From Theoretical to Biological Investigations

International audienceTen metal-based complexes and associated ligands have been synthesized and characterized. One of the metal ligands is a coumarin-phosphine derivative, which displays tunable fluorescence properties. The fluorescence is quenched in the case of the free ligand and ruthenium and osmium complexes, whereas it is strong for the gold complexes and phosphonium derivatives. These trends were rationalized by theoretical calculations, which revealed non-radiative channels involving a dark state for the free ligands that is lower in energy than the emissive state and is responsible for the quenching of fluorescence. For the Ru-II and Os-II complexes, other non-radiative channels involving the manifold of singlet and triplet excited states may play a role. The anti-proliferative properties of all the compounds were evaluated in cancer cell lines (SW480, HCT116, MDA-MB-231 and MCF-7); higher IC50 values were obtained for gold(I) complexes, with the free ligands being only weakly cytotoxic

Gold( i )–BODIPY–imidazole bimetallic complexes as new potential anti-inflammatory and anticancer trackable agents

International audienceTwo new gold(I)–BODIPY–imidazole based trackable therapeutic bimetallic complexes have been synthesized and fully characterized. They display strong antiproliferative properties on several types of cancers including colon, breast, and prostate and one of them presents a significant anti-inflammatory effect. Additionally, the two compounds could be visualised in vitro by confocal microscopy in the submicromolar range

Conception and Evaluation of Fluorescent Phosphine‐Gold Complexes: From Synthesis to in vivo Investigations

International audienceA phosphine gold(I) and phosphine-phosphonium gold(I) complexes bearing a fluorescent coumarin moiety were synthesized and characterized. Both complexes displayed interesting photophysical properties: good molar absorption coefficient, good quantum yield of fluorescence, and ability to be tracked in vitro thanks to two-photon imaging. Their in vitro and in vivo biological properties were evaluated onto cancer cell lines both human and murine and into CT26 tumor-bearing BALB/c mice. They displayed moderate to strong antiproliferative properties and the phosphine-phosphonium gold(I) complex induced significant in vivo anti-cancer effect

TRIM33 prevents pulmonary fibrosis by impairing TGF-β1 signalling.

peer reviewedBACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterised by myofibroblast proliferation and abnormal extracellular matrix accumulation in the lungs. Transforming growth factor (TGF)-β1 initiates key profibrotic signalling involving the SMAD pathway and the small heat shock protein B5 (HSPB5). Tripartite motif-containing 33 (TRIM33) has been reported to negatively regulate TGF-β/SMAD signalling, but its role in fibrogenesis remains unknown. The objective of this study was to elucidate the role of TRIM33 in IPF. METHODS: TRIM33 expression was assessed in the lungs of IPF patients and rodent fibrosis models. Bone marrow-derived macrophages (BMDM), primary lung fibroblasts and 3D lung tissue slices were isolated from Trim33-floxed mice and cultured with TGF-β1 or bleomycin (BLM). Trim33 expression was then suppressed by adenovirus Cre recombinase (AdCre). Pulmonary fibrosis was evaluated in haematopoietic-specific Trim33 knockout mice and in Trim33-floxed mice that received AdCre and BLM intratracheally. RESULTS: TRIM33 was overexpressed in alveolar macrophages and fibroblasts in IPF patients and rodent fibrotic lungs. Trim33 inhibition in BMDM increased TGF-β1 secretion upon BLM treatment. Haematopoietic-specific Trim33 knockout sensitised mice to BLM-induced fibrosis. In primary lung fibroblasts and 3D lung tissue slices, Trim33 deficiency increased expression of genes downstream of TGF-β1. In mice, AdCre-Trim33 inhibition worsened BLM-induced fibrosis. In vitro, HSPB5 was able to bind directly to TRIM33, thereby diminishing its protein level and TRIM33/SMAD4 interaction. CONCLUSION: Our results demonstrate a key role of TRIM33 as a negative regulator of lung fibrosis. Since TRIM33 directly associates with HSPB5, which impairs its activity, inhibitors of TRIM33/HSPB5 interaction may be of interest in the treatment of IPF

PD-1/PD-L1 pathway: an adaptive immune resistance mechanism to immunogenic chemotherapy in colorectal cancer

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Nanofitins targeting heat shock protein 110: An innovative immunotherapeutic modality in cancer.

The presence of an inactivating heat shock protein 110 (HSP110) mutation in colorectal cancers has been correlated with an excellent prognosis and with the ability of HSP110 to favor the formation of tolerogenic (M2-like) macrophages. These clinical and experimental results suggest a potentially powerful new strategy against colorectal cancer: the inhibition of HSP110. In this work, as an alternative to neutralizing antibodies, Nanofitins (scaffold ~7 kDa proteins) targeting HSP110 were isolated from the screening of a synthetic Nanofitin library, and their capacity to bind (immunoprecipitation, biolayer interferometry) and to inhibit HSP110 was analyzed in vitro and in vivo. Three Nanofitins were found to inhibit HSP110 chaperone activity. Interestingly, they share a high degree of homology in their variable domain and target the peptide-binding domain of HSP110. In vitro, they inhibited the ability of HSP110 to favor M2-like macrophages. The Nanofitin with the highest affinity, A-C2, was studied in the CT26 colorectal cancer mice model. Our PET/scan experiments demonstrate that A-C2 may be localized within the tumor area, in accordance with the reported HSP110 abundance in the tumor microenvironment. A-C2 treatment reduced tumor growth and was associated with an increase in immune cells infiltrating the tumor and particularly cytotoxic macrophages. These results were confirmed in a chicken chorioallantoic membrane tumor model. Finally, we showed the complementarity between A-C2 and an anti-PD-L1 strategy in the in vivo and in ovo tumor models. Overall, Nanofitins appear to be promising new immunotherapeutic lead compounds