Etude de l’interaction entre la réponse inflammatoire et la fibrose induite par radiothérapie

La fibrose radio-induite (FRI) est une complication tardive de la radiothérapie souvent associée à une réponse inflammatoire chronique et à un infiltrat de macrophages. Aujourd’hui, les macrophages sont pressentis comme des médiateurs cellulaires important dans le processus de fibrose mais leur rôle n’a jamais été étudié dans le contexte de la FRI. Dans une précédente étude nous avions montré que l’irradiation (IR) induit une polarisation M1 des macrophages cardiaques après irradiation de souris ApoE-/- et est associée un score de fibrose élevé, ce qui suggérait que la polarisation des macrophages pourrait contribuer à la fibrogénèse radio-induite. Afin de valider cette hypothèse, nous avons cherché à caractériser le rôle des macrophages dans la FRI en utilisant un modèle classique de fibrose pulmonaire chez la souris C57Bl/6 induit après IR thoracique à 16Gy. Nous avons caractérisé les phénotypes et la fonction des macrophages alvéolaires (MA) et interstitiels (MI). Durant la phase précoce, les résultats montrent une déplétion des MA accompagnée de la sécrétion de CXCL1, MCP-1 et de MCSF. Cette déplétion est suivie d’une repopulation suite au recrutement et à la prolifération des monocytes/macrophages d’origine médullaire. La nouvelle population de MA présente une polarisation hybride accompagnée d’une augmentation simultanée de la sécrétion de cytokines Th1 et Th2. Durant la phase tardive les MI présentent une polarisation de type M2 accompagnée d’une diminution des cytokines Th1 et d’une augmentation de cytokines Th2 dans le lysat tissulaire. Nous avons ensuite cherché à caractériser la contribution des MA hybrides vs MI M2 dans le processus de fibrose. Nous avons montré que contrairement au MA hybrides, les MI M2 étaient capables d’induire l’activation des fibroblastes in vitro et l’expression de TGF-β1. De plus, la déplétion des MA hybrides avec une administration intranasale de clodronate exacerbe la FRI et induit l’augmentation de l’infiltrat de MI M2. Ensuite, nous nous somme interrogés à la contribution du processus de fibrose dans la polarisation des macrophages. Après 24h de coculture entre fibroblastes irradiés et macrophages pulmonaires non irradiés, une sécrétion de cytokines telles que M-CSF et TIMP-1 qui pourraient stimuler l’activation des fibroblastes est observée. De plus, l’inhibition de la FRI avec de la pravastatine montre que l’inhibition de la fibrose est accompagnée d’une augmentation des MI M1 et d’une diminution des MI M2 dans le poumon. En résumé, nos résultats montrent une contribution opposée des Macrophages Alvéolaires et des Macrophages Interstitiels dans le processus de fibrose radio-induite ainsi qu’une contribution du processus de fibrose dans le type d’activation des Macrophages interstitiels formant ainsi une boucle d’activation fibrogénique chronique.Radiation-induced fibrosis (RIF) is a delayed complication of radiotherapy often associated with chronic inflammatory process and macrophage infiltration. Nowadays, macrophages are suggested to be important cellular contributors to fibrogenic process, but their implication in the context of RIF has never been investigated. In a previous study we have shown that irradiation (IR) induced the polarization of cardiac macrophages into M1 in ApoE-/- mice and was associated with a high fibrosis score in ApoE-/- mice, suggesting that macrophage polarization could drive tissue sensitivity to ionizing radiation. This observation prompted us to investigate the role of macrophages in RIF using a classical experimental model of lung fibrosis developed in C57Bl/6 mice after 16Gy thorax-IR. We profiled both alveolar macrophages (AM) and interstitial macrophages (IM). During the acute phase we found AM depletion associated with CXCL1, MCP-1 and M-CSF secretion, followed by a repopulation phase mediated by recruitment and proliferation of monocytes/macrophages from the bone marrow. Interestingly, the newly recruited AM exhibited a yet never described hybrid polarization (M1/M2), associated with the up-regulation of both Th1 and Th2 cytokines. At delayed times points, IM were M2-polarized and associated with downregulation of Th1 cytokines and upregulation of Th2 cytokines in tissue lysates. These results suggest a differential contribution of hybrid AM vs M2 IM to fibrogenesis. Interestingly, in contrast to activated hybrid AM, activated M2 IM were able to induce fibroblast activation in vitro mediated by an enhanced TGF-β1 expression. Therefore, specific depletion of hybrid AM using intranasal administration of clodrosome increased RIF score and enhanced M2 IM infiltration. We next evaluated if the fibrogenic process can in turn affect macrophage polarization. Interestingly, after coculture of irradiated fibroblast with non-irradiated pulmonary macrophages, secretion of cytokines such as M-CSF and TIMP-1, which can stimulate macrophage activation, was observed. Furthermore, RIF inhibition using pravastatin treatment showed that fibrosis inhibition was associated with a decrease in M2 IM accompanied by an increase in M1 IM, but had no effect on polarization of AM. These present study shows a dual and opposite contribution of alevolar versus intertitial macrophages in RIF and the contribution of the fibrogenic process to IM polarization, resulting thereby in a chronical fibrogenic loop

Radiotherapy–immunotherapy combinations – perspectives and challenges

International audienceIonizing radiation was historically used to treat cancer to kill tumour cells, in particular by inducing DNA damage. This view of radiotherapy (RT) as a simple cytotoxic agent has dramatically changed in the last years, and it is now widely accepted that RT can deeply reshape the tumour environment, in particular by modulating the immune response. Such evidence gives a strong rationale to use immunomodulators to boost the therapeutic value of RT, opening the era of “immunoradiotherapy”. The increasing amount of preclinical and clinical data concerning the combination of immunomodulators with RT, and in particular with immune checkpoints inhibitors as anti-PD1/PDL1 and anti-CTLA4, reflects the interest of the scientific and medical community concerning immunoradiotherapy. The expectations are enormous, since the rational for performing such combinations is strong, and the possibility to use a local treatment as RT to amplify a systemic antitumor response, as in the cases of the abscopal effect. Nevertheless, several points remain to be addresses as the need to find biomarkers to selected patients that will benefit from immunoradiotherapy, the identification of the best sequences/schedules for combination with immunomodulators, how to overcome resistance. Also, the effects of immunoradiotherapy on healthy tissues and related toxicity remains largely unexplored. To answers these critical questions and make immunoradiotherapy keep its promises, large efforts are needed from both the pharmaceutical industry and the academic/governmental research. Moreover, thanks to the work of both these players, the arsenal of available immunomodulators is quickly expanding, thus opening the field to more and more combinations with RT. We thus forecast that the field of immunoradiotherapy will further expand in the next years, and it needs to be supported by appropriate investment plans

Radiotherapy as a means to increase the efficacy of T-cell therapy in solid tumors

ABSTRACTChimeric antigen receptor (CAR)-T cells have demonstrated significant improvements in the treatment of refractory B-cell malignancies that previously showed limited survival. In contrast, early-phase clinical studies targeting solid tumors have been disappointing. This may be due to both a lack of specific and homogeneously expressed targets at the surface of tumor cells, as well as intrinsic properties of the solid tumor microenvironment that limit homing and activation of adoptive T cells. Faced with these antagonistic conditions, radiotherapy (RT) has the potential to change the overall tumor landscape, from depleting tumor cells to reshaping the tumor microenvironment. In this article, we describe the current landscape and discuss how RT may play a pivotal role for enhancing the efficacy of adoptive T-cell therapies in solid tumors. Indeed, by improving homing, expansion and activation of infused T cells while reducing tumor volume and heterogeneity, the use of RT could help the implementation of engineered T cells in the treatment of solid tumors

Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

Background Irradiation (IR) and immune checkpoint inhibitor (ICI) combination is a promising treatment modality. However, local and distance treatment failure and resistance can occur. To counteract this resistance, several studies propose CD73, an ectoenzyme, as a potential target to improve the antitumor efficiency of IR and ICI. Although CD73 targeting in combination with IR and ICI has shown attractive antitumor effects in preclinical models, the rationale for CD73 targeting based on CD73 tumor expression level deserves further investigations.Methods Here we evaluated for the first time the efficacy of two administration regimens of CD73 neutralizing antibody (one dose vs four doses) in combination with IR according to the expression level of CD73 in two subcutaneous tumor models expressing different levels of CD73.Results We showed that CD73 is weakly expressed by MC38 tumors even after IR, when compared with the TS/A model that highly expressed CD73. Treatment with four doses of anti-CD73 improved the TS/A tumor response to IR, while it was ineffective against the CD73 low-expressing MC38 tumors. Surprisingly, a single dose of anti-CD73 exerted a significant antitumor activity against MC38 tumors. On CD73 overexpression in MC38 cells, four doses of anti-CD73 were required to improve the efficacy of IR. Mechanistically, a correlation between a downregulation of iCOS expression in CD4+ T cells and an improved response to IR after anti-CD73 treatment was observed and iCOS targeting could restore an impaired benefit from anti-CD73 treatment.Conclusions These data emphasize the importance of the dosing regimen for anti-CD73 treatment to improve tumor response to IR and identify iCOS as part of the underlying molecular mechanisms. Our data suggest that the selection of appropriate dosing regimen is required to optimize the therapeutic efficacy of immunotherapy–radiotherapy combinations

NADPH oxidase DUOX1 sustains TGF-β1 signalling and promotes lung fibrosis

International audienceInterstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of pulmonary fibrosis, and is governed by transforming growth factor (TGF)-β1/Smad signalling. TGF-β1 and oxidative stress cooperate to drive fibrosis. Cells can produce reactive oxygen species through activation and/or induction of NADPH oxidases, such as dual oxidase (DUOX1/2). Since DUOX enzymes, as extracellular hydrogen peroxide (H 2 O 2­­ )-generating systems, are involved in extracellular matrix formation and in wound healing in different experimental models, we hypothesised that DUOX-based NADPH oxidase plays a role in the pathophysiology of pulmonary fibrosis. Our in vivo data (idiopathic pulmonary fibrosis patients and mouse models of lung fibrosis) showed that the NADPH oxidase DUOX1 is induced in response to lung injury. DUOX1-deficient mice (DUOX1 +/− and DUOX1 −/− ) had an attenuated fibrotic phenotype. In addition to being highly expressed at the epithelial surface of airways, DUOX1 appears to be well expressed in the fibroblastic foci of remodelled lungs. By using primary human and mouse lung fibroblasts, we showed that TGF-β1 upregulates DUOX1 and its maturation factor DUOXA1 and that DUOX1-derived H 2 O 2 promoted the duration of TGF-β1-activated Smad3 phosphorylation by preventing phospho-Smad3 degradation. Analysis of the mechanism revealed that DUOX1 inhibited the interaction between phospho-Smad3 and the ubiquitin ligase NEDD4L, preventing NEDD4L-mediated ubiquitination of phospho-Smad3 and its targeting for degradation. These findings highlight a role for DUOX1-derived H 2 O 2 in a positive feedback that amplifies the signalling output of the TGF-β1 pathway and identify DUOX1 as a new therapeutic target in pulmonary fibrosis

KRASG12C inhibition using MRTX1257: a novel radio-sensitizing partner

Abstract Background KRAS activating mutations are considered the most frequent oncogenic drivers and are correlated with radio-resistance in multiple cancers including non-small cell lung cancer (NSCLC) and colorectal cancer. Although KRAS was considered undruggable until recently, several KRAS inhibitors have recently reached clinical development. Among them, MRTX849 (Mirati Therapeutics) showed encouraging clinical outcomes for the treatment of selected patients with KRAS G12C mutated NSCLC and colorectal cancers. In this work, we explore the ability of MRTX1257, a KRASG12C inhibitor analogous to MRTX849, to radio-sensitize KRAS G12C+/+ mutated cell lines and tumors. Methods Both in vitro and in vivo models of radiotherapy (RT) in association with MRTX1257 were used, with different RAS mutational profiles. We assessed in vitro the radio-sensitizing effect of MRTX1257 in CT26 KRASG12C+/+, CT26 WT, LL2 WT and LL2 NRAS KO (LL2 NRAS−/−) cell lines. In vivo, we used syngeneic models of subcutaneous CT26 KRASG12C+/+ tumors in BALB/c mice and T cell deficient athymic nu/nu mice to assess both the radio-sensitizing effect of MRTX1257 and its immunological features. Results MRTX1257 was able to radio-sensitize CT26 KRASG12C+/+ cells in vitro in a time and dose dependent manner. Moreover, RT in association with MRTX1257 in BALB/c mice bearing CT26 KRASG12C+/+ subcutaneous tumors resulted in an observable cure rate of 20%. However, no durable response was observed with similar treatment in athymic nude mice. The analysis of the immune microenvironment of CT26 KRASG12C+/+ tumors following RT and MRTX1257 showed an increase in the proportion of various cell subtypes including conventional CD4 + T cells, dendritic cells type 2 (cDC2) and inflammatory monocytes. Furthermore, the expression of PD-L1 was dramatically down-regulated within both tumor and myeloid cells, thus illustrating the polarization of the tumor microenvironment towards a pro-inflammatory and anti-tumor phenotype following the combined treatment. Conclusion This work is the first to demonstrate in vitro as in vivo the radio-sensitizing effect of MRTX1257, a potent KRASG12C inhibitor compatible with oral administration, in CT26 KRASG12C mutated cell lines and tumors. This is a first step towards the use of new combinatorial strategies using KRAS inhibitors and RT in KRASG12C mutated tumors, which are the most represented in NSCLC with 14% of patients harboring this mutational profile

TGFβ receptor inhibition unleashes interferon-β production by tumor-associated macrophages and enhances radiotherapy efficacy

International audienceBackground Transforming growth factor-beta (TGFβ) can limit the efficacy of cancer treatments, including radiotherapy (RT), by inducing an immunosuppressive tumor environment. The association of TGFβ with impaired T cell infiltration and antitumor immunity is known, but the mechanisms by which TGFβ participates in immune cell exclusion and limits the efficacy of antitumor therapies warrant further investigations. Methods We used the clinically relevant TGFβ receptor 2 (TGFβR2)-neutralizing antibody MT1 and the small molecule TGFβR1 inhibitor LY3200882 and evaluated their efficacy in combination with RT against murine orthotopic models of head and neck and lung cancer. Results We demonstrated that TGFβ pathway inhibition strongly increased the efficacy of RT. TGFβR2 antibody upregulated interferon beta expression in tumor-associated macrophages within the irradiated tumors and favored T cell infiltration at the periphery and within the core of the tumor lesions. We highlighted that both the antitumor efficacy and the increased lymphocyte infiltration observed with the combination of MT1 and RT were dependent on type I interferon signaling. Conclusions These data shed new light on the role of TGFβ in limiting the efficacy of RT, identifying a novel mechanism involving the inhibition of macrophage-derived type I interferon production, and fostering the use of TGFβR inhibition in combination with RT in therapeutic strategies for the management of head and neck and lung cancer

Low Doses of Radiation Increase the Immunosuppressive Profile of Lung Macrophages During Viral Infection and Pneumonia

International audiencePurpose: Severe pneumonia and acute respiratory distress syndrome (ARDS) have been described in patients with severe coronavirus disease 2019 (COVID-19). Recently, early clinical data reported the feasibility of low doses of radiation therapy (RT) in the treatment of ARDS in patients with severe COVID-19. However, the involved mechanisms remained unknown. Methods and Materials: Here, we used airways-instilled lipopolysaccharide (LPS) and influenza virus (H1N1) as murine models of pneumonia, and toll-like receptor (TLR)-3 stimulation in human lung macrophages. Results: Low doses of RT (0.5-1 Gray) decreased LPS-induced pneumonia, and increased the percentage of nerve- and airway-associated macrophages producing interleukin (IL) 10. During H1N1 viral infection, we observed decreased lung tissue damage and immune cell infiltration in irradiated animals. Low doses of RT increased IL-10 production by infiltrating immune cells into the lung. Irradiation of TLR-3 ligand-stimulated human lung macrophages ex vivo increased IL-10 secretion and decreased interferon γ production in the culture supernatant. The percentage of human lung macrophages producing IL-6 was also decreased. Conclusions: Our data highlight a mechanism by which low doses of RT regulate lung inflammation and skew lung macrophages toward an anti-inflammatory profile. These data provide a preclinical mechanistic support to clinical trials evaluating low doses of RT, such as COVID-19-induced ARDS

Histological analysis of control and irradiated hearts 20, 40 and 60weeks post-irradiation.

<p><b>A</b> and <b>B.</b> Cardiomyocyte surface area measurement was performed on 180–200 cardiomyocytes from 4 serial slides (HES heart sections) of C57Bl6 and ApoE−/− respectively (n = 3–5 animals in each group), 0 Gy 20weeks vs 0 Gy 40weeks; * P<0.05; 0 Gy 20weeks vs 0 Gy 60weeks; * P<0.05; 0 Gy vs irradiated groups at 20weeks; £ P<0.05, ^££ P<0.01; 0 Gy vs irradiated groups at 40weeks; ^μ P<0.05, ^μμ P<0.01, 0 Gy vs irradiated groups at 60weeks; ^$ P<0.05, ^$$$ P<0.001. <b>C</b> and <b>D.</b> Histological assessment of cardiac ventricular pathology by HES staining in C57Bl6 and ApoE−/− respectively (n = 4–6 animals in each group and 2 sections of heart per animal). Original magnification ×400, <b>C</b> in C57Bl6 and <b>D</b> in ApoE−/−.</p