Bioprinting Soft 3D Models of Hematopoiesis using Natural Silk Fibroin-Based Bioink Efficiently Supports Platelet Differentiation.

Hematopoietic stem and progenitor cells (HSPCs) continuously generate platelets throughout one's life. Inherited Platelet Disorders affect ≈ 3 million individuals worldwide and are characterized by defects in platelet formation or function. A critical challenge in the identification of these diseases lies in the absence of models that facilitate the study of hematopoiesis ex vivo. Here, a silk fibroin-based bioink is developed and designed for 3D bioprinting. This bioink replicates a soft and biomimetic environment, enabling the controlled differentiation of HSPCs into platelets. The formulation consisting of silk fibroin, gelatin, and alginate is fine-tuned to obtain a viscoelastic, shear-thinning, thixotropic bioink with the remarkable ability to rapidly recover after bioprinting and provide structural integrity and mechanical stability over long-term culture. Optical transparency allowed for high-resolution imaging of platelet generation, while the incorporation of enzymatic sensors allowed quantitative analysis of glycolytic metabolism during differentiation that is represented through measurable color changes. Bioprinting patient samples revealed a decrease in metabolic activity and platelet production in Inherited Platelet Disorders. These discoveries are instrumental in establishing reference ranges for classification and automating the assessment of treatment responses. This model has far-reaching implications for application in the research of blood-related diseases, prioritizing drug development strategies, and tailoring personalized therapies.

Constructing a model including the cryptic sulfur cycle in Chesapeake Bay requires judicious choices for key processes and parameters.

A new biogeochemical model for Chesapeake Bay has been developed by merging two published models - the ECB model of Da et al. (2018) that has been calibrated for the Bay but only simulates nitrogen, carbon and oxygen and the BioRedoxCNPS model of al Azhar et al. (2014) and Hantsoo et al. (2018) that includes cryptic sulfur cycling. Comparison between these models shows that judicious choices are required for key processes and parameters. This manuscript documents the sources of differences between the two published models in order to select the most realistic configuration for our new model.•This study focuses on three sets of differences-processes only included in ECB (burial and dissolved organic matter), processes only included in BioRedoxCNPS (explicit dynamics for hydrogen sulfide, sulfate and nitrite, light attenuation that does not include CDOM or sediments), and differences in parameters common to the two codes.•Sensitivity studies that highlight particular choices (absorption by dissolved organic matter, nitrification rates, stoichiometric ratios) are also shown.•The new model includes sulfur cycling and has comparable skill in predicting oxygen as ECB, but also has improved simulation of nitrogen species compared with both original codes.

KRASG12C inhibition using MRTX1257: a novel radio-sensitizing partner.

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 KRASG12C mutated NSCLC and colorectal cancers. In this work, we explore the ability of MRTX1257, a KRASG12C inhibitor analogous to MRTX849, to radio-sensitize KRASG12C+/+ 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.

Factors That Predict a Sustained Humoral Response to COVID-19 Vaccines in Kidney Transplant Recipients.

INTRODUCTION: Kidney transplant recipients (KTRs) produce a weak humoral response to coronavirus disease 2019 (COVID-19) vaccines. However, the factors associated with the quality of the serological response to three doses of COVID-19 vaccine have not been unambiguously identified. METHODS: We included KTRs followed in the Nephrology Department at Amiens University Hospital (Amiens, France) between June and December 2021 who had received three doses of a COVID-19 mRNA vaccine (or two doses plus an episode of polymerase chain reaction-confirmed COVID-19). The lack of a humoral response was defined as an antibody titer below 7.1 binding antibody units (BAU)/mL, and an optimal response was defined as an antibody titer above 264 BAU/mL. RESULTS: Of the 371 patients included, 246 (66.3%) were seropositive, and 97 (26.1%) had an optimal response. In a multivariate analysis, the only factor associated with seropositivity was a history of COVID-19 [odds ratio (OR) 87.2; 95% confidence interval (CI) (7.88-965.0); p < 0.0001], while the main factors associated with non-response were female sex [OR 0.28; 95%CI (0.15-0.51); p < 0.0001], less than 36 months between kidney transplantation and vaccination [OR 0.26; 95%CI (0.13-0.52); p < 0.0001], a higher creatinine level [OR 0.33; 95%CI (0.19-0.56); p < 0.0001], the use of tacrolimus [OR 0.23; 95%CI (0.12-0.45); p < 0.0001], the use of belatacept [OR 0.01; 95%CI (0.001-0.20); p = 0.002] and three-drug immunosuppression [OR 0.39; 95%CI (0.19-0.78); p = 0.015]. A history of COVID-19 was associated with an optimal response [OR 4.03; 95%CI (2.09-7.79); p < 0.0001], while an older age at vaccination [OR 0.97; 95%CI (0.95-0.99); p = 0.002], less than 36 months between kidney transplantation and vaccination [OR 0.35; 95%CI (0.18-0.69); p = 0.002], a higher creatinine level [OR 0.60; 95%CI (0.38-0.93); p = 0.02], three-drug immunosuppression [OR 0.45; 95%CI (0.27-0.76); p = 0.003] were associated with a poorer response. CONCLUSION: We identified factors associated with a humoral response to a COVID-19 mRNA vaccine in KTRs. These findings might help physicians to optimize vaccination in KTRs.

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.

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

Chimeric 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.

Radiomics to evaluate interlesion heterogeneity and to predict lesion response and patient outcomes using a validated signature of CD8 cells in advanced melanoma patients treated with anti-PD1 immunotherapy.

PURPOSE: While there is still a significant need to identify potential biomarkers that can predict which patients are most likely to respond to immunotherapy treatments, radiomic approaches have shown promising results. The objectives of this study were to evaluate whether a previously validated radiomics signature of CD8 T-cells could predict progressions at a lesion level and whether the spatial heterogeneity of this radiomics score could be used at a patient level to assess the clinical response and survival of melanoma patients. METHODS: Clinical data from patients with advanced melanoma treated in our center with immunotherapy were retrieved. Radiomic features were extracted and the CD8 radiomics signature was applied. A progressive lesion was defined by an increase in lesion size of 20% or more. Dispersion metrics of the radiomics signature were estimated to evaluate the impact of interlesion heterogeneity on patient's response. Fine-tuned cut-offs for predicting overall survival were evaluated after splitting data into training and test sets. RESULTS: A total of 136 patients were included in this study, with 1120 segmented lesions at baseline, and 1052 lesions at first evaluation. A low CD8 radiomics score at baseline was associated with a significantly higher risk of lesion progression (AUC=0.55, p=0.0091), especially for lesions larger than >1 mL (AUC=0.59 overall, p=0.0035, with AUC=0.75, p=0.002 for subcutaneous lesions, AUC=0.68, p=0.01, for liver lesions and AUC=0.62, p=0.03 for nodes). The least infiltrated lesion according to the radiomics score of CD8 T-cells was positively associated with overall survival (training set HR=0.31, p=0.00062, test set HR=0.28, p=0.016), which remained significant in a multivariate analysis including clinical and biological variables. CONCLUSIONS: These results confirm the predictive value at a lesion level of the biologically inspired CD8 radiomics score in melanoma patients treated with anti-PD1-based immunotherapy and may be interesting to assess the disease spatial heterogeneity to evaluate the patient prognosis with potential clinical implication such as tumor selection for focal ablative therapies.

Nitrogen reductions have decreased hypoxia in the Chesapeake Bay: Evidence from empirical and numerical modeling.

Seasonal hypoxia is a characteristic feature of the Chesapeake Bay due to anthropogenic nutrient input from agriculture and urbanization throughout the watershed. Although coordinated management efforts since 1985 have reduced nutrient inputs to the Bay, oxygen concentrations at depth in the summer still frequently fail to meet water quality standards that have been set to protect critical estuarine living resources. To quantify the impact of watershed nitrogen reductions on Bay hypoxia during a recent period including both average discharge and extremely wet years (2016-2019), this study employed both statistical and three-dimensional (3-D) numerical modeling analyses. Numerical model results suggest that if the nitrogen reductions since 1985 had not occurred, annual hypoxic volumes (O2 < 3 mg L-1) would have been ~50-120% greater during the average discharge years of 2016-2017 and ~20-50% greater during the wet years of 2018-2019. The effect was even greater for O2 < 1 mg L-1, where annual volumes would have been ~80-280% greater in 2016-2017 and ~30-100% greater in 2018-2019. These results were supported by statistical analysis of empirical data, though the magnitude of improvement due to nitrogen reductions was greater in the numerical modeling results than in the statistical analysis. This discrepancy is largely accounted for by warming in the Bay that has exacerbated hypoxia and offset roughly 6-34% of the improvement from nitrogen reductions. Although these results may reassure policymakers and stakeholders that their efforts to reduce hypoxia have improved ecosystem health in the Bay, they also indicate that greater reductions are needed to counteract the ever-increasing impacts of climate change.

Ablation of collagen VI leads to the release of platelets with altered function.

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI-related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI-null mouse model (Col6a1-/-), we found that collagen VI-null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1-/- megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1-/- mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1-/- mice. Peripheral blood platelets of patients affected by collagen VI-related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI-related diseases.

Miniaturized 3D bone marrow tissue model to assess response to Thrombopoietin-receptor agonists in patients.

Thrombocytopenic disorders have been treated with the Thrombopoietin-receptor agonist Eltrombopag. Patients with the same apparent form of thrombocytopenia may respond differently to the treatment. We describe a miniaturized bone marrow tissue model that provides a screening bioreactor for personalized, pre-treatment response prediction to Eltrombopag for individual patients. Using silk fibroin, a 3D bone marrow niche was developed that reproduces platelet biogenesis. Hematopoietic progenitors were isolated from a small amount of peripheral blood of patients with mutations in ANKRD26 and MYH9 genes, who had previously received Eltrombopag. The ex vivo response was strongly correlated with the in vivo platelet response. Induced Pluripotent Stem Cells (iPSCs) from one patient with mutated MYH9 differentiated into functional megakaryocytes that responded to Eltrombopag. Combining patient-derived cells and iPSCs with the 3D bone marrow model technology allows having a reproducible system for studying drug mechanisms and for individualized, pre-treatment selection of effective therapy in Inherited Thrombocytopenias.

Platelets are tiny cell fragments essential for blood to clot. They are created and released into the bloodstream by megakaryocytes, giant cells that live in the bone marrow. In certain genetic diseases, such as Inherited Thrombocytopenia, the bone marrow fails to produce enough platelets: this leaves patients extremely susceptible to bruising, bleeding, and poor clotting after an injury or surgery. Certain patients with Inherited Thrombocytopenia respond well to treatments designed to boost platelet production, but others do not. Why these differences exist could be investigated by designing new test systems that recreate the form and function of bone marrow in the laboratory. However, it is challenging to build the complex and poorly understood bone marrow environment outside of the body. Here, Di Buduo et al. have developed an artificial three-dimensional miniature organ bioreactor system that recreates the key features of bone marrow. In this system, megakaryocytes were grown from patient blood samples, and hooked up to a tissue scaffold made of silk. The cells were able to grow as if they were in their normal environment, and they could shed platelets into an artificial bloodstream. After treating megakaryocytes with drugs to stimulate platelet production, Di Buduo et al. found that the number of platelets recovered from the bioreactor could accurately predict which patients would respond to these drugs in the clinic. This new test system enables researchers to predict how a patient will respond to treatment, and to tailor therapy options to each individual. This technology could also be used to test new drugs for Inherited Thrombocytopenias and other blood-related diseases; if scaled-up, it could also, one day, generate large quantities of lab-grown blood cells for transfusion.

Effects of reduced shoreline erosion on Chesapeake Bay water clarity.

Shoreline erosion supplies sediments to estuaries and coastal waters, influencing water clarity and primary production. Globally, shoreline erosion sediment inputs are changing with anthropogenic alteration of coastlines in populated regions. Chesapeake Bay, a prime example of such a system where shoreline erosion accounts for a large proportion of sediments entering the estuary, serves here as a case study for investigating the effects of changing sediment inputs on water clarity. Long-term increases in shoreline armoring have contributed to decreased erosional sediment inputs to the estuary, changing the composition of suspended particles in surface waters. This study examined the impact of shoreline erosion on water clarity using a coupled hydrodynamic-biogeochemical model. Experiments were conducted to simulate realistic shoreline conditions representative of the early 2000s, increased shoreline erosion, and highly armored shorelines. Together, reduced shoreline erosion and the corresponding reduced rates of resuspension result in decreased concentrations of inorganic particles, improving water clarity particularly in the lower Bay and in dry years where and when riverine sediment influence is low. This clarity improvement relaxed light limitation, which increased organic matter production. Differences between the two extreme experiments revealed that in the mid-estuary in February to April, surface inorganic suspended sediment concentrations decreased 3-7 mg L-1, while organic suspended solids increased 1-3 mg L-1. The resulting increase in the organic-to-inorganic ratio often had opposite effects on clarity according to different metrics, improving clarity in mid-Bay central channel waters in terms of light attenuation depth, but simultaneously degrading clarity in terms of Secchi depth because the resulting increase in organic suspended solids decreased the water's transparency. This incongruous water clarity effect, the spatial extent of which is defined here as an Organic Fog Zone, was present in February to April in all years studied, but occurred farther south in wet years.

Challenges in Quantifying Air-Water Carbon Dioxide Flux Using Estuarine Water Quality Data: Case Study for Chesapeake Bay.

Estuaries play an uncertain but potentially important role in the global carbon cycle via CO2 outgassing. The uncertainty mainly stems from the paucity of studies that document the full spatial and temporal variability of estuarine surface water partial pressure of carbon dioxide ( pCO2). Here, we explore the potential of utilizing the abundance of pH data from historical water quality monitoring programs to fill the data void via a case study of the mainstem Chesapeake Bay (eastern United States). We calculate pCO2 and the air-water CO2 flux at monthly resolution from 1998 to 2018 from tidal fresh to polyhaline waters, paying special attention to the error estimation. The biggest error is due to the pH measurement error, and errors due to the gas transfer velocity, temporal sampling, the alkalinity mixing model, and the organic alkalinity estimation are 72%, 27%, 15%, and 5%, respectively, of the error due to pH. Seasonal, interannual, and spatial variability in the air-water flux and surface pCO2 is high, and a correlation analysis with oxygen reveals that this variability is driven largely by biological processes. Averaged over 1998-2018, the mainstem bay is a weak net source of CO2 to the atmosphere of 1.2 (1.1, 1.4) mol m-2 yr-1 (best estimate and 95% confidence interval). Our findings suggest that the abundance of historical pH measurements in estuaries around the globe should be mined in order to constrain the large spatial and temporal variability of the CO2 exchange between estuaries and the atmosphere.

Mechanisms of platelet release: in vivo studies and in vitro modeling.

Mechanisms related to platelet release in the context of the bone marrow niche are not completely known. In this review we discuss what has been discovered about four critical aspects of this process: 1) the bone marrow niche organization, 2) the role of the extracellular matrix components, 3) the mechanisms by which megakaryocytes release platelets and 4) the novel approaches to mimic the bone marrow environment and produce platelets ex vivo.

Improvement in Tuberculosis Outcomes With a Combined Medical and Social Approach.

Setting: Studies performed locally in Switzerland in the late eighties reported unsatisfactory treatment outcomes. Better outcomes were observed since the introduction of directly observed therapy (DOT) in the late nineties and improvement in social support in recent years. Design: retrospective study of treatment outcomes for all tuberculosis (TB) patients notified in Vaud County (VD), Switzerland, between, 1st of January 2010 and 31st of December of 2014. Results: 375 patients were notified in VD during the study period. The global outcome was successful in 90.1% of patients (338/375). In 183 culture and PCR positive pulmonary TB, the documented cure rate was 57.9% (106/183), and the treatment completion was 59/183 (32.2%), i.e., a treatment success of 90.2%. DOT was applied globally in 234/375 (62.4%) and in 64/67 of the asylum seekers (AS) (95.5%) followed at the dispensary. Treatment outcomes were successful in 60/67 (89.6%) AS. Discussion: Improvements in tuberculosis outcomes resulted not only from the introduction of DOT in VD in the nineties but also from a change in the management, with increased attention to the social problems faced by the migrants. Conclusion: A combined medical and social approach of TB care in VD improved treatment outcomes.

Optimized fractionated radiotherapy with anti-PD-L1 and anti-TIGIT: a promising new combination.

PURPOSE/OBJECTIVE: Radiotherapy (RT) induces an immunogenic antitumor response, but also some immunosuppressive barriers. It remains unclear how different fractionation protocols can modulate the immune microenvironment. Clinical studies are ongoing to evaluate immune checkpoint inhibitors (ICI) in association with RT. However, only few trials aim to optimize the RT fractionation to improve efficacy of these associations. Here we sought to characterize the effect of different fractionation protocols on immune response with a view to associating them with ICI. MATERIALS/METHODS: Mice bearing subcutaneous CT26 colon tumors were irradiated using a SARRP device according to different radiation schemes with a same biologically effective dose. Mice were monitored for tumor growth. The radiation immune response (lymphoid, myeloid cells, lymphoid cytokines and immune checkpoint targets) was monitored by flow cytometry at different timepoints after treatment and by RNA sequencing analysis (RNAseq). The same radiation protocols were performed with and without inhibitors of immune checkpoints modulated by RT. RESULTS: In the absence of ICI, we showed that 18x2Gy and 3x8Gy induced the longest tumor growth delay compared to 1×16.4Gy. While 3x8Gy and 1×16.4Gy induced a lymphoid response (CD8+ T-cells, Regulators T-cells), 18x2Gy induced a myeloid response (myeloid-derived suppressor cells, tumor-associated macrophages 2). The secretion of granzyme B by CD8+ T cells was increased to a greater extent with 3x8Gy. The expression of PD-L1 by tumor cells was moderately increased by RT, but most durably with 18x2Gy. T cell immunoreceptor with Ig and ITIM domains (TIGIT) expression by CD8+ T-cells was increased with 3x8Gy, but decreased with 18x2Gy. These results were also observed with RNAseq. RT was dramatically more effective with 3x8Gy compared to all the other treatments schemes when associated with anti-TIGIT and anti-PD-L1 (9/10 mice in complete response). The association of anti-PD-L1 and RT was also effective in the 18x2Gy group (8/12 mice in complete response). CONCLUSION: Each fractionation scheme induced different lymphoid and myeloid responses as well as various modulations of PD-L1 and TIGIT expression. Furthermore, 3x8Gy was the most effective protocol when associated with anti-PD-L1 and anti-TIGIT. This is the first study combining RT and anti-TIGIT with promising results; further studies are warranted.

The 6th R of Radiobiology: Reactivation of Anti-Tumor Immune Response.

Historically, the 4Rs and then the 5Rs of radiobiology explained the effect of radiation therapy (RT) fractionation on the treatment efficacy. These 5Rs are: Repair, Redistribution, Reoxygenation, Repopulation and, more recently, intrinsic Radiosensitivity. Advances in radiobiology have demonstrated that RT is able to modify the tumor micro environment (TME) and to induce a local and systemic (abscopal effect) immune response. Conversely, RT is able to increase some immunosuppressive barriers, which can lead to tumor radioresistance. Fractionation and dose can affect the immunomodulatory properties of RT. Here, we review how fractionation, dose and timing shape the RT-induced anti-tumor immune response and the therapeutic effect of RT. We discuss how immunomodulators targeting immune checkpoint inhibitors and the cGAS/STING (cyclic GMP-AMP Synthase/Stimulator of Interferon Genes) pathway can be successfully combined with RT. We then review current trials evaluating the RT/Immunotherapy combination efficacy and suggest new innovative associations of RT with immunotherapies currently used in clinic or in development with strategic schedule administration (fractionation, dose, and timing) to reverse immune-related radioresistance. Overall, our work will present the existing evidence supporting the claim that the reactivation of the anti-tumor immune response can be regarded as the 6th R of Radiobiology.

Impact of PI3Kα (Phosphoinositide 3-Kinase Alpha) Inhibition on Hemostasis and Thrombosis.

Objective- PI3Kα (phosphoinositide 3-kinase alpha) is a therapeutic target in oncology, but its role in platelets and thrombosis remains ill characterized. In this study, we have analyzed the role of PI3Kα in vitro, ex vivo, and in vivo in 2 models of arterial thrombosis. Approach and Results- Using mice selectively deficient in p110α in the megakaryocyte lineage and isoform-selective inhibitors, we confirm that PI3Kα is not mandatory but participates to thrombus growth over a collagen matrix at arterial shear rate. Our data uncover a role for PI3Kα in low-level activation of the GP (glycoprotein) VI-collagen receptor by contributing to ADP secretion and in turn full activation of PI3Kß and Akt/PKB (protein kinase B). This effect was no longer observed at high level of GP VI agonist concentration. Our study also reveals that over a vWF (von Willebrand factor) matrix, PI3Kα regulates platelet stationary adhesion contacts under arterial flow through its involvement in the outside-in signaling of vWF-engaged αIIbß3 integrin. In vivo, absence or inhibition of PI3Kα resulted in a modest but significant decrease in thrombus size after superficial injuries of mouse mesenteric arteries and an increased time to arterial occlusion after carotid lesion, without modification in the tail bleeding time. Considering the more discrete and nonredundant role of PI3Kα compared with PI3Kß, selective PI3Kα inhibitors are unlikely to increase the bleeding risk at least in the absence of combination with antiplatelet drugs or thrombopenia. Conclusions- This study provides mechanistic insight into the role of PI3Kα in platelet activation and arterial thrombosis.