Superior antitumor immune response achieved with proton over photon immunoradiotherapy is amplified by the nanoradioenhancer NBTXR3.

Recent findings suggest that immunoradiotherapy (IRT), combining photon radiotherapy (XRT) or proton radiotherapy (PRT) with immune checkpoint blockade, can enhance systemic tumor control. However, the comparative efficacy of XRT and PRT in IRT remains understudied. To address this, we compared outcomes between XRT + αPD1 and PRT + αPD1 in murine αPD1-resistant lung cancer (344SQR). We also assessed the impact of the nanoparticle radioenhancer NBTXR3 on both XRT + αPD1 and PRT + αPD1 for tumor control and examined the tumor immune microenvironment using single-cell RNA sequencing (scRNAseq). Additionally, mice cured by NBTXR3 + PRT + αPD1 were rechallenged with three lung cancer cell lines to evaluate memory antitumor immunity. PRT + αPD1 showed superior local tumor control and abscopal effects compared to XRT + αPD1. NBTXR3 + PRT + αPD1 significantly outperformed NBTXR3 + XRT + αPD1 in tumor control, promoting greater infiltration of antitumor lymphocytes into irradiated tumors. Unirradiated tumors treated with NBTXR3 + PRT + αPD1 had more NKT cells, CD4 T cells, and B cells, with fewer Tregs, than those treated with NBTXR3 + XRT + αPD1. NBTXR3 + PRT + αPD1 also stimulated higher expression of IFN-γ, GzmB, and Nkg7 in lymphocytes, reduced the TGF-ß pathway, and increased tumor necrosis factor alpha expression compared to NBTXR3 + XRT + αPD1. Moreover, NBTXR3 + PRT + αPD1 resulted in greater M1 macrophage polarization in both irradiated and unirradiated tumors. Mice achieving remission through NBTXR3 + PRT + αPD1 exhibited a robust memory immune response, effectively inhibiting growth of subsequent tumors from three distinct lung cancer cell lines. Proton IRT combined with NBTXR3 offers enhanced tumor control and survival rates over photon-based treatments in managing αPD1-resistant lung cancer, indicating its potential as a potent systemic therapy.

Position paper of diagnosis and treatment of post-extubation laryngitis in children: a multidisciplinary expert-based opinion.

OBJECTIVES: To make recommendations on the diagnosis and treatment of post-extubation laryngitis (PEL) in children with or without other comorbidities. METHODS: A three-iterative modified Delphi method was applied. Specialists were recruited representing pediatric otolaryngologists, pediatric and neonatal intensivists. Questions and statements approached topics encompassing definition, diagnosis, endoscopic airway evaluation, risk factors, comorbidities, management, and follow-up. A consensus was defined as a supermajority >70%. RESULTS: Stridor was considered the most frequent symptom and airway endoscopy was recommended for definitive diagnosis. Gastroesophageal reflux and previous history of intubation were considered risk factors. Specific length of intubation did not achieve a consensus as a risk factor. Systemic corticosteroids should be part of the medical treatment and dexamethasone was the drug of choice. No consensus was achieved regarding dosage of corticosteroids, although endoscopic findings help defining dosage and length of treatment. Non-invasive ventilation, laryngeal rest, and use of comfort sedation scales were recommended. Indications for microlaryngoscopy and bronchoscopy under anesthesia were symptoms progression or failure to improve after the first 72-h of medical treatment post-extubation, after two failed extubations, and/or suspicion of severe lesions on flexible fiberoptic laryngoscopy. CONCLUSIONS: Management of post-extubation laryngitis is challenging and can be facilitated by a multidisciplinary approach. Airway endoscopy is mandatory and impacts decision-making, although there is no consensus regarding dosage and length of treatment.

Radiotherapy-activated NBTXR3 nanoparticles promote ferroptosis through induction of lysosomal membrane permeabilization.

PURPOSE: Radiotherapy-activated NBTXR3 (NBTXR3 + RT) has demonstrated superior efficacy in cancer cell destruction and tumor growth control, compared to radiotherapy (RT), in preclinical and clinical settings. Previous studies highlighted the immunomodulatory properties of NBTXR3 + RT, such as modification of tumor cell immunogenicity/adjuvanticity, producing an effective local tumor control and abscopal effect, related to an enhanced antitumor immune response. Furthermore, NBTXR3 + RT has shown potential in restoring anti-PD1 efficacy in a refractory tumor model. However, the early events leading to these results, such as NBTXR3 endocytosis, intracellular trafficking and primary biological responses induced by NBTXR3 + RT remain poorly understood. METHODS: We analyzed by transmission electron microscopy endocytosis and intracellular localization of NBTXR3 nanoparticles after endocytosis in various cell lines, in vitro and in vivo. A kinetic of NBTXR3 endocytosis and its impact on lysosomes was conducted using LysoTracker staining, and a RNAseq analysis was performed. We investigated the ability of NBTXR3 + RT to induce lysosomal membrane permeabilization (LMP) and ferroptosis by analyzing lipid peroxidation. Additionally, we evaluated the recapture by cancer cells of NBTXR3 released from dead cells. RESULTS: NBTXR3 nanoparticles were rapidly internalized by cells mainly through macropinocytosis and in a less extend by clathrin-dependent endocytosis. NBTXR3-containing endosomes were then fused with lysosomes. The day following NBTXR3 addition, we measured a significant increase in LysoTracker lysosome labeling intensity, in vitro as in vivo. Following RT, a significant lysosomal membrane permeabilization (LMP) was measured exclusively in cells treated with NBTXR3 + RT, while RT had no effect. The day post-irradiation, a significant increase in lipid peroxidation, a biomarker of ferroptosis, was measured with NBTXR3 + RT compared to RT. Moreover, we demonstrated that NBTXR3 nanoparticles released from dead cells can be recaptured by cancer cells. CONCLUSIONS: Our findings provide novel insights into the early and specific biological effects induced by NBTXR3 + RT, especially LMP, not induced by RT in our models. The subsequent significant increase in lipid peroxidation partially explains the enhanced cancer cell killing capacity of NBTXR3 + RT compared to RT, potentially by promoting ferroptosis. This study improves our understanding of the cellular mechanisms underlying NBTXR3 + RT and highlights its potential as an agnostic therapeutic strategy for solid cancers treatment.

Position paper of diagnosis and treatment of post-extubation laryngitis in children: a multidisciplinary expert-based opinion

Abstract Objectives To make recommendations on the diagnosis and treatment of post-extubation laryngitis (PEL) in children with or without other comorbidities. Methods A three-iterative modified Delphi method was applied. Specialists were recruited representing pediatric otolaryngologists, pediatric and neonatal intensivists. Questions and statements approached topics encompassing definition, diagnosis, endoscopic airway evaluation, risk factors, comorbidities, management, and follow-up. A consensus was defined as a supermajority >70%. Results Stridor was considered the most frequent symptom and airway endoscopy was recommended for definitive diagnosis. Gastroesophageal reflux and previous history of intubation were considered risk factors. Specific length of intubation did not achieve a consensus as a risk factor. Systemic corticosteroids should be part of the medical treatment and dexamethasone was the drug of choice. No consensus was achieved regarding dosage of corticosteroids, although endoscopic findings help defining dosage and length of treatment. Non-invasive ventilation, laryngeal rest, and use of comfort sedation scales were recommended. Indications for microlaryngoscopy and bronchoscopy under anesthesia were symptoms progression or failure to improve after the first 72-h of medical treatment post-extubation, after two failed extubations, and/or suspicion of severe lesions on flexible fiberoptic laryngoscopy. Conclusions Management of post-extubation laryngitis is challenging and can be facilitated by a multidisciplinary approach. Airway endoscopy is mandatory and impacts decision-making, although there is no consensus regarding dosage and length of treatment.

Nanoparticle-enhanced proton beam immunoradiotherapy drives immune activation and durable tumor rejection.

The combination of radiation therapy (RT) and immunotherapy has emerged as a promising treatment option in oncology. Historically, x-ray radiation (XRT) has been the most commonly used form of RT. However, proton beam therapy (PBT) is gaining recognition as a viable alternative, as it has been shown to produce similar outcomes to XRT while minimizing off-target effects. The effects of PBT on the antitumor immune response have only just begun to be described, and to our knowledge no studies to date have examined the effect of PBT as part of a combinatorial immunoradiotherapeutic strategy. Here, using a 2-tumor model of lung cancer in mice, we show that PBT in tandem with an anti-PD1 antibody substantially reduced growth in both irradiated and unirradiated tumors. This was accompanied by robust activation of the immune response, as evidenced by whole-tumor and single-cell RNA sequencing showing upregulation of a multitude of immune-related transcripts. This response was further significantly enhanced by the injection of the tumor to be irradiated with NBTXR3 nanoparticles. Tumors of mice treated with the triple combination exhibited increased infiltration and activation of cytotoxic immune cells. This triple combination eradicated both tumors in 37.5% of the treated mice and showed robust long-term immunity to cancer.

NBTXR3 improves the efficacy of immunoradiotherapy combining nonfucosylated anti-CTLA4 in an anti-PD1 resistant lung cancer model.

The efficacy of immunoradiotherapy consisting of radiation therapy and immune checkpoint blockade relies on effectively promoting the systemic antitumor immune response's activation while simultaneously reducing local factors favoring immune suppression. We previously demonstrated that NBTXR3, a nanoparticle radioenhancer, significantly improved immune responses in a murine anti-PD1-resistant metastatic lung cancer model. We hypothesize that radioactivated-NBTXR3 addition to anti-PD1 and a second-generation anti-CTLA4 could improve treatment effectiveness. To test this hypothesis, we inoculated mice with 344SQR cells in the right and left legs to establish primary and secondary tumors. The primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7, followed by three fractions of 12 Gy radiation on days 8, 9, and 10. The secondary tumors received two fractions of 1Gy radiation on days 13 and 14. Multiple rounds of anti-PD1, anti-CTLA4 or nonfucosylated anti-CTLA4 were given to the mice. Immune profiling of the tumors revealed that the combination of NBTXR3 with immunoradiotherapy significantly upregulated the activities of a wide range of antitumor immune pathways and reduced the abundance of regulatory suppressor T cells. This combination effectively eradicated the primary and secondary tumors and increased animal survival to 75%. Remarkably, previously treated with NBTXR3-containing treatment, the survivor mice exhibited a long-lasting antitumor memory immune response. This data provides compelling evidence of the efficacy of NBTXR3 to synergize with the immunoradiotherapy approach when combined with an anti-PD1 and multiple checkpoints such as a second generation anti-CTLA4 and show the potential for clinical uses of antitumor immunomodulatory effects of NBTXR3.

Radiotherapy-activated NBTXR3 nanoparticles modulate cancer cell immunogenicity and TCR repertoire.

BACKGROUND: Radiotherapy is a powerful and widely used technique for the treatment of solid tumors. Beyond its ability to destroy tumor cells, it has been demonstrated that radiotherapy can stimulate the anti-tumor immune response. Unfortunately, this effect is mainly restricted to the irradiated lesion, as tumor control outside the treated field (called the 'abscopal effect') is rarely obtained. In addition, many pro-tumoral factors prevent this anti-tumor immune response from being sustained and efficient. We previously reported that radiotherapy-activated NBTXR3 produced a significant CD8-dependent abscopal effect in immunocompetent mice bearing CT26.WT tumors, while radiotherapy failed to generate such a response. METHODS: To identify the mechanisms that may explain this response, we evaluated the capacity of radiotherapy-activated NBTXR3 to modulate the immunogenicity of tumor cells by analysis of immunogenic cell death biomarkers and immunopeptidome sequencing. In vivo, we analyzed treated tumors for CD4+, CD8 + and CD68 + cell infiltrates by immunohistochemistry and digital pathology and sequenced the T cell receptor (TCR) repertoire in both treated and untreated distant tumors. RESULTS: We showed that NBTXR3 activated by radiotherapy both increased immunogenic cell death biomarkers and modulated the immunopeptidome profile of CT26.WT cells. Immunohistochemistry analysis of treated tumors revealed a significant increase in CD4+, CD8 + and CD68 + cell infiltrates for NBTXR3 activated by radiotherapy group, compared to radiotherapy. We also measured significant modifications in TCR repertoire diversity in the radiotherapy-activated NBTXR3 group, both in treated and distant untreated tumors, compared to radiotherapy alone. CONCLUSIONS: These results indicate that radiotherapy-activated NBTXR3 can act as an effective immunomodulator, modifying tumor cell immunogenicity and impacting the lymphocyte population.

Nucleophilicity of cysteine and related biothiols and the development of fluorogenic probes and other applications.

Biothiols such as l-cysteine, l-homocysteine, and glutathione play essential roles in many biological processes, and are directly associated with several health conditions. Therefore, the development of fast, selective, sensitive, and inexpensive methods for quantitatively analyzing biothiols in aqueous solution, but especially in biological samples, is a very attractive research field. In this feature review, we have approached the relevance of biothiols' nucleophilicity to develop selective fluorogenic probes. Since biothiols have considerable structural similarity, relevant strategies are in full development, including several fluorescent molecular platforms, specific receptor sites, reaction conditions, and optical responses. All of these features are properly presented and discussed. Biothiol sensing protocols are based on traditional organic chemistry reactions such as (hetero)aromatic nucleophilic substitution, addition, and substitution at carbonyl carbon, conjugate addition, and nucleophilic substitution at saturated carbon, amongst others including combined processes; furthermore, mechanistic aspects are detailed herein, including some interesting historical contexts. The feasibility of related fluorogenic probes is illustrated by analysis in complex matrices such as serum, cells, tissues, and animal models. Applications of these reactions in more complex systems such as sulfhydryl-based peptides and proteins are also presented, aiming at functionalizing and detecting these nucleophiles. Most literature cited in this review is recent; however, some other prominent works are also detailed. It is believed that this review may be accessible for many academic levels and may efficiently contribute not only to popularizing science but also to the rational development of fluorogenic probes for biothiol sensing.

FHITlow /pHER2high signature in non-small cell lung cancer is predictive of anti-HER2 molecule efficacy.

Despite its efficacy in solid tumours, in particular HER2+ breast cancer, HER2-targeted therapy has given rise to disappointing results in non-small cell lung cancer (NSCLC). With the aim of refining the target population for anti-HER2 therapies in NSCLC, we investigated the relationships between HER2 and the tumour suppressor fragile histidine triad (FHIT) in lung tumour cells. First, we observed a negative correlation between FHIT expression and the activated form of HER2 (pHER2) in NSCLC samples and in lung tumour cell lines. Moreover, the silencing or overexpression of FHIT in lung cell lines led to an increase or decrease of HER2 activity, respectively. We also demonstrated that two anti-HER2 drugs, irbinitinib and trastuzumab, restore a more epithelial phenotype and counteract cell invasiveness and growth of FHIT-silenced tumour cell lines. Finally, we showed that the FHITlow /pHER2high phenotype predicts sensitivity to an anti-HER2 therapy in primary tumour cells from NSCLC patients. Our results show that FHIT regulates the activity of HER2 in lung tumour cells and that FHIT-inactivated tumour cells are sensitive to HER2 inhibitors. A new subclass of patients with NSCLC may be eligible for an anti-HER2 therapy. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Inflammatory response and macrophage polarization using different physicochemical biomaterials for oral and maxillofacial reconstruction.

Knowledge about the action of immune system in the recognition of biomaterials has been extremely helpful when it comes about understanding host response and biomaterials' fate in human body. This study aimed to investigate inflammatory response and macrophage polarization during bone healing process of rat's calvaria critical defects using different bone materials in order to evaluate their influence on bone repair and on the quality of the newly formed bone tissue. Eighty male albinus Wistar rats underwent surgical procedure for the confectioning of a 5-mm diameter bone defect in their right parietal bone, and divided in four groups (n = 20 each), according the biomaterial: AG - Control, particulate intramembranous autogenous bone graft, HA/TCP - particulate biphasic calcium phosphate with HA/TCP (60/40), DBB - particulate deproteinized bovine bone, VC - particulate bioactive vitroceramic. After 3, 7, 21, and 45 days, the specimens were removed and prepared for microcomputed tomography (microCT), light and polarized microscopy, immunohistochemical analysis, and histomorphometry. No significant differences were detected considering percentage of leukocytes among the groups and periods, as well as in relation to immunolabeling for inflammatory (M1) and reparative (M2) macrophages. However, immunolabeling for bone marker indicated a delayed osteoblast differentiation in VC group, resulting in a decrease in mineralized bone matrix parameters in this group, revealed by microCT. In addition, AG and HA/TCP presented a satisfactory bone collagenous content. Despite the distinct origins and physicochemical properties of the tested biomaterials, they presented similar immune-inflammatory responses in the present experimental model, influencing bone-related proteins and bone quality, which must be considered according to their use.

Tumour cell blebbing and extracellular vesicle shedding: key role of matrikines and ribosomal protein SA.

BACKGROUND: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases. METHODS: EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA. RESULTS: We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding. CONCLUSIONS: Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.

Structural characterization and in vivo pro-tumor properties of a highly conserved matrikine.

Elastin-derived peptides (EDPs) exert protumor activities by increasing tumor growth, migration and invasion. A number of studies have highlighted the potential of VGVAPG consensus sequence-derived elastin-like polypeptides whose physicochemical properties and biocompatibility are particularly suitable for in vivo applications, such as drug delivery and tissue engineering. However, among the EDPs, the influence of elastin-derived nonapeptides (xGxPGxGxG consensus sequence) remains unknown. Here, we show that the AGVPGLGVG elastin peptide (AG-9) present in domain-26 of tropoelastin is more conserved than the VGVAPG elastin peptide (VG-6) from domain-24 in mammals. The results demonstrate that the structural features of AG-9 and VG-6 peptides are similar. CD, NMR and FTIR spectroscopies show that AG-9 and VG-6 present the same conformation, which includes a mixture of random coils and ß-turn structures. On the other hand, the supraorganization differs between peptides, as demonstrated by AFM. The VG-6 peptide gathers in spots, whereas the AG-9 peptide aggregates into short amyloid-like fibrils. An in vivo study showed that AG-9 peptides promote tumor progression to a greater extent than do VG-6 peptides. These results were confirmed by in vitro studies such as 2D and 3D proliferation assays, migration assays, adhesion assays, proteinase secretion studies and pseudotube formation assays to investigate angiogenesis. Our findings suggest the possibility that the AG-9 peptide present in patient sera may dramatically influence cancer progression and could be used in the design of new, innovative antitumor therapies.