Correlation Between Serum and Urine Biomarkers and the Intensity of Acute Radiation Cystitis in Patients Treated With Radiation Therapy for Localized Prostate Cancer: Protocol for the Radiotoxicity Bladder Biomarkers (RABBIO) Study.

BACKGROUND: Despite improvements in radiation techniques, pelvic radiotherapy is responsible for acute and delayed bladder adverse events, defined as radiation cystitis. The initial symptoms of bladder injury secondary to pelvic irradiation are likely to occur during treatment or within 3 months of radiotherapy in approximately 50% of irradiated patients, and have a significant impact on their quality of life. The pathophysiology of radiation cystitis is not well understood, particularly because of the risk of complications associated with access to bladder tissue after irradiation, which limits our ability to study this process and develop treatments. OBJECTIVE: It is an original study combining digital data collection to monitor patients' symptoms and biological markers during irradiation. The main objective of our study is to evaluate the correlation of biological biomarkers with the intensity of acute radiation cystitis and the quality of life of patients, assessed with the digital telemonitoring platform Cureety. METHODS: Patients with intermediate-risk localized prostate cancer who are eligible for localized radiotherapy will be included. Inflammatory biomarkers will be analyzed in urine and blood samples before the start of radiotherapy and at weeks 4, 12, and 48 of irradiation, through quantitative methods such as a multiplex Luminex assay, flow cytometry, and enzyme-linked immunosorbent assay. We will also characterize the patients' gut and urine microbiota composition using 16S ribosomal RNA sequencing technology. Between sample collection visits, patients will complete various questionnaires related to radiation cystitis symptoms (using the International Prostate Symptom Score), adverse events, and quality of life (using the Functional Assessment of Cancer Therapy-Prostate questionnaire), using the Cureety digital remote monitoring platform. Upon receipt of the questionnaires, an algorithm will process the information and classify patients in accordance with the severity of symptoms and adverse events reported on the basis of Common Terminology Criteria for Adverse Events and International Prostate Symptom Score standards. This will allow us to correlate levels of urinary, blood, and fecal biomarkers with the severity of acute radiation cystitis symptoms and patient-reported quality of life. RESULTS: The study started in March 2022. We estimate a recruitment period of approximately 18 months, and the final results are expected in 2024. CONCLUSIONS: This prospective study is the first to explore the overexpression of inflammatory proteins in fluid biopsies from patients with symptoms of acute radiation cystitis. In addition, the 1-year follow-up after treatment will allow us to predict which patients are at risk of late radiation cystitis and to refer them for radioprotective treatment. The results of this study will allow us to develop strategies to limit radiation damage to the bladder and improve the quality of life of patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT05246774; https://clinicaltrials.gov/ct2/show/NCT05246774?term=NCT05246774. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/38362.

Effectiveness of a digital telemonitoring platform for cancer care of older patients: The ConnectElderlyPatientToDoctor study.

While telemedicine has been shown to improve the quality of care for cancer patients, it remains underused for older patients (OP), partly due to the assumption that OPs are unabled or unwilling to use digital tools. However, more than 50% of new cancers are diagnosed in people over 70. The ConnectElderlyPatientToDoctor study aimed to evaluate the OP compliance with the use of the digital telemonitoring platform Cureety in oncology. All cancer patients followed at the Military Hospital Bégin were eligible for the study. Patients were invited to respond to a symptomatology questionnaire personalized to their pathology and treatment. An algorithm evaluated the health status of the patient based on the reported adverse events. The population was divided into two groups, OP and younger patients (YP), based on a cut-off at 70 years. The primary endpoint was to assess the compliance of OPs with the use of the digital oncology platform Cureety, compared to YP. From July 2020 to September 2021, a total of 117 patients were included in our study. We found that 66% of the patients were compliant, with no difference between the two groups (71.2% of YP, 61.7% of OP, P = .29). In OPs, progression free survival (PFS) ratio at 6-months was 64.6% in the tolerant patients vs 23.4% in the nontolerant patients (HR = 0.1980, 95% CI = 0.04431-0.8845, P = .0339). The median PFS was 23.3 months in the tolerant group vs 3.3 months in the nontolerant group (P = .0339). The data of overall survival are immature. OPs had a clear benefit from using this platform, similar to what was observed for YP. Patients felt less isolated and felt that they benefited from personalized care with early ambulatory medical care of adverse events. We also found that the health indicators collected with the platform in the first month of treatment are predictive of the progression of the disease. This solution makes it possible to streamline and improve the care pathway of OP.

Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium.

Background: Radiation cystitis (RC) results from chronic inflammation, fibrosis, and vascular damage. The urinary symptoms it causes have a serious impact on patients' quality of life. Despite the improvement in irradiation techniques, the incidence of radiation cystitis remains stable over time, and the therapeutic possibilities remain limited. Mesenchymal stem/stromal cells (MSC) appear to offer2 a promising therapeutic approach by promoting tissue repair through their paracrine action via extracellular vesicles (MSC-EVs) or conditioned medium from human mesenchymal stromal cells (MSC-CM). We assess the therapeutic potential of MSC-EVs or MSC-CM in an in vitro model of RC. Methods:in vitro RC was induced by irradiation of human bladder fibroblasts (HUBF) with the small-animal radiation research platform (SARRP). HUBF were induced towards an RC phenotype after 3 × 3.5 Gy irradiation in the presence of either MSC-EVs or MSC-CM, to assess their effect on fibrosis, angiogenesis, and inflammatory markers. Results: Our data revealed in vitro a higher therapeutic potential of MSC-EVs and MSC-CM in prevention of RC. This was confirmed by down-regulation of α-SMA and CTGF transcription, and the induction of the secretion of anti-fibrotic cytokines, such as IFNγ, IL10 and IL27 and the decrease in the secretion of pro-fibrotic cytokines, IGFBP2, IL1ß, IL6, IL18, PDGF, TNFα, and HGF, by irradiated HUBFs, conditioned with MSC-EVs or MSC-CM. The secretome of MSC (MSC-CM) or its subsecretome (MSC-EVs) are proangiogenic, with the ability to induce vessels from HUVEC cells, ensuring the management of bladder vascular lesions induced by irradiation. Conclusion: MSC-EVs and MSC-CM appear to have promising therapeutic potential in the prevention of RC in vitro, by targeting the three main stages of RC: fibrosis, inflammation and vascular damage.

The Use of Telemedicine in Cancer Clinical Trials: Connect-Patient-to-Doctor Prospective Study.

BACKGROUND: Telemedicine is currently being adopted for the management of patients in routine care. However, its use remains limited in the context of clinical trials. OBJECTIVE: This study aimed to demonstrate the feasibility of telemonitoring and patient-reported outcomes collection in the context of clinical trials. METHODS: The patients who were included in an interventional oncology clinical trial were eligible. The patients were registered with a digital tool to respond to a patient-reported outcomes questionnaire (ePRO) based on CTCAE (The Common Terminology Criteria for Adverse Events, National Cancer Institute), version 5.0, personalized to their pathology and treatment. An algorithm evaluated the health status of the patient based on the reported adverse events, with a classification in 4 different states (correct, compromise, state to be monitored, or critical state). The main objective was to evaluate the feasibility of remote monitoring via a connected platform of patients included in a clinical trial. RESULTS: From July 1, 2020, to March 31, 2021, 39 patients were included. The median age was 71 years (range 41-94); 74% (n=29) were male, and 59% (n=23) had metastatic disease. Out of the 969 ePRO questionnaires completed over the course of the study, 77.0% (n=746) were classified as "correct," 10.9% (n=106) as "compromised," and 12.1% (n=117) as "to be monitored" or "critical." The median response time was 7 days (IQR 7-15.5), and 76% (25/33) of the patients were compliant. Out of the 35 patients who answered a satisfaction questionnaire, 95% (n=33) were satisfied or very satisfied with the tool, and 85% (n=30) were satisfied with their relationship with the health care team. There were 5 unscheduled hospitalizations during the study period. CONCLUSIONS: Remote monitoring in clinical trials is feasible, with a high level of patient participation and satisfaction. It benefits patients, but it also ensures the high quality of the trial through the early management of adverse events and better knowledge of the tolerance profile of experimental treatments. This e-technology will likely be deployed more widely in our clinical trials.

FRET-based measurement of GPCR conformational changes.

The C-termini of G protein-coupled receptors (GPCRs) interact with specific kinases and arrestins in an agonist-dependent manner suggesting that conformational changes induced by ligand binding within the transmembrane domains are transmitted to the C-terminus. Förster resonance energy transfer (FRET) can be used to monitor changes in distance between two protein domains if each site can be specifically and efficiently labeled with a donor or acceptor fluorophore. In order to probe GPCR conformational changes, we have developed a FRET technique that uses site-specific donor and acceptor fluorophores introduced by two orthogonal labeling chemistries. Using this strategy, we examined ligand-induced changes in the distance between two labeled sites in the beta(2) adrenoceptor (beta(2)-AR), a well-characterized GPCR model system. The donor fluorophore, LumioGreen, is chelated by a CCPGCC motif [Fluorescein Arsenical Helix or Hairpin binder (FlAsH) site] introduced through mutagenesis. The acceptor fluorophore, Alexa Fluor 568, is attached to a single reactive cysteine (C265). FRET analyses revealed that the average distance between the intracellular end of transmembrane helix (TM) six and the C-terminus of the beta(2)-AR is 62 A. This relatively large distance suggests that the C-terminus is extended and unstructured. Nevertheless, ligand-specific conformational changes were observed (1). The results provide new insight into the structure of the beta(2)-AR C-terminus and ligand-induced conformational changes that may be relevant to arrestin interactions. The FRET labeling technique described herein can be applied to many GPCRs (and other membrane proteins) and is suitable for conformational studies of domains other than the C-terminus.

A monoclonal antibody for G protein-coupled receptor crystallography.

G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins.

Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic group II chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.

Coupling ligand structure to specific conformational switches in the beta2-adrenoceptor.

G protein-coupled receptors (GPCRs) regulate a wide variety of physiological functions in response to structurally diverse ligands ranging from cations and small organic molecules to peptides and glycoproteins. For many GPCRs, structurally related ligands can have diverse efficacy profiles. To investigate the process of ligand binding and activation, we used fluorescence spectroscopy to study the ability of ligands having different efficacies to induce a specific conformational change in the human beta2-adrenoceptor (beta2-AR). The 'ionic lock' is a molecular switch found in rhodopsin-family GPCRs that has been proposed to link the cytoplasmic ends of transmembrane domains 3 and 6 in the inactive state. We found that most partial agonists were as effective as full agonists in disrupting the ionic lock. Our results show that disruption of this important molecular switch is necessary, but not sufficient, for full activation of the beta2-AR.

Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells.

Molecular chaperones assist the folding of newly translated and stress-denatured proteins. In prokaryotes, overlapping sets of chaperones mediate both processes. In contrast, we find that eukaryotes evolved distinct chaperone networks to carry out these functions. Genomic and functional analyses indicate that in addition to stress-inducible chaperones that protect the cellular proteome from stress, eukaryotes contain a stress-repressed chaperone network that is dedicated to protein biogenesis. These stress-repressed chaperones are transcriptionally, functionally, and physically linked to the translational apparatus and associate with nascent polypeptides emerging from the ribosome. Consistent with a function in de novo protein folding, impairment of the translation-linked chaperone network renders cells sensitive to misfolding in the context of protein synthesis but not in the context of environmental stress. The emergence of a translation-linked chaperone network likely underlies the elaborate cotranslational folding process necessary for the evolution of larger multidomain proteins characteristic of eukaryotic cells.

Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states.

The beta2 adrenoreceptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified beta2AR. Here we show that upon catecholamine binding, beta2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions.

Lessons from constitutively active mutants of G protein-coupled receptors.

In the past decade, the concept of constitutive activity has profoundly modified our understanding of G protein-coupled-receptors (GPCRs). Here, we review the contribution of constitutively active mutants (CAMs) to our understanding of three aspects of GPCR physiopathology: (1) GPCR activation is a complex mechanism involving both the release of inactive state conformational constraints, mimicked by most CAMs, and the creation of new interactions that stabilize the active state and are mimicked by a restricted set of CAMs; (2) GPCR phosphorylation, internalization and desensitization processes are activated by receptor conformations, which partly overlap those activating G protein; (3) natural CAMs, mostly affecting GPCRs of the endocrine system, are found in several hereditary and acquired diseases, including cancers. One major remaining question is how CAMs recapitulate the different structural modifications of the agonist-induced active conformation(s) of the wild-type receptor. This characterization is a prerequisite for further use of CAMs as ligand-free models of active GPCRs in structural, cellular and physiological studies.

Constitutive internalization of constitutively active agiotensin II AT(1A) receptor mutants is blocked by inverse agonists.

As constitutively active mutants (CAMs) mimic an active conformation, they can be used to characterize the process of G protein-coupled receptor activation. Here, we used CAMs to study the link between activation and internalization of the angiotensin II AT(1A) receptor. The cellular localization of fluorescently tagged N111A, I245T, and L305Q mutants was determined by confocal microscopy. In the absence of ligand, CAMs were mostly located in intracellular vesicles, whereas the wild-type AT(1A) was found at the cell surface. After 2 h incubation with inverse agonist, losartan, CAMs were translocated to the plasma membrane. Similar observations were made in H295, a human adrenocortical cell line which expresses physiologically the AT(1) receptor. This phenomenon, which was not dependent on protein synthesis and the pharmacology and kinetics of which were similar to the recycling of the wild-type receptor, was called "externalization". After externalization and losartan removal, the L305Q CAM underwent rapid ligand-independent endocytosis, with the same kinetics and temperature sensitivity as the angiotensin II-induced internalization of the wild-type AT(1A). Moreover, the addition of a second mutation known to block internalization (Delta 329 truncation) prevented intracellular localization of the CAM. These data show that AT(1A) CAMs are constitutively and permanently internalized and recycled. This mechanism is different from the down-regulation observed for CAMs of other G protein-coupled receptors and thus defines a new paradigm for the cellular regulation of CAMs.