Revolutionizing Radiotoxicity Management with Mesenchymal Stem Cells and Their Derivatives: A Focus on Radiation-Induced Cystitis.

Although radiation therapy plays a crucial role in cancer treatment, and techniques have improved continuously, irradiation induces side effects in healthy tissue. Radiation cystitis is a potential complication following the therapeutic irradiation of pelvic cancers and negatively impacts patients' quality of life (QoL). To date, no effective treatment is available, and this toxicity remains a therapeutic challenge. In recent times, stem cell-based therapy, particularly the use of mesenchymal stem cells (MSC), has gained attention in tissue repair and regeneration due to their easy accessibility and their ability to differentiate into several tissue types, modulate the immune system and secrete substances that help nearby cells grow and heal. In this review, we will summarize the pathophysiological mechanisms of radiation-induced injury to normal tissues, including radiation cystitis (RC). We will then discuss the therapeutic potential and limitations of MSCs and their derivatives, including packaged conditioned media and extracellular vesicles, in the management of radiotoxicity and RC.

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.

Effects of preconditioning with TNFα and IFNγ in angiogenic potential of mesenchymal stromal cell-derived extracellular vesicles.

Introduction: Mesenchymal stromal cells (MSCs) have demonstrated therapeutic properties both in vitro and in vivo to treat various diseases, including anti-inflammatory, immunomodulatory and pro-angiogenic effects. These therapeutic effects are mediated by their secretome composed of soluble factors and extracellular vesicles (EVs). The composition of EVs reflects the molecular and functional characteristics of parental cells. MSC preconditioning can alter the composition of EVs, thereby influencing their therapeutic potential. Methods: MSCs were subjected to preconditioning with two cytokines, TNFα and IFNγ. Following 24 h of preconditioning, MSC-EVs secreted into the culture supernatant were isolated through tangential filtration. Particle concentration and size distribution were measured by nanoparticle tracking analysis, and the surface antigen expression of the EV-specific CD63 was quantified via Enzyme Linked ImmunoSorbent Assay. The angiogenic potential of MSCEVs obtained after preconditioning MSCs was assessed by the analysis of their protein composition and their influence on human umbilical vein endothelial cell (HUVECs) proliferation, migration, and tube-forming ability. Results: Preconditioning with TNFα and IFNγ did not influence the MSC-EV profile but did induce changes in their protein content. Indeed, the expression of pro-angiogenic proteins increased in EVs from preconditioned MSCs compared to EVs from no-preconditioned MSCs. EVs from preconditioned MSCs tend to stimulate HUVEC migration, proliferation and tubeforming ability. These observations imply the presence of a pro-angiogenic potential in EVs obtained after preconditioning of MSCs with TNFα and IFNγ. Discussion: In conclusion, it appears that the pro-angiogenic potential of EVs is enhanced through preconditioning of MSCs with TNFα and IFNγ. The use of these MSCs-EVs in therapy would circumvent the limitations of current cell-based therapies. Indeed, the therapeutic potential of MSC-EVs presents an attractive strategy for exploiting the clinical benefits of MSC therapy. For example, in the field of regenerative medicine, the exploitation of cell-free therapy using highly pro-angiogenic MSC-EVs is of great interest.

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.

Long-Term Anxiety-like Behavior and Microbiota Changes Induced in Mice by Sublethal Doses of Acute Sarin Surrogate Exposure.

Anxiety disorder is one of the most reported complications following organophosphorus (OP) nerve agent (NA) exposure. The goal of this study was to characterize the long-term behavioral impact of a single low dose exposure to 4-nitrophenyl isopropyl methylphosphonate (NIMP), a sarin surrogate. We chose two different sublethal doses of NIMP, each corresponding to a fraction of the median lethal dose (one mild and one convulsive), and evaluated behavioral changes over a 6-month period following exposure. Mice exposed to both doses showed anxious behavior which persisted for six-months post-exposure. A longitudinal magnetic resonance imaging examination did not reveal any anatomical changes in the amygdala throughout the 6-month period. While no cholinesterase activity change or neuroinflammation could be observed at the latest timepoint in the amygdala of NIMP-exposed mice, important modifications in white blood cell counts were noted, reflecting a perturbation of the systemic immune system. Furthermore, intestinal inflammation and microbiota changes were observed at 6-months in NIMP-exposed animals regardless of the dose received. This is the first study to identify long-term behavioral impairment, systemic homeostasis disorganization and gut microbiota alterations following OP sublethal exposure. Our findings highlight the importance of long-term care for victims of NA exposure, even in asymptomatic cases.

COVID-19-Associated Pneumonia: Radiobiological Insights.

The evolution of SARS-CoV-2 pneumonia to acute respiratory distress syndrome is linked to a virus-induced "cytokine storm", associated with systemic inflammation, coagulopathies, endothelial damage, thrombo-inflammation, immune system deregulation and disruption of angiotensin converting enzyme signaling pathways. To date, the most promising therapeutic approaches in COVID-19 pandemic are linked to the development of vaccines. However, the fight against COVID-19 pandemic in the short and mid-term cannot only rely on vaccines strategies, in particular given the growing proportion of more contagious and more lethal variants among exposed population (the English, South African and Brazilian variants). As long as collective immunity is still not acquired, some patients will have severe forms of the disease. Therapeutic perspectives also rely on the implementation of strategies for the prevention of secondary complications resulting from vascular endothelial damage and from immune system deregulation, which contributes to acute respiratory distress and potentially to long term irreversible tissue damage. While the anti-inflammatory effects of low dose irradiation have been exploited for a long time in the clinics, few recent physiopathological and experimental data suggested the possibility to modulate the inflammatory storm related to COVID-19 pulmonary infection by exposing patients to ionizing radiation at very low doses. Despite level of evidence is only preliminary, these preclinical findings open therapeutic perspectives and are discussed in this article.

Chronic Inflammation and Radiation-Induced Cystitis: Molecular Background and Therapeutic Perspectives.

Radiation cystitis is a potential complication following the therapeutic irradiation of pelvic cancers. Its clinical management remains unclear, and few preclinical data are available on its underlying pathophysiology. The therapeutic strategy is difficult to establish because few prospective and randomized trials are available. In this review, we report on the clinical presentation and pathophysiology of radiation cystitis. Then we discuss potential therapeutic approaches, with a focus on the immunopathological processes underlying the onset of radiation cystitis, including the fibrotic process. Potential therapeutic avenues for therapeutic modulation will be highlighted, with a focus on the interaction between mesenchymal stromal cells and macrophages for the prevention and treatment of radiation cystitis.

Exposure of Human Skin Organoids to Low Genotoxic Stress Can Promote Epithelial-to-Mesenchymal Transition in Regenerating Keratinocyte Precursor Cells.

For the general population, medical diagnosis is a major cause of exposure to low genotoxic stress, as various imaging techniques deliver low doses of ionizing radiation. Our study investigated the consequences of low genotoxic stress on a keratinocyte precursor fraction that includes stem and progenitor cells, which are at risk for carcinoma development. Human skin organoids were bioengineered according to a clinically-relevant model, exposed to a single 50 mGy dose of γ rays, and then xeno-transplanted in nude mice to follow full epidermis generation in an in vivo context. Twenty days post-xenografting, mature skin grafts were sampled and analyzed by semi-quantitative immuno-histochemical methods. Pre-transplantation exposure to 50 mGy of immature human skin organoids did not compromise engraftment, but half of xenografts generated from irradiated precursors exhibited areas displaying focal dysplasia, originating from the basal layer of the epidermis. Characteristics of epithelial-to-mesenchymal transition (EMT) were documented in these dysplastic areas, including loss of basal cell polarity and cohesiveness, epithelial marker decreases, ectopic expression of the mesenchymal marker α-SMA and expression of the EMT promoter ZEB1. Taken together, these data show that a very low level of radiative stress in regenerating keratinocyte stem and precursor cells can induce a micro-environment that may constitute a favorable context for long-term carcinogenesis.

MSC-Derived Extracellular Vesicles: New Emergency Treatment to Limit the Development of Radiation-Induced Hematopoietic Syndrome?

Nuclear accidents or acts of terrorism involving radioactive sources might lead to mass casualties irradiation. The hematopoietic system is one of the most critical and radiation-sensitive tissues because the limited life span of blood cells requires the continuous division of hematopoietic stem cells (HSCs) into the bone marrow. The radiation-induced hematopoietic syndrome, RI-HS, is an impairment of the hematopoiesis that will result in pancytopenia of various degrees. In fact, treatment with granulocyte-colony stimulating factor (G-CSF) is considered as a valuable adjunct to treatment controls in some irradiated patients. Nevertheless, these overexposed patients with bone marrow suppression have minimal medullary territories that do not allow complete recovery of hematopoiesis but lead to significant immunoreactivity following allogeneic hematopoietic stem cell transplantation (HSCT). The high morbidity and mortality of these overexposed patients is a reminder of the lack of effective treatment for hematopoietic syndrome. During the last 20 y, a therapeutic approach for mesenchymal stem cells (MSC) has been proposed for the management of accidentally irradiated victims. Many preclinical animal studies have shown that MSC, mainly by their secretory activity, in particular extracellular vesicles (EVs), contribute to the control of inflammation and promote regeneration of tissues by accelerating angiogenesis and re-epithelialization processes. Therefore, we investigated the potential effect of EVs on the reduction of early bone marrow ionization toxicity, early anti-apoptotic therapy, and vascular protection in the RI-HS model. The main purpose is to propose an innovative treatment of non-patient-specific RI-HS emergency treatment in order to limit allogeneic HSC.

KLF4 inhibition promotes the expansion of keratinocyte precursors from adult human skin and of embryonic-stem-cell-derived keratinocytes.

Expanded autologous skin keratinocytes are currently used in cutaneous cell therapy, and embryonic-stem-cell-derived keratinocytes could become a complementary alternative. Regardless of keratinocyte provenance, for efficient therapy it is necessary to preserve immature keratinocyte precursors during cell expansion and graft processing. Here, we show that stable and transient downregulation of the transcription factor Krüppel-like factor 4 (KLF4) in keratinocyte precursors from adult skin, using anti-KLF4 RNA interference or kenpaullone, promotes keratinocyte immaturity and keratinocyte self-renewal in vitro, and enhances the capacity for epidermal regeneration in mice. Both stable and transient KLF4 downregulation had no impact on the genomic integrity of adult keratinocytes. Moreover, transient KLF4 downregulation in human-embryonic-stem-cell-derived keratinocytes increased the efficiency of skin-orientated differentiation and of keratinocyte immaturity, and was associated with improved generation of epidermis. As a regulator of the cell fate of keratinocyte precursors, KLF4 could be used for promoting the ex vivo expansion and maintenance of functional immature keratinocyte precursors.

Biological features of herpes simplex virus type 1 latency in mice according to experimental conditions and type of neurones.

PURPOSE: To compare the biological patterns of viral transcripts during herpes simplex virus type 1 (HSV1) latent infection according to experimental conditions. METHODS: Two types of murine models of HSV1 infection were used: the corneal scarification model, often used in studies of HSV1 latency, and the oro-ocular murine model. Two strains of HSV1 were used for the inoculation: SC16, a wild type strain considered as highly neuroinvasive, and KOS, previously described as poorly neurovirulent. The amounts of viral genomes, and those of four types of viral transcripts (immediate-early, early and late, together with latency-associated transcripts [LATs]), were measured by quantitative PCR and RT-PCR in the main sites of HSV1 latent infection at 6 days, 1 and 3 months post inoculation, and the number of LAT-expressing neurons was assessed by in-situ hybridization on histological sections of trigeminal ganglia (TG). RESULTS: Using the SC16 strain of HSV1 in the oro-ocular model, immediate-early transcripts were still present at 1 month post inoculation (early stage of latent infection), but were not detected at 3 months (late stage of latent infection). In both cases, early and late viral genes transcripts were not detected, demonstrating the latent nature of the infection with this combination of experimental conditions. In contrast, such progress in the viral gene expression was not observed in the corneal scarification model, particularly when the KOS strain of HSV1 was used. CONCLUSIONS: These results highlight that the behavior of the virus in the nervous system depends on the method inoculation, and the viral strain. All these parameters are likely to influence the establishment of latent infection.

Spectral-domain optical coherence tomography of the rodent eye: highlighting layers of the outer retina using signal averaging and comparison with histology.

Spectral-Domain Optical Coherence Tomography (SD-OCT) is a widely used method to observe retinal layers and follow pathological events in human. Recently, this technique has been adapted for animal imaging. This non-invasive technology brings a cross-sectional visualization of the retina, which permits to observe precisely each layer. There is a clear expansion of the use of this imaging modality in rodents, thus, a precise characterization of the different outer retinal layers observed by SD-OCT is now necessary to make the most of this technology. The identification of the inner strata until the outer nuclear layer has already been clearly established, while the attribution of the layers observed by SD-OCT to the structures corresponding to photoreceptors segments and retinal pigment epithelium is much more questionable. To progress in the understanding of experimental SD-OCT imaging, we developed a method for averaging SD-OCT data to generate a mean image allowing to better delineate layers in the retina of pigmented and albino strains of mice and rats. It allowed us to locate precisely the interface between photoreceptors and retinal pigment epithelium and to identify unambiguously four layers corresponding to the inner and outer parts of photoreceptors segments. We show that the thickness of the various layers can be measured as accurately in vivo on SD-OCT images, than post-mortem by a morphometric analysis of histological sections. We applied SD-OCT to different models and demonstrated that it allows analysis of focal or diffuse retinal pathological processes such as mutation-dependent damages or light-driven modification of photoreceptors. Moreover, we report a new method of combined use of SD-OCT and integration to quantify laser-induced choroidal neovascularization. In conclusion, we clearly demonstrated that SD-OCT represents a valuable tool for imaging the rodent retina that is at least as accurate as histology, non-invasive and allows longitudinal follow-up of the same animal.