Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Breast milk transmission and involvement of mammary glands in tick-borne flavivirus infected mice.

Tick-borne flaviviruses (TBFs) are transmitted to humans through milk and tick bites. Although a case of possible mother-to-child transmission of tick-borne encephalitis virus (TBEV) through breast milk has been reported, this route has not been confirmed in experimental models. Therefore, in this study, using type I interferon receptor-deficient A129 mice infected with Langat virus (LGTV), we aimed to demonstrate the presence of infectious virus in the milk and mammary glands of infected mice. Our results showed viral RNA of LGTV in the pup's stomach milk clots (SMCs) and blood, indicating that the virus can be transmitted from dam to pup through breast milk. In addition, we observed that LGTV infection causes tissue lesions in the mammary gland, and viral particles were present in mammary gland epithelial cells. Furthermore, we found that milk from infected mice could infect adult mice via the intragastric route, which has a milder infection process, longer infection time, and a lower rate of weight loss than other modes of infection. Specifically, we developed a nano-luciferase-LGTV reporter virus system to monitor the dynamics of different infection routes and observed dam-to-pup infection using in vivo bioluminescence imaging. This study provides comprehensive evidence to support breast milk transmission of TBF in mice and has helped provide useful data for studying TBF transmission routes.IMPORTANCETo date, no experimental models have confirmed mother-to-child transmission of tick-borne flavivirus (TBF) through breastfeeding. In this study, we used a mouse model to demonstrate the presence of infectious viruses in mouse breast milk and mammary gland epithelial cells. Our results showed that pups could become infected through the gastrointestinal route by suckling milk, and the infection dynamics could be monitored using a reporter virus system during breastfeeding in vivo. We believe our findings have provided substantial evidence to understand the underlying mechanism of breast milk transmission of TBF in mice, which has important implications for understanding and preventing TBF transmission in humans.

Imaging CAR-NK cells targeted to HER2 ovarian cancer with human sodium-iodide symporter-based positron emission tomography.

Chimeric antigen receptor (CAR) cell therapies utilize CARs to redirect immune cells towards cancer cells expressing specific antigens like human epidermal growth factor receptor 2 (HER2). Despite their potential, CAR T cell therapies exhibit variable response rates and adverse effects in some patients. Non-invasive molecular imaging can aid in predicting patient outcomes by tracking infused cells post-administration. CAR-T cells are typically autologous, increasing manufacturing complexity and costs. An alternative approach involves developing CAR natural killer (CAR-NK) cells as an off-the-shelf allogeneic product. In this study, we engineered HER2-targeted CAR-NK cells co-expressing the positron emission tomography (PET) reporter gene human sodium-iodide symporter (NIS) and assessed their therapeutic efficacy and PET imaging capability in a HER2 ovarian cancer mouse model.NK-92 cells were genetically modified to express a HER2-targeted CAR, the bioluminescence imaging reporter Antares, and NIS. HER2-expressing ovarian cancer cells were engineered to express the bioluminescence reporter Firefly luciferase (Fluc). Co-culture experiments demonstrated significantly enhanced cytotoxicity of CAR-NK cells compared to naive NK cells. In vivo studies involving mice with Fluc-expressing tumors revealed that those treated with CAR-NK cells exhibited reduced tumor burden and prolonged survival compared to controls. Longitudinal bioluminescence imaging demonstrated stable signals from CAR-NK cells over time. PET imaging using the NIS-targeted tracer 18F-tetrafluoroborate ([18F]TFB) showed significantly higher PET signals in mice treated with NIS-expressing CAR-NK cells.Overall, our study showcases the therapeutic potential of HER2-targeted CAR-NK cells in an aggressive ovarian cancer model and underscores the feasibility of using human-derived PET reporter gene imaging to monitor these cells non-invasively in patients.

A rare olive compound oleacein functions as a TrkB agonist and mitigates neuroinflammation both in vitro and in vivo.

BACKGROUND: Neuroinflammation is widely acknowledged as a characteristic feature of almost all neurological disorders and specifically in depression- and anxiety-like disorders. In recent years, there has been significant attention on natural compounds with potent anti-inflammatory effects due to their potential in mitigating neuroinflammation and neuroplasticity. METHODS: In the present study, we aimed to evaluate the neuroprotective effects of oleacein (OC), a rare secoiridoid derivative found in extra virgin olive oil. Our goal was to explore the BDNF/TrkB neurotrophic activity of OC and subsequently assess its potential for modulating neuroinflammatory response using human neuroblastoma cells (SH-SY5Y cells) and an in vivo model of depression induced by lipopolysaccharide (LPS)-mediated inflammation. RESULTS: In SH-SY5Y cells, OC exhibited a significant dose-dependent increase in BDNF expression. This enhancement was absent when cells were co-treated with inhibitors of BDNF's receptor TrkB, as well as downstream molecules PI3K and MEK. Whole-transcriptomics analysis revealed that OC upregulated cell cycle-related genes under normal conditions, while downregulating inflammation-associated genes in LPS-induced conditions. Furthermore, surface plasmon resonance (SPR) assays demonstrated that OC exhibited a stronger and more stable binding affinity to TrkB compared to the positive control, 7,8-dihydroxyflavone. Importantly, bioluminescence imaging revealed that a single oral dose of OC significantly increased BDNF expression in the brains of Bdnf-IRES-AkaLuc mice. Furthermore, oral administration of OC at a dosage of 10 mg/kg body weight for 10 days significantly reduced immobility time in the tail suspension test compared to the LPS-treated group. RT-qPCR analysis revealed that OC significantly decreased the expression of pro-inflammatory cytokines Tnfα, Il6, and Il1ß, while simultaneously enhancing Bdnf expression, as well as both pro and mature BDNF protein levels in mice hippocampus. These changes were comparable to those induced by the positive control antidepressant drug fluoxetine. Additionally, microarray analysis of mouse brains confirmed that OC could counteract LPS-induced inflammatory biological events. CONCLUSION: Altogether, our study represents the first report on the potential antineuroinflammatory and antidepressant properties of OC via modulation of BDNF/TrkB neurotrophic activity. This finding underscores the potential of OC as a natural therapeutic agent for depression- and anxiety-related disorders.

X-ray irradiation effectively inactivated lymphocytes in transfusion in vivo monitored by the bioluminescence transfusion-associated graft-versus-host disease model.

BACKGROUND AND OBJECTIVES: Transfusion of cold-stored whole blood is the preferred resuscitation method for trauma patients but may cause transfusion-associated graft-versus-host disease (TA-GVHD). Standard clinical practice to prevent this is to irradiate blood components with gamma-rays. X-ray irradiations are also a safe and effective alternative to gamma-ray irradiation. We established a visual mouse model of TA-GVHD to compare the viability and function of lymphocytes exposed to gamma- and x-ray irradiation. MATERIALS AND METHODS: A haploidentical transplantation mouse model was established to simulate TA-GVHD with Balb/c mice as donors and hybrid F1 CB6 mice (Balb/c × C57) as recipients. Spleen cells from Tg-Fluc+ Balb/c mice were isolated and irradiated with gamma-rays and x-rays. Lymphocyte activation, apoptosis and proliferation post phorbol 1 2-myristate 1 3-acetate (PMA) stimulation were evaluated. After transfusion, we monitored Fluc+ lymphocytes daily by bioluminescence imaging. Recipients were euthanized on day 21, and tissues were examined pathologically and for inflammatory cytokines. RESULTS: The viability of gamma- or x-ray irradiated lymphocytes decreased significantly with slight changes in proliferation in vivo after transfusion. Compared with the non-irradiated group, both the gamma- and x-ray irradiated groups showed significantly decreased clinical scoring and inflammatory cytokine levels. The fluorescence intensity of the body and target organs was reduced after irradiation. CONCLUSION: No recipients acquired TA-GVHD after lymphocyte transfusion subjected to gamma- or x-rays, showing that x-rays inactivate as well as gamma rays and are suitable for irradiating whole blood.

Emerging Synthetic Bioluminescent Reactions for Non-Invasive Imaging of Freely Moving Animals.

Bioluminescence imaging (BLI) is an indispensable technique for visualizing the dynamics of diverse biological processes in mammalian animal models, including cancer, viral infections, and immune responses. However, a critical scientific challenge remains: non-invasively visualizing homeostatic and disease mechanisms in freely moving animals to understand the molecular basis of exercises, social behavior, and other phenomena. Classical BLI relies on prolonged camera exposure to accumulate the limited number of photons that traveled from deep tissues in anesthetized or constrained animals. Recent advancements in synthetic bioluminescence reactions, utilizing artificial luciferin-luciferase pairs, have considerably increased the number of detectable photons from deep tissues, facilitating high-speed BLI to capture moving objects. In this review, I provide an overview of emerging synthetic bioluminescence reactions that enable the non-invasive imaging of freely moving animals. This approach holds the potential to uncover unique physiological processes that are inaccessible with current methodologies.

In vivo optical imaging of tumor stromal cells with hypoxia-inducible factor activity.

Tumors contain various stromal cells, such as immune cells, endothelial cells, and fibroblasts, which contribute to the development of a tumor-specific microenvironment characterized by hypoxia and inflammation, and are associated with malignant progression. In this study, we investigated the activity of intratumoral hypoxia-inducible factor (HIF), which functions as a master regulator of the cellular response to hypoxia and inflammation. We constructed the HIF activity-monitoring reporter gene hypoxia-response element-Venus-Akaluc (HVA) that expresses the green fluorescent protein Venus and modified firefly luciferase Akaluc in a HIF activity-dependent manner, and created transgenic mice harboring HVA transgene (HVA-Tg). In HVA-Tg, HIF-active cells can be visualized using AkaBLI, an ultra-sensitive in vivo bioluminescence imaging technology that produces an intense near-infrared light upon reaction of Akaluc with the D-luciferin analog AkaLumine-HCl. By orthotopic transplantation of E0771, a mouse triple negative breast cancer cell line without a reporter gene, into HVA-Tg, we succeeded in noninvasively monitoring bioluminescence signals from HIF-active stromal cells as early as 8 days after transplantation. The HIF-active stromal cells initially clustered locally and then spread throughout the tumors with growth. Immunohistochemistry and flow cytometry analyses revealed that CD11b+ F4/80+ macrophages were the predominant HIF-active stromal cells in E0771 tumors. These results indicate that HVA-Tg is a useful tool for spatiotemporal analysis of HIF-active tumor stromal cells, facilitating investigation of the roles of HIF-active tumor stromal cells in tumor growth and malignant progression.

Quantitative Assessment on Optical Properties as a Basis for Bioluminescence Imaging: An Experimental and Numerical Approach to the Transport of Optical Photons in Phantom Materials.

Bioluminescence imaging (BLI) is a widely used technique in preclinical scientific research, particularly in the development of mRNA-based medications and non-invasive tumor diagnostics. It has become an essential tool in current science. However, the current state of bioluminescence imaging is primarily qualitative, making it challenging to obtain quantitative measurements and to draw accurate conclusions. This fact is caused by the unique properties of optical photons and tissue interactions. In this paper, we propose an experimental setup and Geant4-simulations to gain a better understanding of the optical properties and processes involved in bioluminescence imaging. Our goal is to advance the field towards more quantitative measurements. We will discuss the details of our experimental setup, the data we collected, the outcomes of the Geant4-simulations, and additional information on the underlying physical processes.

Visualizing the Intermittent Gating of Na+ /H+ Antiporters in Single Native Bioluminescent Bacteria.

While the intermittent gating of ion channels has been well studied for decades, dynamics of the action of secondary transporters, another major pathway for ion transmembrane transports, remains largely unexplored in living cells. Herein, intermittent blinking of the spontaneous bioluminescence (BL) from single native bacteria, P. phosphoreum, was reported, investigated and attributed to the intermittent gating of sodium/proton antiporters (NhaA) between the active and inactive conformations. Each gating event caused the rapid depolarization and recovery of membrane potential within several seconds, accompanying with the apparent BL blinking due to the transient inhibitions on the activity of the respiratory chain. Temperature-dependent measurements further obtained an activation energy barrier of the conformational change of 20.3 kJ mol-1 .

Visualization of osteocalcin and bone morphogenetic protein 2 (BMP2) secretion from osteoblastic cells by bioluminescence imaging.

The secretions of osteocalcin and bone morphogenetic protein 2 (BMP2) from living osteoblastic cells were visualized for the first time using a method of video-rate bioluminescence imaging. The fusion proteins with Gaussia luciferase (GLase) for mouse osteocalcin and BMP2 (OC-GLase and BMP2-GLase, respectively) expressed in osteoblastic MC3T3-E1 cells were correctly processed and secreted. In the video images of exocytotic secretion, the luminescence spots of OC-GLase and BMP2-GLase disappeared rapidly and gradually, respectively, indicating different manners of these proteins in diffusion. Notably, a deletion mutant of BMP2 (Δ3BMP2-GLase) lacking three basic amino acid residues in the N-terminal region for binding to heparan sulfate showed rapidly disappearing luminescence spots. In our imaging conditions, the half-life of luminescence for the spots of Δ3BMP2-GLase (1.61 ± 0.20 s) was similar to that of OC-GLase (1.22 ± 0.14 s) but not to that of BMP2-GLase (4.31 ± 0.41 s). These results suggest that, in contrast to osteocalcin, the diffusion of BMP2 from cells occurred slowly after exocytosis. Thus, our bioluminescence imaging method is useful to study the diffusion properties of secreted proteins in exocytosis.

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications.

BACKGROUND: Currently, there is no regenerative therapy for patients with neurological and neurodegenerative disorders. Cell-therapies have emerged as a potential treatment for numerous brain diseases. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. METHODS: We generated good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. NPCs were characterized in vitro for their differentiation potential and in vivo after transplantation into wild type as well as genetically immunosuppressed mice. RESULTS: Generated NPCs had a stable gene-expression over at least 15 passages and could be scaled for up to 1018 cells per initially seeded 106 cells. After withdrawal of growth factors in vitro, cells adapted a neural fate and mainly differentiated into active neurons. To ensure a pure NPC population for in vivo applications, we reduced the risk of iPSC contamination by applying micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted NPCs have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month, while no residual pluripotent cells were detectable. CONCLUSION: We describe the generation of transgene- and xeno-free NPCs. This simple differentiation protocol combined with the ability of in vivo cell tracking presents a valuable tool to develop safe and effective cell therapies for various brain injuries.

Firefly luciferase offers superior performance to AkaLuc for tracking the fate of administered cell therapies.

INTRODUCTION: A novel, red-shifted bioluminescence imaging (BLI) system called AkaBLI has been recently developed for cell tracking in preclinical models and to date, limited data is available on how it performs in relation to existing systems. PURPOSE: To systematically compare the performance of AkaBLI and the standard Firefly luciferase (FLuc) systems to monitor the biodistribution and fate of cell therapies in rodents. METHODS: Umbilical cord mesenchymal stromal cells (MSCs) were transduced to produce two genetically engineered populations, expressing either AkaLuc or the engineered FLuc luc2. The bioluminescence of AkaLuc+ and FLuc+ cells was assessed both in vitro (emission spectra, saturation kinetics and light emission per cell) and in vivo (substrate kinetics following intraperitoneal and subcutaneous administration and biodistribution of the cells up to day 7). RESULTS: Introduction of the reporter genes has no effect on MSC phenotype. For BLI, the FLuc system is superior to AkaBLI in terms of (i) light output, producing a stronger signal after subcutaneous substrate delivery and more consistent signal kinetics when delivered intraperitoneally; (ii) absence of hepatic background; and (iii) safety, where the AkaLuc substrate was associated with a reaction in the skin of the mice in vivo. CONCLUSION: We conclude that there is no advantage in using the AkaBLI system to track the biodistribution of systemically administered cell-based regenerative medicine therapies in vivo.

Molecular fluorophores for in vivo bioimaging in the second near-infrared window.

PURPOSE: This systematic review aims to summarize the current developments of fluorescence and chemi/bioluminescence imaging based on the molecular fluorophores for in vivo imaging in the second near-infrared window. METHODS AND RESULTS: By investigating most of the relevant references on the web of science and some journals, this review firstly begins with an overview of the background of fluorescence and chemi/bioluminescence imaging. Secondly, the chemical and optical properties of NIR-II dyes are discussed, such as water solubility, chemostability and photo-stability, and brightness. Thirdly, the bioimaging based on NIR-II fluorescence emission is outlined, including the in vivo imaging of polymethine dyes, donor - acceptor - donor (D - A - D) chromophores, and lanthanide complexes. Fourthly, we demonstrate the chemi/bioluminescence in vivo imaging in the second near-infrared window. Fifthly, the clinical application and translation of near-infrared fluorescence imaging are presented. Finally, the current challenges, feasible strategies and potential prospects of the fluorophores and in vivo bioimaging are discussed. CONCLUSIONS: Based on the above literature research on the applications of molecular fluorescent and chemi/bioluminescent probes in the second near-infrared window in recent years, this review weighs the advantages and disadvantages of fluorescence and chemi/bioluminescence imaging, and NIR-II fluorophores based on polymethine dyes, D - A - D chromophores, and lanthanide complexes. Besides, this review also provides a very important guidance for expanding the imaging applications of molecular fluorophores in the second near-infrared window.

Bioluminescence imaging of fibroblast activation protein-alpha in vivo and human plasma with highly sensitive probe.

Fibroblast activation protein-alpha (FAPα) has emerged as a biomarker of tumor stromal fibroblasts. FAP was overexpressed in stromal fibroblasts of human malignancies and positively correlated with the depth of tumor invasion, lymphatic metastasis, distant metastases, high TNM stage and poor prognosis. However, the circulating FAP levels in the plasma of gastric cancer patients and the relationship between FAP levels and gastric cancer remain unknown. Moreover, probes with super selectivity, extremely high sensitivity, and excellent performance in quantitative detection are still lacking. Herein, we developed the bioluminescent probe BL-FAP for sensitive detection and imaging of endogenous FAP in gastric cancer cells and tissues and plasma from gastric cancer patients. The probe exhibited the high signal-to-noise ratio (15000∼fold), the excellent selectivity (FAP/DPP IV ratio and FAP/PREP ratio = 50000∼ fold), and the high sensitivity (18.1 pg/mL). BL-FAP facilitates monitoring of endogenous FAP in living cells and nude mice bearing MGC-803-luc tumors. More importantly, this probe was successfully applied to the measurement of FAP activity levels in plasma from gastric cancer patients for the first time. A significant enhancement in FAP levels was observed in patients with gastric cancer, suggesting that the FAP level may be a potential diagnostic parameter for gastric cancer.

CT/bioluminescence dual-modal imaging tracking of stem cells labeled with Au@PEI@PEG nanotracers and RfLuc in nintedanib-assisted pulmonary fibrosis therapy.

Mesenchymal stem cells (MSCs) are promising in idiopathic pulmonary fibrosis (IPF) therapy. However, low survival rate and ambiguous behavior of MSCs after transplantation impede their clinical translation. To this end, we have developed a new strategy to improve the survival rate and monitor the behavior of the transplanted MSCs simultaneously. In our strategy, nintedanib, a tyrosine kinase inhibitor, is employed to protect the human MSCs (hMSCs) from excessive oxidative stress responses and inflammatory environment in the damaged lung. Moreover, by labeling of the transplanted hMSCs with a computed tomography (CT) nanotracer, Au nanoparticles functionalized with polyethylenimine (PEI) and polyethylene glycol (PEG) (Au@PEI@PEG), in combination with red-emitting firefly luciferase (RfLuc), in vivo CT/bioluminescence (BL) dual-modal imaging tracking of the location, distribution, and survival of the transplanted hMSCs in presence of nintedanib were achieved, which facilitates the profound understanding of the role the stem cells play in IPF therapy.

Bioluminescence imaging of mouse monocyte chemoattractant protein-1 expression in inflammatory processes.

Monocyte chemoattractant protein-1 (MCP-1) plays a crucial role in various inflammatory diseases. To reveal the impact of MCP-1 during diseases and to develop anti-inflammatory agents, we establish a transgenic mouse line. The firefly luciferase gene is incorporated into the mouse genome and driven by the endogenous MCP-1 promoter. A bioluminescence photographing system is applied to monitor luciferase levels in live mice during inflammation, including lipopolysaccharide-induced sepsis, concanavalin A-induced T cell-dependent liver injury, CCl 4-induced acute hepatitis, and liver fibrosis. The results demonstrate that the luciferase signal induced in inflammatory processes is correlated with endogenous MCP-1 expression in mice. Furthermore, the expressions of MCP-1 and the luciferase gene are dramatically inhibited by administration of the anti-inflammatory drug dexamethasone in a septicemia model. Our results suggest that the transgenic MCP-1-Luc mouse is a useful model to study MCP-1 expression in inflammation and disease and to evaluate the efficiency of anti-inflammatory drugs in vivo.

Fibronectin modified alginate coating enhances cell targeting and homing to bone marrow in BALB/c mice.

Stem cell homing to bone marrow (BM) suffers from premature differentiation of transfused cells within the blood stream, thereby reducing the efficiency of stem cell transplantation (SCT). This work is attempted to enhance the homing of cells in BM. Fibronectin modified alginate (A-F) was prepared and used to coat the cells. Biodistribution and survival advantage provided by A-F coating was evaluated in BALB/c mice. The A-F conjugate showed characteristic FT-IR peaks of alginate at 3308 cm-1 and 1634 cm-1, and Fibronectin peak at 675 cm-1. The A-F coating prevented antibodies from binding to their respective cell surface receptors. The A-F coat abolished haemagglutination. Significant distribution of coated cells was observed in BM after 24 h. This provided protection to 7 Gy irradiated mice. The A-F coating showed no histological toxicity in vivo. The coating formulation is likely to be useful for shielding clinically relevant cell types to improve targeting for organ regeneration.

MAEBL Contributes to Plasmodium Sporozoite Adhesiveness.

The sole currently approved malaria vaccine targets the circumsporozoite protein-the protein that densely coats the surface of sporozoites, the parasite stage deposited in the skin of the mammalian host by infected mosquitoes. However, this vaccine only confers moderate protection against clinical diseases in children, impelling a continuous search for novel candidates. In this work, we studied the importance of the membrane-associated erythrocyte binding-like protein (MAEBL) for infection by Plasmodium sporozoites. Using transgenic parasites and live imaging in mice, we show that the absence of MAEBL reduces Plasmodium berghei hemolymph sporozoite infectivity to mice. Moreover, we found that maebl knockout (maebl-) sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. The maebl- defective phenotypes in mosquito salivary gland and liver infection were reverted by genetic complementation. Using a parasite line expressing a C-terminal myc-tagged MAEBL, we found that MAEBL levels peak in midgut and hemolymph parasites but drop after sporozoite entry into the salivary glands, where the labeling was found to be heterogeneous among sporozoites. MAEBL was found associated, not only with micronemes, but also with the surface of mature sporozoites. Overall, our data provide further insight into the role of MAEBL in sporozoite infectivity and may contribute to the design of future immune interventions.

Enhanced Survival of High-Risk Medulloblastoma-Bearing Mice after Multimodal Treatment with Radiotherapy, Decitabine, and Abacavir.

Children with high-risk SHH/TP53-mut and Group 3 medulloblastoma (MB) have a 5-year overall survival of only 40%. Innovative approaches to enhance survival while preventing adverse effects are urgently needed. We investigated an innovative therapy approach combining irradiation (RT), decitabine (DEC), and abacavir (ABC) in a patient-derived orthotopic SHH/TP53-mut and Group 3 MB mouse model. MB-bearing mice were treated with DEC, ABC and RT. Mouse survival, tumor growth (BLI, MRT) tumor histology (H/E), proliferation (Ki-67), and endothelial (CD31) staining were analyzed. Gene expression was examined by microarray and RT-PCR (Ki-67, VEGF, CD31, CD15, CD133, nestin, CD68, IBA). The RT/DEC/ABC therapy inhibited tumor growth and enhanced mouse survival. Ki-67 decreased in SHH/TP53-mut MBs after RT, DEC, RT/ABC, and RT/DEC/ABC therapy. CD31 was higher in SHH/TP53-mut compared to Group 3 MBs and decreased after RT/DEC/ABC. Microarray analyses showed a therapy-induced downregulation of cell cycle genes. By RT-PCR, no therapy-induced effect on stem cell fraction or immune cell invasion/activation could be shown. We showed for the first time that RT/DEC/ABC therapy improves survival of orthotopic SHH/TP53-mut and Group 3 MB-bearing mice without inducing adverse effects suggesting the potential for an adjuvant application of this multimodal therapy approach in the human clinic.

Tracking Immature Testicular Tissue after Vitrification In Vitro and In Vivo for Pre-Pubertal Fertility Preservation: A Translational Transgenic Mouse Model.

Pediatric cancer survivors experiencing gonadotoxic chemoradiation therapy may encounter subfertility or permanent infertility. However, previous studies of cryopreservation of immature testicular tissue (ITT) have mainly been limited to in vitro studies. In this study, we aim to evaluate in vitro and in vivo bioluminescence imaging (BLI) for solid surface-vitrified (SSV) ITT grafts until adulthood. The donors and recipients were transgenic and wild-type mice, respectively, with fresh ITT grafts used as the control group. In our study, the frozen ITT grafts remained intact as shown in the BLI, scanning electron microscopy (SEM) and immunohistochemistry (IHC) analyses. Graft survival was analyzed by BLI on days 1, 2, 5, 7, and 31 after transplantation. The signals decreased by quantum yield between days 2 and 5 in both groups, but gradually increased afterwards until day 31, which were significantly stronger than day 1 after transplantation (p = 0.008). The differences between the two groups were constantly insignificant, suggesting that both fresh and SSV ITT can survive, accompanied by spermatogenesis, until adulthood. The ITT in both groups presented similar BLI intensity and intact cells and ultrastructures for spermatogenesis. This translational model demonstrates the great potential of SSV for ITT in pre-pubertal male fertility preservation.