e14573 Background: Stromal rich tumours pose a significant challenge to the effectiveness of systemic anticancer therapies, including immunotherapies and oncolytic viruses. This is due to the abundance of cancer associated fibroblasts (CAFs) that suppress immune cells and limit drug penetration. THEO-260 is a potent, mechanistically novel oncolytic virus therapy intrinsically capable of lysing both cancer cells and CAFs, and is currently in development for late stage relapsed refractory ovarian cancer. Methods: The efficacy and safety of THEO-260 was assessed in multicellular spheroids, in patient-derived ex vivo fresh ovarian samples and in mouse models of human stromal tumours. Results: Unpassaged ascites and tumour samples collected from cytoreductive surgeries for advanced ovarian cancer (n=24 independent patients) were treated with a clinically-relevant dose of THEO-260. THEO-260 effectively killed the cancer cell (EpCAM+ EGFR+PDL1+ CA125+) and CAF (FAP+) populations, being efficacious in treatment-naive and platinum-resistant patient samples. Importantly, the T cell population in these patients, in addition to primary normal fibroblasts (including intestinal, pulmonary, dermal, cardiac, uterine fibroblasts) from healthy donors, were not damaged. Ex vivo studies show THEO-260 stimulates effector T cell responses upon tumour lysis as indicated by IFNγ release. Demonstrating suitability as an IV delivered agent, THEO-260 was active in pooled human serum and whole human blood (n = 10 donors). THEO-260 excels in low nutrient, low proliferative environments, typical of human solid tumours and has been developed with a view to persist in the tumour microenvironment without the need for repeated cycles of delivery. THEO-260 is well tolerated in vivo following a clinically relevant intravenous dosing regimen. In PARP inhibitor and platinum resistant ovarian cancer murine models containing human CAFs and cancer cells, THEO-260 shows antitumor efficacy, with a complete reduction in tumour volume in subcutaneous and intraperitoneal systems, and superiority to SOC. Virus activity is not restricted to ovarian cancer, with cell line data demonstrating killing in additional solid tumour types, including pancreatic, colorectal, lung, and breast cancer. THEO-260 exhibits genetic and temperature based stability, and GMP manufacturing to high yields is underway. Conclusions: Taken together, we present a preclinical data package for THEO-260 supporting progression to Phase I clinical trials.

Sickle cell disease (SCD) is an inherited blood disorder characterized by sickled red blood cells (RBCs), which are more sensitive to haemolysis and can contribute to disease pathophysiology. Although treatment of SCD can include RBC transfusion, patients with SCD have high rates of alloimmunization. We hypothesized that RBCs from patients with SCD have functionally active mitochondria and can elicit a type 1 interferon response. We evaluated blood samples from more than 100 patients with SCD and found elevated frequencies of mitochondria in reticulocytes and mature RBCs, as compared to healthy blood donors. The presence of mitochondria in mature RBCs was confirmed by flow cytometry, electron microscopy, and proteomic analysis. The mitochondria in mature RBCs were metabolically competent, as determined by enzymatic activities and elevated levels of mitochondria-derived metabolites. Metabolically-active mitochondria in RBCs may increase oxidative stress, which could facilitate and/or exacerbate SCD complications. Coculture of mitochondria-positive RBCs with neutrophils induced production of type 1 interferons, which are known to increase RBC alloimmunization rates. These data demonstrate that mitochondria retained in mature RBCs are functional and can elicit immune responses, suggesting that inappropriate retention of mitochondria in RBCs may play an underappreciated role in SCD complications and be an RBC alloimmunization risk factor.

Aging and obesity independently contribute toward an endothelial dysfunction that results in an imbalanced VWF to ADAMTS13 ratio. In addition, plasma thrombin and plasmin generation are elevated and reduced, respectively, with increasing age and also with increasing body mass index (BMI). The severity risk of Corona Virus Disease 2019 (COVID-19) increases in adults older than 65 and in individuals with certain pre-existing health conditions, including obesity (>30 kg/m2). The present cross-sectional study focused on an analysis of the VWF/ADAMTS13 axis, including measurements of von Willebrand factor (VWF) antigen (VWF:AG), VWF collagen binding activity (VWF:CBA), Factor VIII antigen, ADAMTS13 antigen, and ADAMTS13 activity, in addition to thrombin and plasmin generation potential, in a demographically diverse population of COVID-19 negative (−) (n = 288) and COVID-19 positive (+) (n = 543) patient plasmas collected at the time of hospital presentation. Data were analyzed as a whole, and then after dividing patients by age (<65 and ≥65) and independently by BMI [<18.5, 18.5–24.9, 25–29.9, >30 (kg/m2)]. These analyses suggest that VWF parameters (i.e., the VWF/ADAMTS13 activity ratio) and thrombin and plasmin generation differed in COVID-19 (+), as compared to COVID-19 (−) patient plasma. Further, age (≥65) more than BMI contributed to aberrant plasma indicators of endothelial coagulopathy. Based on these findings, evaluating both the VWF/ADAMTS13 axis, along with thrombin and plasmin generation, could provide insight into the extent of endothelial dysfunction as well as the plasmatic imbalance in coagulation and fibrinolysis potential, particularly for at-risk patient populations.

Cancer spheroids are in vitro 3D models that became crucial in nanomaterials science thanks to the possibility of performing high throughput screening of nanoparticles and combined nanoparticle-drug therapies on in vitro models. However, most of the current spheroid analysis methods involve manual steps. This is a time-consuming process and is extremely liable to the variability of individual operators. For this reason, rapid, user-friendly, ready-to-use, high-throughput image analysis software is necessary. In this work, we report the INSIDIA 2.0 macro, which offers researchers high-throughput and high content quantitative analysis of in vitro 3D cancer cell spheroids and allows advanced parametrization of the expanding and invading cancer cellular mass. INSIDIA has been implemented to provide in-depth morphologic analysis and has been used for the analysis of the effect of graphene quantum dots photothermal therapy on glioblastoma (U87) and pancreatic cancer (PANC-1) spheroids. Thanks to INSIDIA 2.0 analysis, two types of effects have been observed: In U87 spheroids, death is accompanied by a decrease in area of the entire spheroid, with a decrease in entropy due to the generation of a high uniform density spheroid core. On the other hand, PANC-1 spheroids' death caused by nanoparticle photothermal disruption is accompanied with an overall increase in area and entropy due to the progressive loss of integrity and increase in variability of spheroid texture. We have summarized these effects in a quantitative parameter of spheroid disruption demonstrating that INSIDIA 2.0 multiparametric analysis can be used to quantify cell death in a non-invasive, fast, and high-throughput fashion.

Failure of immune tolerance can lead to autoantibody production resulting in autoimmune diseases, a broad spectrum of organ-specific or systemic disorders. Immune tolerance mechanisms regulate autoreactive T and B cells, yet some lymphocytes escape and promote autoantibody production. CD4+ T cell dysregulation, characterized by decreased or impaired regulatory cells (Tregs) and/or accumulation of memory and effector T cells such as TH17, plays a crucial role in the pathogenesis of these diseases. Antinuclear antibody (ANAs) testing is used as a first step for the diagnosis of autoimmune disorders, although most ANA-positive individuals do not have nor will develop an autoimmune disease. Studying the differences of T cell compartment among healthy blood donors, ANA-negative patients and ANA-positive patients, in which loss of tolerance have not led to autoimmunity, may improve our understanding on how tolerance mechanisms fail. Herein, we report that ANA-positive patients exhibit a distinct distribution of T cell subsets: significantly reduced frequencies of recent thymic emigrants (RTE) and naïve T cells, and significantly increased frequencies of central memory T cells, TH2 and TH17 cells; modulations within the T cell compartment are most profound within the 18-40 year age range. Moreover, CD4+ T cells in ANA-positive patients are metabolically active, as determined by a significant increase in mTORC1 and mTORC2 signals, compared to ANA-negative patients and healthy blood donors. No significant impairment of Treg numbers or pro-inflammatory cytokine production was observed. These results identify a unique T cell signature associated with autoantibody production in the absence of autoimmune disease.

Investigating the metabolic effects of radiation is critical to understand the impact of radiotherapy, space travel, and exposure to environmental radiation. In patients undergoing hemopoietic stem cell transplantation, iron overload is a common risk factor for poor outcomes. However, no studies have interrogated the multiorgan effects of these treatments concurrently. Herein, we use a model that recapitulates transfusional iron overload, a condition often observed in chronically transfused patients. We applied an omics approach to investigate the impact of both the iron load and irradiation on the host metabolome. The results revealed dose-dependent effects of irradiation in the red blood cells, plasma, spleen, and liver energy and redox metabolism. Increases in polyamines and purine salvage metabolites were observed in organs with high oxygen consumption including the heart, kidneys, and brain. Irradiation also impacted the metabolism of the duodenum, colon, and stool, suggesting a potential effect on the microbiome. Iron infusion affected the response to radiation in the organs and blood, especially in erythrocyte polyamines and spleen antioxidant metabolism, and affected glucose, methionine, and glutathione systems and tryptophan metabolism in the liver, stool, and the brain. Together, the results suggest that radiation impacts metabolism on a multiorgan level with a significant interaction of the host iron status.

The Corona Virus Disease 2019 (COVID-19) pandemic represents an ongoing worldwide challenge. The present large study sought to understand independent and overlapping metabolic features of samples from acutely ill patients (n = 831) that tested positive (n = 543) or negative (n = 288) for COVID-19. High-throughput metabolomics analyses were complemented with antigen and enzymatic activity assays on plasma from acutely ill patients collected while in the emergency department, at admission, or during hospitalization. Lipidomics analyses were also performed on COVID-19-positive or -negative subjects with the lowest and highest body mass index (n = 60/group). Significant changes in amino acid and fatty acid/acylcarnitine metabolism emerged as highly relevant markers of disease severity, progression, and prognosis as a function of biological and clinical variables in these patients. Further, machine learning models were trained by entering all metabolomics and clinical data from half of the COVID-19 patient cohort and then tested on the other half, yielding ~78% prediction accuracy. Finally, the extensive amount of information accumulated in this large, prospective, observational study provides a foundation for mechanistic follow-up studies and data sharing opportunities, which will advance our understanding of the characteristics of the plasma metabolism in COVID-19 and other acute critical illnesses.

Loss of humoral tolerance to red blood cells (RBCs) can lead to autoimmune hemolytic anemia (AIHA), a severe, and sometimes fatal disease. Patients with AIHA present with pallor, fatigue, decreased hematocrit, and splenomegaly. While secondary AIHA is associated with lymphoproliferative disorders, infections, and more recently, as an adverse event secondary to cancer immunotherapy, the etiology of primary AIHA is unknown. Several therapeutic strategies are available; however, there are currently no licensed treatments for AIHA and few therapeutics offer treatment-free durable remission. Moreover, supportive care with RBC transfusions can be challenging as most autoantibodies are directed against ubiquitous RBC antigens; thus, virtually all RBC donor units are incompatible. Given the severity of AIHA and the lack of treatment options, understanding the cellular and molecular mechanisms that facilitate the breakdown in tolerance would provide insight into new therapeutics. Herein, we report a new murine model of primary AIHA that reflects the biology observed in patients with primary AIHA. Production of anti-erythrocyte autoantibodies correlated with sex and age, and led to RBC antigen modulation, complement fixation, and anemia, as determined by decreased hematocrit and hemoglobin values and increased reticulocytes in peripheral blood. Moreover, autoantibody-producing animals developed splenomegaly, with altered splenic architecture characterized by expanded white pulp areas and nearly diminished red pulp areas. Additional analysis suggested that compensatory extramedullary erythropoiesis occurred as there were increased frequencies of RBC progenitors detectable in the spleen. No significant correlations between AIHA onset and inflammatory status or microbiome were observed. To our knowledge, this is the first report of a murine model that replicates observations made in humans with idiopathic AIHA. Thus, this is a tractable murine model of AIHA that can serve as a platform to identify key cellular and molecular pathways that are compromised, thereby leading to autoantibody formation, as well as testing new therapeutics and management strategies.

Glioblastoma (GB) remains an aggressive malignancy with an extremely poor prognosis. Discovering new candidate drug targets for GB remains an unmet medical need. Caveolin-1 (Cav-1) has been shown to act variously as both a tumour suppressor and tumour promoter in many cancers. The implications of Cav-1 expression in GB remains poorly understood. Using clinical and genomic databases we examined the relationship between tumour Cav-1 gene expression (including its spatial distribution) and clinical pathological parameters of the GB tumour and survival probability in a TCGA cohort (n=155) and CGGA cohort (n=220) of GB patients. High expression of Cav-1 represented a significant independent predictor of shortened survival (HR = 2.985, 5.1 vs 14.9 months) with a greater statistically significant impact in female patients and in the Proneural and Mesenchymal GB subtypes. High Cav-1 expression correlated with other factors associated with poor prognosis: IDH w/t status, high histological tumour grade and low KPS score. A total of 4879 differentially expressed genes (DEGs) in the GB tumour were found to correlate with Cav-1 expression (either positively or negatively). Pathway enrichment analysis highlighted an over-representation of these DEGs to certain biological pathways. Focusing on those that lie within a framework of epithelial to mesenchymal transition and tumour cell migration and invasion we identified 27 of these DEGs. We then examined the prognostic value of Cav-1 when used in combination with any of these 27 genes and identified a subset of combinations (with Cav-1) indicative of co-operative synergistic mechanisms of action. Overall, the work has confirmed Cav-1 can serve as an independent prognostic marker in GB, but also augment prognosis when used in combination with a panel of biomarkers or clinicopathologic parameters. Moreover, Cav-1 appears to be linked to many signalling entities within the GB tumour and as such this work begins to substantiate Cav-1 or its associated signalling partners as candidate target for GB new drug discovery.

Optical neural networks process information at the speed of light and are energetically efficient. Photonic artificial intelligence allows speech recognition, image classification, and Ising machines. Modern machine learning paradigms, as extreme learning machines, reveal that disordered and biological materials may realize optical neural networks with thousands of nodes trained only at the input and at the readout. May we use living matter for machine learning? Here, we employ living three-dimensional tumor brain models to demonstrate a random optical learning machine (ROM) for the investigation of glioblastoma. The tumor spheroid act as a computational reservoir. The ROM detects cancer morphodynamics by laser-induced hyperthermia, quantifies chemotherapy, and cell metabolism. The ROM is a sensitive noninvasive smart probe for cytotoxicity assay and enables real-time investigation of tumor dynamics. We hence design and demonstrate a novel bio-hardware for optical computing and the study of light/complex matter interaction. Can living systems function as artificial neural networks for biophysical applications? Here, the authors show that living tumor spheroids can be employed as random optical learning machines and used to investigate cancer morphodynamics and quantify the effect of chemotherapy.

Optical neuromorphic computing processes information at the speed of light, but requires a careful design and fabrication of the deep layers, which strongly hampers the development of large-scale photonic learning machines [1,2]. New paradigms, as reservoir computing [3], suggest that brain-inspired complex systems such as disordered and biological materials may realize artificial neural networks with thousands of computational nodes trained only at the input and at the readout.

We employ living three-dimensional tumour brain models to demonstrate a bio-inspired optical neural network trained via image transmission to detect cancer mor-phodynamics inaccessible by optical imaging.

Glioblastoma multiforme (GBM) is a malignant and highly aggressive form of brain tumour, with extremely poor prognosis. One of its features is the ability of the tumour to invade through normal brain resulting in tumour relapse. Our hypothesis was that Caveolin-1 (Cav-1), a major component of the caveolae and recognized to be involved in a number of signalling pathways, has a key pro-invasive role in GBM. We pursued our hypothesis by inhibiting the expression of Cav-1 in different adult GBM cell lines using different genetic techniques (liposome shRNA, lentiviral shRNA and CRISPR). We found that Cav-1 drives clonogenicity (CHAPTER 3) and invasion in a combination of two- and three-dimensional models (CHAPTER 5). We focused our research on the invasion phenomenon and, in order to provide a robust quantification approach to study invasion in 3D spheroid assays, we developed (CHAPTER 4) a open-source semi-automated script, INSIDIA, available for all researchers in the community to use. This tool was used to quantify the impact of Cav-1 on invasive capacity. In in-vitro systems, we explored the impact of Cav-1 expression upon molecules associated with the invasion phenomenon (CHAPTER 5). We found Cav-1 to be associated with CTSB, MMP1 and UPA and receptors like UPAR and CD44, as well as AKT activation. Interrogating the “The Cancer Genome Atlas” (TCGA) database, we confirmed that Cav-1 is an independent biomarker of poor prognosis in GBM patients (CHAPTER 6). This clinical data also found association of genes that may cooperate with Cav-1, including CD44, ITGA3, VIM, CTSB, CTSL, TSP-1, TIMP1 and MT1MMP. Collectively this thesis provides strong in vitro and clinical data supporting that Cav-1 as a key molecule promoting GBM invasion, and further identifying Cav-1 as a potential drug discovery target in GBM.

Glioblastoma multiforme (GBM) cells display a highly invasive phenotype, a hallmark which counters effective surgical and radiotherapy strategies. Caveolin-1 (Cav-1) is the main structural and functional component of caveolae. The impact of the expression of Cav-1 within a range of tumour and tumour-associated stromal cells is variable with both oncogenic and tumour suppressive roles reported which appear to be both disease-specific and context-dependent. Our hypothesis is that Cav-1 serves as promoter of invasion of GBM cells. To investigate our hypothesis we used a lentiviral shRNA approach to silence Cav-1 in three GBM cell lines (U87, UP007, UP029) derived from adult brain tumours. We employed an in-vitro 3D cell-sprouting invasion assay with GBM cell spheres embedded in Matrigel. Quantification of invasion was undertaken using a novel image analysis tool or 3D systems, INSIDIA (ImageJ Macro for High-throughput Spheroid Invasion Analysis). Parallel migration and invasion studies were performed using a Boyden Chamber approach, as well as cell-cell adhesion assays. Activation of signalling pathways in 2D and 3D cultures were performed by proteomic array and Western Blot analysis. GBM cells expressing Cav-1 (Cav-1 +ve) displayed a higher invasive capacity compared cells where Cav-1 had been silenced Cav-1 –ve), the latter also showing increased cell-cell adhesion. A significant finding from the signalling analysis was an inverse association between Cav-1 silencing and activation of AKT evidenced by increased phosphorylation at both Ser473 and Thr308 sites. Ongoing studies are exploring this signalling axis and its relationship to the invasive phenotype. CM and MG acknowledge Cancer Research Wales support. GP and HF acknowledge Brain Tumour Research support.

The new era of artificial intelligence demands large-scale ultrafast hardware for machine learning. Optical artificial neural networks process classical and quantum information at the speed of light, and are compatible with silicon technology, but lack scalability and need expensive manufacturing of many computational layers. New paradigms, as reservoir computing and the extreme learning machine, suggest that disordered and biological materials may realize artificial neural networks with thousands of computational nodes trained only at the input and at the readout. Here we employ biological complex systems, i.e., living three-dimensional tumour brain models, and demonstrate a random neural network (RNN) trained to detect tumour morphodynamics via image transmission. The RNN, with the tumour spheroid as a three-dimensional deep computational reservoir, performs programmed optical functions and detects cancer morphodynamics from laser-induced hyperthermia inaccessible by optical imaging. Moreover, the RNN quantifies the effect of chemotherapy inhibiting tumour growth. We realize a non-invasive smart probe for cytotoxicity assay, which is at least one order of magnitude more sensitive with respect to conventional imaging. Our random and hybrid photonic/living system is a novel artificial machine for computing and for the real-time investigation of tumour dynamics.

Time-series image capture of in vitro 3D spheroidal cancer models embedded within an extracellular matrix affords examination of spheroid growth and cancer cell invasion. However, a customizable, comprehensive and open source solution for the quantitative analysis of such spheroid images is lacking. Here, the authors describe INSIDIA (INvasion SpheroID ImageJ Analysis), an open-source macro implemented as a customizable software algorithm running on the FIJI platform, that enables high-throughput high-content quantitative analysis of spheroid images (both bright-field gray and fluorescent images) with the output of a range of parameters defining the spheroid "tumor" core and its invasive characteristics.

INTRODUCTION: The poor prognosis associated with Glioblastoma multiforme (GBM) is multifactorial but includes the capacity of residual tumour cells not removed by surgery and resistant to radio-/chemo-therapy undergoing diffuse invasion into the surrounding brain tissue. Caveolin-1 (Cav-1) is the major structural and functional component of caveolae. In a number of tumour types Cav-1 is recognised to participate in cytoskeletal rearrangement, integrin-mediated adhesion and/or matrix remodelling. We proposed Cav-1 serves to promote invasion of GBM cells. To investigate this we have employed in an in-vitro 3D cellular invasion assay. METHOD: The human GBM cell lines, UP007 and UP029 established from primary cultures of biopsy-derived brain tumours (University of Portsmouth), U-87 MG (ECACC) and U-373 MG (ECACC) were genetically modified to stably knock-out Cav-1 using a Lentiviral Cav-1 shRNA approach; corresponding stably transfected non-target (NT) shRNA cell lines were generated as controls. Neuropheres were formed and embedded within an extracellular matrix (Matrigel™). Over a two-/four-day period (depending on cell line) the migration of cells away from the neurophere core (CORE) was quantified by image capture and processing (Image J) using a custom-developed MatLab script for pixel density analysis indicative of the density of migrating cellular material. RESULTS: Cav-1 knockout resulted in significant (P<0.01) reductions (42–54%) in cell invasion (reduced area of invasion and cell density within this area) of UP007, UP029 and U-87 MG; reductions comparable in magnitude to that of exposure to high concentrations of the Src inhibitor – saracatinib (2 μM). The wild-type and control (NT) U-373 MG showed very little invasive capacity with the corresponding Cav-1 negative cells showing only a consistent trend (P>0.05) towards reduced invasion. Depending upon the cell line the Cav-1 knockdown also resulted in reduced size and cellular density of the neurosphere core (UP007 and UP029) indicative of reduced proliferation and/or cell survival capacity. CONCLUSION: Using an in-vitro 3D cellular invasion assay we have found Cav-1 expression in a series of three GBM cell lines to promote cellular invasion capacity. Ongoing studies are addressing signalling mechanisms and the influence of the microenvironment. ACKNOWLEDGEMENTS: CM and MG acknowledge support from the Cancer Research Wales Ed Evans Scholarship for CM. GP and HF acknowledge support from Brain Tumour Research.

Metastatic growth in breast cancer (BC) has been proposed as an exclusive property of cancer stem cells (CSCs). However, formal proof of their identity as cells of origin of recurrences at distant sites and the molecular events that may contribute to tumor cell dissemination and metastasis development are yet to be elucidated. In this study, we analyzed a set of patient-derived breast cancer stem cell (BCSC) lines. We found that in vitro BCSCs exhibit a higher chemoresistance and migratory potential when compared with differentiated, nontumorigenic, breast cancer cells (dBCCs). By developing an in vivo metastatic model simulating the disease of patients with early BC, we observed that BCSCs is the only cell population endowed with metastatic potential. Gene-expression profile studies comparing metastagenic and non-metastagenic cells identified TAZ, a transducer of the Hippo pathway and biomechanical cues, as a central mediator of BCSCs metastatic ability involved in their chemoresistance and tumorigenic potential. Overexpression of TAZ in low-expressing dBCCs induced cell transformation and conferred tumorigenicity and migratory activity. Conversely, loss of TAZ in BCSCs severely impaired metastatic colonization and chemoresistance. In clinical data from 99 BC patients, high expression levels of TAZ were associated with shorter disease-free survival in multivariate analysis, thus indicating that TAZ may represent a novel independent negative prognostic factor. Overall, this study designates TAZ as a novel biomarker and a possible therapeutic target for BC.

Noise exposure causes damage of multiple cochlear cell types producing permanent hearing loss with important social consequences. In mammals, no regeneration of either damaged hair cells or auditory neurons has been observed and no successful treatment is available to achieve a functional recovery. Loads of evidence indicate adipose-derived stem cells (ASCs) as promising tools in diversified regenerative medicine applications, due to the high degree of plasticity and trophic features. This study was aimed at identifying the path of in vivo cell migration and expression of trophic growth factors, upon ASCs transplantation into the cochlea, following noise-induced injury. ASCs were isolated in primary culture from the adipose tissue of a guinea pig, transduced using a viral vector to express the green fluorescent protein, and implanted into the scala tympani of deafened animals. Auditory function was assessed 3 and 7 days after surgery. The expression of trophic growth factors was comparatively analyzed using real-time PCR in control and noise-injured cochlear tissues. Immunofluorescence was used to assess the in vivo localization and expression of trophic growth factors in ASCs and cochleae, 3 and 7 days following homologous implantation. ASC implantation did not modify auditory function. ASCs migrated from the perilymphatic to the endolymphatic compartment, during the analyzed time course. Upon noise exposure, the expression of chemokine ligands and receptors related to the PDGF, VEGF, and TGFbeta pathways, increased in the cochlear tissues, possibly guiding in vivo cell migration. Immunofluorescence confirmed the increased expression, which appeared to be further strengthened by ASCs' implantation. These results indicated that ASCs are able to migrate at the site of tissue damage and express trophic factors, upon intracochlear implantation, providing an original proof of principle, which could pave the way for further developments of ASC-based treatments of deafness.