Background: Graves’ disease (GD) is an autoimmune condition in which autoantibodies to the thyrotropin receptor (TSHR) cause hyperthyroidism. About 50% of GD patients also have Graves’ orbitopathy (GO), an intractable disease in which expansion of the orbital contents causes diplopia, proptosis and even blindness. Murine models of GD/GO, developed in different centres, demonstrated significant variation in gut microbiota composition which correlated with TSHR-induced disease heterogeneity. To investigate whether correlation indicates causation, we modified the gut microbiota to determine whether it has a role in thyroid autoimmunity. Female BALB/c mice were treated with either vancomycin, probiotic bacteria, human fecal material transfer (hFMT) from patients with severe GO or ddH2O from birth to immunization with TSHR-A subunit or beta-galactosidase (βgal; age ~ 6 weeks). Incidence and severity of GD (TSHR autoantibodies, thyroid histology, thyroxine level) and GO (orbital fat and muscle histology), lymphocyte phenotype, cytokine profile and gut microbiota were analysed at sacrifice (~ 22 weeks).Results: In ddH2O-TSHR mice, 84% had pathological autoantibodies, 67% elevated thyroxine, 77% hyperplastic thyroids and 70% orbital pathology.Firmicuteswere increased, andBacteroidetesreduced relative to ddH2O-βgal; CCL5 was increased. The random forest algorithm at the genus level predicted vancomycin treatment with 100% accuracy but 74% and 70% for hFMT and probiotic, respectively. Vancomycin significantly reduced gut microbiota richness and diversity compared with all other groups; the incidence and severity of both GD and GO also decreased; reduced orbital pathology correlated positively withAkkermansiaspp. whilst IL-4 levels increased. Mice receiving hFMT initially inherited their GO donors’ microbiota, and the severity of induced GD increased, as did the orbital brown adipose tissue volume in TSHR mice. Furthermore, genusBacteroides, which is reduced in GD patients, was significantly increased by vancomycin but reduced in hFMT-treated mice. Probiotic treatment significantly increased CD25⁺Treg cells in orbital draining lymph nodes but exacerbated induced autoimmune hyperthyroidism and GO.Conclusions: These results strongly support a role for the gut microbiota in TSHR-induced disease. Whilst changes to the gut microbiota have a profound effect on quantifiable GD endocrine and immune factors, the impact on GO cellular changes is more nuanced. The findings have translational potential for novel, improved treatments.

The treatment of uveal melanoma and its metastases has not evolved sufficiently over the last decades in comparison to other tumour entities, posing a great challenge in the field of ocular oncology. Despite improvements in the conventional treatment regime and new discoveries about the genetic and molecular background of the primary tumour, effective treatment strategies to either prevent tumours or treat patients with advanced or metastatic disease are still lacking. New therapeutic options are necessary in order to achieve satisfactory local tumour control, reduce the risk of metastasis development, and preserve the eyeball and possibly the visual function of the eye. The development of in vivo model systems remains crucial for the identification and investigation of potential novel treatment modalities. The aim of this study was the optimisation of the chorioallantoic membrane (CAM) model for uveal melanoma research. We analysed the established CAM assay and its modification after the implantation of three-dimensional spheroids. The chorioallantoic membrane of a chick embryo was used to implant uveal melanoma-cell-line-derived spheroids in order to study their growth rate, angiogenic potential, and metastatic capability. Using the UM 92.1, UPMD2, UPMM3, and Mel270 cell lines, we were able to improve the viability of the embryos from 20% to >80% and to achieve up to a fourfold volume increase of the transplanted spheroid masses. The results point to the value of an optimised chicken embryo assay as an in vivo model for testing novel therapies for uveal melanoma by simplifying the research conditions and by contributing to a considerable reduction in animal experiments.

Graves’ orbitopathy (GO) is the most common extra thyroidal complication of Graves’ disease (GD) and occurs predominantly in women but more severe in men. The reason for this effect of gender on GO is unknown. Herein we studied the manifestation of GO in both sexes of an induced mouse model in absence of additional risk factors present in patients like advanced age, genetic variabilities or smoking. Male and female mice were immunized with human TSHR A-subunit encoding plasmid. Both sexes comparably developed autoimmune hyperthyroidism characterized by TSHR stimulating autoantibodies, elevated T4 values, hyperplastic thyroids and hearts. Autoimmune mice developed inflammatory eye symptoms and proptosis, although males earlier than females. Serial in vivo¹H/¹⁹F-magnetic resonance imaging revealed elevated inflammatory infiltration, increased fat volume and glycosaminoglycan deposition in orbits of both sexes but most significantly in female mice. Histologically, infiltration of T-cells, extension of brown fat and overall collagen deposition were characteristics of GO in male mice. In contrast, female mice developed predominately macrophage infiltration in muscle and connective tissue, and muscle hypertrophy. Apart from sex-dependent variabilities in pathogenesis, disease classification revealed minor sex-differences in incidence and total outcome. In conclusion, sex does not predispose for autoimmune hyperthyroidism and associated GO.

Uveal melanoma (UM) is the most common primary cancer of the eye in adults. A new systemic therapy is needed to reduce the high metastasis and mortality rate. As β-blockers are known to have anti-tumor effects on various cancer entities, this study focuses on investigating the effect of β1-selective blockers atenolol, celiprolol, bisoprolol, metoprolol, esmolol, betaxolol, and in particular, nebivolol on UM. The study was performed on 3D tumor spheroids as well as 2D cell cultures, testing tumor viability, morphological changes, long-term survival, and apoptosis. Flow cytometry revealed the presence of all three β-adrenoceptors with a dominance of β2-receptors on cell surfaces. Among the blockers tested, solely nebivolol concentration-dependently decreased viability and altered 3D tumor spheroid structure. Nebivolol blocked the repopulation of cells spreading from 3D tumor spheroids, indicating a tumor control potential at a concentration of ≥20 µM. Mechanistically, nebivolol induced ATP depletion and caspase-3/7 activity, indicating that mitochondria-dependent signaling is involved. D-nebivolol or nebivolol combined with the β2-antagonist ICI 118.551 displayed the highest anti-tumor effects, suggesting a contribution of both β1- and β2-receptors. Thus, the present study reveals the tumor control potential of nebivolol in UM, which may offer a perspective for co-adjuvant therapy to reduce recurrence or metastasis.

Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite local tumor control, no effective therapy has been found to prevent metastasis, resulting in a high mortality rate. In the present study, we evaluated the anti-tumor potential of non-selective ß-blockers in 3D tumor spheroids grown from UM cell lines. Of the various ß-blockers tested, carvedilol and its enantiomers were most potent in decreasing the viability of Mel270 spheroids. Carvedilol at a concentration of 10–50 µM significantly elicited cytotoxicity and induced apoptosis in spheroid cells. In result, carvedilol inhibited tumor spheroid growth and compactness, and furthermore prevented the long-term survival and repopulation of spreading spheroid cells. The drug sensitivity of the different spheroids grown from Mel270, 92-1, UPMD2, or UPMM3 cell lines was dependent on 3D morphology rather than on high-risk cytogenetic profile or adrenergic receptor expression levels. In fact, the monosomy-3-containing UPMM3 cell line was most responsive to carvedilol treatment compared to the other cell lines. The concurrent treatment of UPMM3 spheroids with carvedilol and 5 or 10 Gy irradiation revealed additive cytotoxic effects that provided tumor control. Collectively, our data demonstrate the anti-tumor properties of carvedilol and its enantiomers, which may serve as candidates for the co-adjuvant therapy of UM.

Electrochemotherapy (ECT) is emerging as a complementary treatment modality for local tumor control in various cancer entities. Irradiation is an established therapeutic option for oncologic patients, which is commonly combined with chemotherapy due to its insufficient targeting ability. The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. ECT can potentiate the radiosensitizing effect of chemotherapeutic agents such as bleomycin. The present study aims to evaluate the radiosensitizing effect of concomitant ECT with bleomycin on 3D tumor spheroids with primary and radioresistant uveal melanoma cell lines (UPMD2, UPMM3, UM92.1, Mel270) and irradiation. The changes in the spheroid growth and the cell viability as well the cytotoxic long-term effect of the combination treatment were evaluated with various combinations of electroporation settings and bleomycin concentrations as well as radiotherapy doses. A broad range of radiosensitivity was documented among the spheroids from different uveal melanoma cell lines. The primary cell lines showed a higher radiosensitivity and required lower irradiation and bleomycin doses. The maximal tumor control with a reduction of cell survival <10% was achieved with a 5 Gy irradiation only in the primary uveal melanoma cell lines and in combination with all tested ECT settings, whereas the same result could be obtained in UM92.1 spheroids only after ECT with 20 Gy irradiation. Based on the spheroid growth and the measurement of the cross-sectional area, the Mel270 spheroids, originating from a previously irradiated recurrent uveal melanoma, required higher doses of bleomycin and ECT settings after irradiation with 5 Gy in order to achieve a significant growth reduction. No significant difference could be demonstrated for the reduction of cell viability in the combination therapy with 20 Gy and 1000 V/cm between 1 and 2.5 µg/mL bleomycin even in Mel270 spheroids, underlying the importance of a drug delivery system to potentiate the radiosensitizing effect of agents in lower doses. ECT should be further assessed for its applicability in clinical settings as a therapeutic radiosensitizing option for radioresistant tumors and a sufficient local tumor control with lower chemotherapy and irradiation doses.