The aim of the study was to assess the efficacy of a treatment protocol that combines photodynamic therapy (PDT) and nitroglycerin (NG) on human retinoblastoma tumors xenografted on nude mice.

Purposes: The aim of the study was to assess the efficacy of a treatment protocol that combines photodynamic therapy (PDT) and nitroglycerin (NG) on human retinoblastoma tumors xenografted on mice. We aimed to increase the PDT efficiency (in our least treatment-responsive retinoblastoma line) with better PS delivery to the tumor generated by NG, which is known to dilate vessels and enhance the permeability and retention of macromolecules in solid tumors. Methods: In vivo follow-up of the therapeutic effects was performed by sodium MRI, which directly monitors variations in sodium concentrations non-invasively and can be used to track the tumor response to therapy. NG ointment was applied one hour before PDT. The PDT protocol involves double-tumor targeting, i.e., cellular and vascular. The first PS dose was injected followed by a second one, separated by a 3 h interval. The timelapse allowed the PS molecules to penetrate tumor cells. Ten minutes after the second dose, the PS was red-light-activated. Results: In this study, we observed that the PDT effect was enhanced by applying nitroglycerin ointment to the tumor-bearing animal's skin. PDT initiates the bystander effect on retinoblastomas, and NG increases this effect by increasing the intratumoral concentration of PS, which induces a higher production of ROS in the illuminated region and thus increases the propagation of the cell death signal deeper into the tumor (bystander effect).

Retinoblastoma is a rare intraocular malignancy in children. Current treatments have many adverse effects. New therapeutic approaches like intravitreal injections of chemotherapies are currently being developed but their toxicities need to be evaluated on animal models. This study compares the efficacy and toxicity of intravitreal melphalan, topotecan and carboplatin, alone or in combination (sequential administration), in the LHBetaTag retinoblastoma mice.

Choroidal melanoma is the most frequent malignant intraocular tumor. Radiotherapy using various modalities is the standard conservative treatment for confined lesions. It allows globe preservation in most cases, but 50% to 60% of irradiated eyes develop radiation retinopathy, secondary to multiple retinal vascular occlusions, leading in the most severe cases to neovascular glaucoma and secondary enucleation. The purpose of this experimental study was to create and test a new model of radiation retinopathy in the rat retina, and to explore potential pathways involved in the early pathogenesis of radiation retinopathy. We used the Small Animal Radiation Research Platform (SARRP, Xstrahl, UK) to build a dose-response model of retinal irradiation. Long-Evans rats were irradiated with an X-ray source, after exorbiting the eye. Only one eye was irradiated for ethical reasons. There were 4 groups of 4 animals according to the radiation dose received. Group 1 received one 15-Gray fraction of, Group 2 received two 15-Gray fractions, Group 3 received three 15-Gray fractions, and Group 4, the control group, received one sham irradiation fraction. Multimodal imaging with OCT, color fundus photograph, and fluoresceine angiography (Micron 4, Phoenix Researchlabs, USA) was performed at baseline and seven days after irradiation. Animals were sacrificed 8 days after irradiation. Eyes were either fixated for inclusion and sections, fixated for flat-mounts of retina/RPE, or milled for Western Blot analysis. Irradiating of rat retinas using SARRP was successful. At day 7, there were no specific features on angiography, but OCT revealed inconsistent features in the peripheral retina of one animal (Group 3 fractions). In two animals, exorbitation was not possible due to variations of periocular morphology (Groups 2 and 3 fractions). Both animals developed corneal opacifications attributed to lacrymal gland damage. SARRP is a simple and efficient tool to model radiation retinopathy in small animals. Further work is required to improve our understanding of this potentially blinding complication of ocular irradiation References Ramos MS, Echegaray JJ, Kuhn-Asif S, Wilkinson A, Yuan A, Singh AD, Browne AW.Animal models of radiation retinopathy - From teletherapy to brachytherapy.Exp Eye Res. 2019 Apr;181:240-251. Rothschild PR, Salah S, Berdugo M, Gélizé E, Delaunay K, Naud MC, Klein C, Moulin A, Savoldelli M, Bergin C, Jeanny JC, Jonet L, Arsenijevic Y, Behar-Cohen F, Crisanti P. ROCK-1 mediates diabetes-induced retinal pigment epithelial and endothelial cell blebbing: Contribution to diabetic retinopathy. Sci Rep. 2017 Aug 18;7(1):8834. Rousseau M, Gaugler M-H, Rodallec A, Bonnaud S, Paris F, Corre I. RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration. Biochem Biophys Res Commun. 2011;414(4):750-755.

Abstract Background: Porphyrins are a group of heterocyclic macrocycle organic compounds. They have shown good efficiency in the photodynamic treatment of tumors in the last twenty years. The photodynamic therapy could be an alternative treatment for retinoblastoma using a photosensitizer with low dark toxicity and low mutagenic properties unlike the conventional chemotherapies. The objectives of this study were to develop a photosensitizer in our lab which is a diethylene glycol mannoconjugated tetraphenyl porphyrin (TPP, MW: 1413.6) and to determine the optimal interval time for photodynamic therapy using the concentration of TPP in eye and blood samples of mice. Materials and method: Transgenic mice developed bilateral retinoblastoma from the retina since 4 weeks of age to 16 weeks of age (tumors fill the ocular globe and the mice will be sacrificed for ethic purpose). The evaluation of the biodistribution of our TPP was realized to know the best drug-light interval to perform the photodynamic therapy. For this purpose, an intraperitoneal injection of 200 µl at 5.25 mg/ml in PEG 400/physiological serum (1:1, v/v) was performed on the LHbetaTag transgenic mice. At different time intervals (8h, 24h, 40h, 48h, and 72h) following injection, the mice were sacrificed and the eyes and blood were collected. TPP in eye samples were extracted in methanol and the level of TPP was measured by ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) analysis (0.5-100 ng/mL). The level of TTP in blood samples was quantified after protein precipitation by UPLC-MS/MS method (5.00-1000 ng/mL). Results: We observed a time dependency accumulation of TPP in eyes with observed Tmax=48 hours. TPP serum concentrations decreased by the same manner up to 48 h. The determination of drug-light interval allowed us to perform the light delivery when the TPP level is highest (48 h) in the ocular tumor tissue (retinoblastoma). Conclusions: Our TPP had a good in vitro photocytotoxicity at very low doses and showed a good efficacy in vivo with subcutaneous patient derived retinoblastoma xenografts in immunodeficient mice. The knowledge of Tmax and this interval is essential to realize the photodynamic therapy with the better efficiency, the final goal being to eradicate the retinoblastoma from the retina. Citation Format: Florent Poyer, Samuel Hugeut, Stéphanie Lemaitre, Olivier Madar, Carole D Thomas, Philippe Maillard, Marie-Paule Teulade-Fichou, Nathalie Cassoux, François Doz, Keyvan Rezai. Evaluation of porphyrin biodistribution for use in photodynamic therapy of retinoblastoma in transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3213.

BACKGROUND:Due to the presence of speckle Poisson noise, the interpretation of spectral domain-optical coherence tomography (SD-OCT) images frequently requires the use of data averaging to improve the signal-to-noise ratio. This implies long acquisition times and requires patient sedation in some cases. Iterative variance stabilizing transformation (VST) is a possible approach by which to remove speckle Poisson noise on single images.METHODS:We used SD-OCT images of human and murine (LH Beta-Tag mouse model) retinas with and without retinoblastoma acquired with 2 different imaging devices (Bioptigen and Micron IV). These images were processed using a denoising workflow implemented in Matlab.RESULTS:We demonstrated the presence of speckle Poisson noise, which can be removed by a VST-based approach. This approach is robust as it works in all used imaging devices and in both human and mouse retinas, independently of the tumor status. The implemented algorithm is freely available from the authors on demand.CONCLUSIONS:On a single denoised image, the proposed method provides results similar to those expected from the SD-OCT averaging. Because of the friendly user interface, it can be easily used by clinicians and researchers in ophthalmology.

PurposeBecause retinoblastoma therapies have many adverse effects, new approaches must be developed and evaluated on animal models. We describe orthotopic xenograft models of retinoblastoma using different strains of mice, suitable for this purpose.MethodsHuman retinoblastoma tumors were established on immunodeficient mice by subcutaneous engraftment of tumors from enucleated eyes. The orthotopic model was obtained by subretinal injections of suspension cells into the right eye of immunodeficient (Swiss-nude, severe combined immunodeficiency [SCID]) and immunocompetent mice (C57BL/6N, B6Albino). In vivo tumor growth was monitored by fundus and spectral-domain optical coherence tomography (SD-OCT) imaging and compared with histology.ResultsRetinal and vitreal tumor growth was achieved both in immunocompetent and immunodeficient strains after the subretinal injection of tumor cells. The best tumor engraftment rate was obtained in the SCID mice (68.8%). No tumor growth was observed in the C57BL/6N strain. Chronic retinal detachment may occur in most strains after the subretinal injection, in particular the Swiss-nude strain, which exhibits retinal degeneration.ConclusionsThe setting up of an orthotopic mouse model depends mainly on the choice of the engrafted cells (cell lines or patient-derived xenografts) but it can also depend on the xenografted mouse strain. Severe combined immunodeficiency mice (an immunodeficient strain) achieved the best tumor engraftment rate (68.8%). However, intraocular tumor growth was also satisfactory (50%) in the immunocompetent strain B6Albino, and this strain will allow to exploit the immune response after a tumor treatment. Both of these strains may therefore be recommended when setting up orthotopic retinoblastoma xenografts.

Depuis plusieurs milliers d'années, la lumière qu'elle soit naturelle ou artificielle a été utilisée comme traitement de maladies de la peau, des yeux ou contre le cancer. Ce n'est qu'au début du XXe siècle que la science s'est intéressée aux interactions « lumière-vivant ». La compréhension de l'action conjuguée, dénommée « photothérapie dynamique », d'un médicament non toxique à l'obscurité, de l'oxygène et de la lumière a permis de mettre au point un protocole d'éradication de tumeurs bénignes ou malignes et d'anomalies vasculaires. Depuis environ 30 ans, on voit apparaître de nombreuses molécules photoactivables de plus en plus efficaces. Peu de molécules photosensibles ont encore atteint le stade clinique, mais les résultats publiés dans la littérature scientifique montrent que la photothérapie dynamique est en plein développement et a un avenir certain [1-3].

BACKGROUND:PDT represents a very localized and non-mutagen antitumoral treatment using a photosensitive molecule (porphyrin family) light activated. The first way of cell damage is a direct one, active on the very site where ROSs have been produced. The second one is indirect by activating and transmitting the processes of cellular death signaling. In order to seek for a better characterization of the photo-biology involved in in vivo PDT and to better understand the differences on the treatment outcome, we have used three different human retinoblastomas xenografted on mice.METHODS:Mice were treated according to the double targeting protocol exposed in a previous paper. One i.v. dose (0.6 mg/kg) of PS was followed by a second dose, separated by a 3 h interval (double targeting PDT). As a consequence both cancer cells and blood vessels were targeted. The treatment was repeated two times, at 4 days interval.RESULTS:First of all, sodium MRI revealed qualitative differences in the sodium average content of the three retinoblastoma lines before treatment. After the PDT treatments the tumor responses were different between the lines as revealed by sodium MRI and later on by histology.CONCLUSIONS:We have put into evidence that PDT is accompanied by a bystander effect that may propagate the cellular death triggered by the initial photoreaction. This effect is highly dependent on the cellular density of the tissue; therefore this factor is to be taken into account in clinical PDT protocols.

An overexpression of MRC2 and CD209 mannose receptors was revealed in retinoblastoma and antibodies against these receptors were grafted to multifunctional nanoparticles for targeting of imaging and photodynamic therapy.

BackgroundPrevious in vivo studies on photodynamic therapy (PDT)-treated, high cellular density tumors showed evidences of a bystander effect accompanying the therapy, cellular death continuing beyond the limits of the photochemical reactions in time and space. This process is generated by the initially damaged cells on the light pathway. The aim of this study was to determine if the bystander effect may be induced as well in colorectal xenografted tumors (less compact structure) and if the cellular signaling depends primarily on cellular proximity or not.MethodsThe photosensitizer was a glycoconjugated, meso substituted porphyrin derivative synthesized at Institut Curie. The longitudinal follow-up of the tumors was carried out by 23Na/1H MRI, ideal imaging modality for mapping the extracellular compartment. Two regimens were followed in order to target either blood vessels alone or blood vessels and cancer cells simultaneously.ResultsThe antivascular PDT did not succeed to arrest the tumors growth at the end of the follow-up. For double targeting PDT, we managed to stop the tumoral evolution. Sodium MRI evidenced a bystander effect.ConclusionThe results obtained showed that the bystander effect is more difficult to induce for the type of colorectal tumors used in this work. It needs a double treatment, 4 days apart, in order to be promoted.

Current treatments for choroidal neovascularization, a major cause of blindness for patients with age-related macular degeneration, treat symptoms but not the underlying causes of the disease. Inflammation has been strongly implicated in the pathogenesis of choroidal neovascularization. We examined the inflammatory role of Toll-like receptor 2 (TLR2) in age-related macular degeneration. TLR2 was robustly expressed by the retinal pigment epithelium in mouse and human eyes, both normal and with macular degeneration/choroidal neovascularization. Nuclear localization of NF-κB, a major downstream target of TLR2 signaling, was detected in the retinal pigment epithelium of human eyes, particularly in eyes with advanced stages of age-related macular degeneration. TLR2 antagonism effectively suppressed initiation and growth of spontaneous choroidal neovascularization in a mouse model, and the combination of anti-TLR2 and antivascular endothelial growth factor receptor 2 yielded an additive therapeutic effect on both area and number of spontaneous choroidal neovascularization lesions. Finally, in primary human fetal retinal pigment epithelium cells, ligand binding to TLR2 induced robust expression of proinflammatory cytokines, and end products of lipid oxidation had a synergistic effect on TLR2 activation. Our data illustrate a functional role for TLR2 in the pathogenesis of choroidal neovascularization, likely by promoting inflammation of the retinal pigment epithelium, and validate TLR2 as a novel therapeutic target for reducing choroidal neovascularization.

Photodynamic therapy (PDT) is a recent approach for the treatment of small cancerous tumours, on-surface or accessible by endoscopy in which a dye (usually a tetrapyrrolic macrocycle) absorbs light and generates cytotoxic reactive oxygen species leading to cellular damage. Retinoblastoma (Rb) is a rare intraocular tumour of childhood. All the multifocal forms are hereditary and constitute a syndrome of genetic predisposition in the cancer. The current treatments with etoposide or carboplatine expose the patient to the late risk of second cancer. The use of PDT as cancer therapy is particularly attractive due to the use of few mutagenic and non-toxic photosensitizers (PS) prior light excitation and to the localized tumour illumination. The photoefficiency towards Rb of a glycoconjugated porphyrin is discussed and compared with the results obtained with a second-generation photosensitizer, the Foscan. Some in vivo results on an animal model of Rb are presented by a point of view of photoefficiency, biodistribution, pharmacokinetic and longitudinal follow-up of the PDT effect using a new non-invasive method of magnetic resonance imaging of real-time. Photodynamic treatments in association with non-invasive sodium imaging open ways for new treatment tailoring or treatment individualization of retinoblastoma in clinic.

La photothérapie dynamique (PDT) est une approche récente du traitement de petites tumeurs cancéreuses de surface ou accessibles par endoscopie, associant l’action d’un photosensibilisateur, non toxique à l’obscurité, concentré dans la tumeur et d’une lumière focalisée sur celle-ci, de longueur d’onde appropriée. Le rétinoblastome (Rb) est une tumeur maligne intraoculaire rare de l’enfant. Toutes les formes multifocales sont héréditaires et constituent un syndrome de prédisposition génétique au cancer. Les chimiothérapies actuelles par étoposide ou carboplatine exposent le patient au risque tardif de deuxième cancer. Il serait donc souhaitable de développer des traitements alternatifs par des composés peu mutagènes. La photothérapie dynamique utilisant une molécule photoactivable localement est une alternative possible aux chimiothérapies. L’efficacité photodynamique contre Rb d’un photosensibilisateur glycoconjugué est discutée et comparée aux résultats obtenus avec un photosensibilisateur de seconde génération, le Foscan®. Quelques résultats in vivo sur un modèle animal de Rb sont présentés d’un point de vue photoefficacité, biodistribution, pharmacocinétique et suivi longitudinal de l’effet de la PDT grâce à une nouvelle méthode d’imagerie de résonance magnétique en temps réel. Un traitement par PDT associé à une méthode d’imagerie non invasive ouvre la voie à la possibilité clinique de nouveaux traitements individualisés du rétinoblastome.

BACKGROUND:Photodynamic therapy is an established cancer treatment in which a photosensitizing agent is activated by exposure to light thus generating cytotoxic reactive oxygen species that cause cellular damage.METHODS:A new photosensitizer synthesized at Curie Institute was used to treat retinoblastoma xenografts in mice, a glycoconjugated meso substituted porphyrin derivative, that showed some retinoblastoma cell affinity. The longitudinal follow-up of the tumors was carried out by (23)Na MRI (without adding exogenous contrast agents) to map the extracellular compartment and to characterize cell packing. Two regimens were followed to target either blood vessels alone or blood vessels and cancer cells simultaneously.RESULTS AND CONCLUSIONS:Only the protocol targeting both cancer cells and blood vessels effectively induces cellular death, confirmed by histology at the end of the experiment. Sodium MRI evidences a huge change in the cellular density of tumors only 24h after a double targeting (vascular and cellular) PDT treatment. We suggest that this change was possibly due to a bystander effect that can be promoted by the intercellular signaling favored by the high cellular density of retinoblastoma. These results indicate that non-invasive (23)Na imaging (which detects the tumor response to treatment from very early stages) in association with non-mutagenic therapies represents an effective option for tailored and individualized clinical treatments.

Sodium is the second best nucleus in living systems MRI after proton, but it is the first one concerning its sensitivity to the electrical configuration of the environment, due to its quadrupolar relaxation properties. Retrieving accurate relaxation data from low signal-to-noise ratio (SNR) experiments implies using post-processing procedures in order to fit correctly the decaying relaxation curves. The conventional image reconstruction method is introducing a positive bias due to the rician noise contribution. Phasing the images restores its initial Gaussian characteristic. Linear prediction (LP) forward of the decays improves the SNR of the last points and calculates missing points. An objective fitting method, based on singular value decomposition (SVD) allows us to determine the number of exponentials existing in the decay and its accurate fitting parameters. Sodium ions' spin-spin relaxation holds great potential to become an endogeneous marker for tumor diagnosis and for therapy response, since different regions of a mouse tumoral liver bear distinguishable relaxation parameters as compared to control.