Application Of Optogenetic Techniques In The Treatment Of Uveal Melanoma

Objective Uveal melanoma is the most common intraocular primary malignancy with a very high fatality rate in adults.Effective,non-invasive,and non-interventional treatment options for uveal melanoma are in urgent need.Optogenetics is an emerging technique that can control the activation of signaling components via irradiation with visible light.The clinical translation of optogenetics has been limited because of the need for surgical implantation of electrodes and relatively shallow tissue penetration.As visible light easily penetrates the eyes,this technology has its unique advantages for the treatment of ophthalmic diseases,so we hypothesized that an optogenetics approach with visible light can be an effective treatment of uveal melanoma without surgery.We evaluated the feasibility of this strategy by using a genetically encoded optogenetic system based on reversible blue light-induced binding pairs between Fas-CIB1-EGFP and CRY2-mCherry-FADD,thus activating the intracellular apoptosis signal pathway.In this study,we observed whether the optogenetic system has a therapeutic effect in vivo and in vitro,so as to find a potential novel therapeutic strategy for uveal melanoma.Methods 1.Transfection and detection of transfection efficiency: The Fas-CIB1-EGFP(green)and CRY2-mCherry-FADD(red)plasmids were co-transfected into B16 cells by Lipofectamine 3000.The efficiency of cell transfection was determined by imaging EGFP and mCherry with excitation at 488 nm and 561 nm,respectively,using fluorescence microscopy.2.Live cell imaging:After blue light exposure(488 nm),blue light-activated translocation of FADD to Fas in B16 cells was photoed in real time with confocal microscope.3.Detection of the blue light-induced B16 apoptosis by the optogenetic system in vitro: B16 cells were divided into four groups: blue-light group,STS(positive control)group,plasmids plus blue-light group,plasmids group.After 24 h post-blue light exposure,Annexin V-FITC/PI apoptosis detection assay was used to test B16 cell apoptosis efficiency by the optogenetic system.Hoechst-PI double staining was for detecting changes in the B16 cell nucleus of apoptotic cells and the percentage of the apoptotic cells in each group.Use caspase-3 and cleaved caspase-3 antibody for western blot analysis.4.Intraocular melanoma animal model: B16 cell suspension was injected into the sub-retina of mice.On the 7th day after modeling,paraffin sections of the model eyes and hematoxylin-eosin(HE)staining were made to observe the growth of melanoma in the eyes of mice.5.Therapy of optogenetic nanosystems controlled by blue-light: On the 7th day after the injection of B16 cells into the eyes of 48 mice,they were randomly divided into four group: group of blue-light,group of plasmid injection,group of plasmid injection plus blue light,and group of Hanks’ solution.There are 12 mice in each group.With the help of the self-developed blue-light automatic control system,it is adjusted to illuminate 10 second per minute for 2 hours per day,for 7 days.6.Test of the binding of optogenetic system induced by blue light irradiation in vivo:On the 7th day,24 hours after intratumoral injection of optogenetic nanosystem,the eyes were irradiated with blue light for 10 seconds per minute for 2 hours.Frozen sections were made quickly,excited by laser with wavelength of 488 nm and 561 nm,under confocal microscopy.7.In vivo imaging and quantification of TUNEL-positive cells:After 1 week of treatment,the tumors were collected.Histological analyses and quantification of TUNEL-positive cells were performed using terminal deoxynucleotidyl d UTP nick-end labeling(TUNEL)staining.8.Assess the degree of uveal melanoma apoptosis: Eyeballs of mice in different experimental groups were harvested and photographed,and their sizes were compared.Then complete the frozen sections and HE staining,the volumes of eyeball tumors in each group were measured and compared.Results 1.B16 cell transfection was successful.Transfection efficiency of the optogenetic nanosystem in vitro was about 40%.2.Confocal microscopy images through a ×63 oil immersion objective showed CRY2-mCherry-FADD recruitment to the Fas-CIB1-EGFP on the cell membrane after blue light exposure(488 nm)and dissociation from the cell membrane in the dark.3.Flow cytometry analysis revealed that the ratios of early and late apoptotic cells were much higher in blue-light plus plasmids group than in blue-light group and plasmids group(~*~*~*P < 0.001).4.Hoechst-propidium iodide(PI)double staining demonstrated the percentage of apoptotic cells were much higher in blue-light plus plasmids group than in bluelight group and plasmids group(~*~*P < 0.001).5.Western blot analysis showed that caspase-3 cleavage occurred in the cells of blue-light plus plasmids group but not in those of group of blue-light and group of plasmids at 24 h after blue light exposure.6.Intraocular melanoma animal models were established successfully.7.After the excitation of 488 nm and 561 nm laser,only in blue-light plus plasmids group,the tumor cells expressed the green marked fusion protein Fas-CIB1-EGFP and the red marked fusion protein CRY2-mCherry-FADD,and the merge images showed that they coincided.8.The results of TUNEL staining showed higher TUNEL-positive rates for bluelight plus plasmids group,much higher than those in blue-light group and plasmids group(~*~*P < 0.01).9.An obvious reduction in both the eyeball and tumor volume of blue-light plus plasmids group,compared with in blue-light group and plasmids group was observed.Conclusion In summary,we demonstrated the efficacy of using a Fas-CIB1-EGFP and CRY 2-mCherry-FADD light-controllable system for the treatment of uveal melanoma both in vitro and in vivo.These results provide a novel therapeutic strategy for treating uveal melanoma or other ocular diseases.