Efficacy And Mechanism Of Chlorin Derivative Photodynamic Therapy For Cutaneous Squamous Cell Carcinoma

Background Cutaneous squamous cell carcinoma(cSCC)accounts for about 20% of all skin cancers and is the second most common non-melanoma skin cancer in humans.cSCC is a malignant tumor that occurs in epithelial keratinocytes and often occurs in the skin of elderly people in long-term exposure sites.The classic treatment is surgical resection,but for large lesions in the head and face or other special parts of the skin,surgical resection is destructive,especially for advanced or multiple skin lesions,surgical treatment is obviously difficult to implement.Photodynamic therapy(PDT)is a combination of drugs and devices for non-invasive tumor treatment.In recent years,photodynamic therapy mediated by photosensitizer chlorin has become a research hotspot,especially in tumor treatment.The aim of this study is to investigate the effect and mechanism of STBF,a novel chlorin derivative,in the treatment of cutaneous squamous cell carcinoma.Objective In this study,two types of human squamous cell carcinoma lines and SKH-1 mouse squamous cell carcinoma implantation tumor models were used to evaluate the killing effect of STBF-PDT on squamous cell carcinoma cells and the therapeutic effect of STBF-PDT on squamous cell carcinoma in mice.Methods1.The UV absorption spectrum and subcellular localization of chlorin were determined.The STBF-Na HCO3 gradient solution(10.8/5.4/2.7/1.35/0.675μM)was prepared according to the instructions,and the absorbance spectrum of the photosensitizer was detected by UV-visible spectrophotometer.STBF fluorescence was directly observed under WOOD excitation.Determination of cell absorption curve;Finally,Lyso Tracker Green DND-26 lysosome green fluorescent probe was used for subcellular localization.2.Study on the activity,reactive oxygen species,clonogenesis ability and apoptosis of squamous cell cells in skin.The dark toxicity and phototoxicity of porphin were detected by CCK-8 method.Intracellular reactive oxygen species(ROS)levels were detected using ROS kits.Colony formation assay was used to evaluate the change of clonogenesis ability.Finally,the generation and degree of apoptosis was determined by TUNEL-DAPI double dyeing method.3.Transcriptome sequencing and WB assay were used to determine the antitumor mechanism.Firstly,differentially expressed genes were identified by cell transcriptome sequencing.GO enrichment analysis and KEGG enrichment analysis were performed on transcriptome sequencing data to demonstrate sensitive signaling pathways.The mechanism of apoptosis was further verified at the protein level.4.Animal experiments were conducted to further verify the antitumor efficacy.The SKH-1 mouse xenograft tumor model was established.First,the fluorescence intensity in the tumor after local injection was measured.Then,the mice in the experimental group were treated with STBF-PDT for 4 times,and the anti-tumor effect of STBF-PDT was further verified by the changes in tumor size,gross photographs,and histopathological examination.Results1.Fluorescence characteristics and intracellular localization of STBF.STBF had the characteristics of ultraviolet absorption and fluorescence,which conformed to the general characteristics of the photosensitizer.The excitation wavelength is located at about 405 nm,and the emission wavelength is located at about 665 nm.Furthermore,according to the cellular absorption curve,it can enter the cell in a relatively short time,and it was confirmed that it mainly tends to localize in the cell lysosome.2.Photodynamic inhibition of cSCC cell growth by STBF-PDT.The results of reactive oxygen species detection showed that after STBF-PDT,the level of intracellular reactive oxygen species increased with the increase of light dose.Subsequently,related cell function experiments showed that after STBF-PDT,cell viability and colony formation ability decreased with the increase of light dose,while the proportion of apoptotic cells increased with the increase of light dose,in a gradient dependent manner.3.STBF-PDT induces apoptosis of cSCC cells through Akt/mTOR signaling pathway.Transcriptome sequencing analysis found that differentially expressed genes were related to apoptosis,cell cycle and other signals.We mainly select mTOR signaling pathway for verification.WB results showed that porphin photodynamics induced apoptosis of squamous cell carcinoma cells by inhibiting the expression of protein related to Akt/mTOR signaling pathway.Moreover,the degree of apoptosis depends on the light dose,which is consistent with the change law of the function experiment in the above part,which again confirms the anti-squamous cell carcinoma effect of STBF-PDT from the aspect of mechanism.4.STBF-PDT inhibited the tumor growth in SKH-1 mice.In the animal experiment,the fluorescence intensity in the tumor was the highest at half an hour after local injection.After photodynamic therapy,the tumors in the experimental group almost disappeared compared with those in the control group,and there was no significant difference in the weight change between the two groups.The histological results showed that a large number of tumor cell necrosis and local infiltration were observed in the photodynamic treatment group,and no obvious tumor margin was observed.Conclusion The photosensitizer used in this study was extracted from spirulina and processed and transformed.It has the photosensitive characteristics of general photosensitizers,and its photodynamic therapeutic effect has been proved by various experiments.Our study shows that STBF-PDT is a very effective cSCC therapy,which is expected to play a greater value in clinical application in the future.