Study On The Regulation Of Anti-tumor Immune Function Of Cervical Cancer CD8~+ T Cells By Immunophotothermal Therapy

Objective:Cervical cancer is the fourth most common malignant tumor in women globally,causing over 300,000 deaths annually.The treatment outcomes and cure rates for early-stage cervical cancer are relatively high,reaching over 85% to 95%.However,for advanced-stage cervical cancer,the treatment outcomes and cure rates are relatively lower,typically ranging between 30% to 50%.Recurrent cervical cancer often exhibits characteristics of drug resistance and increased invasiveness,with cure rates ranging from 10% to 30%.Therefore,there is a need to explore new treatment methods to improve the survival rates of patients with advanced/recurrent cervical cancer and enhance their quality of life.Methods:Part 1:The first part,the Cervical cancer（1）the transcriptome sequencing data acquisition: the Gene Expression Omnibus（GEO）database（https://www.ncbi.nlm.nih.gov/geo/）in the Cervical cancer "for the keyword search.After screening,the GSE63514 dataset was selected as the research object for the subsequent analysis of CD8⁺ T cell immune infiltration in the cervical cancer microenvironment.（2）Cervical cancer single cell sequencing data acquisition: Using "cervical cancer" and "single cell RNA seq" as keywords in the GEO database,cervical cancer single cell sequencing data set with ID GSE208654 was retrieved.（3）Immunohistochemical methods were then used to detect and analyze the expression difference and correlation of CD8⁺ T cells in cervical cancer and adjacent tissues,analyze the correlation between CD8⁺ T cells and clinicopathological features,and compare the expression changes of CD8⁺ T cells in cancer tissues with different differentiation degrees and different clinical stages（FIGO stages）.Part Two:（1）In this study,immune photothermal nanomaterials FA@LIPO@ICG@CpG NPs（FLIC NPs）were synthesized by thin film dispersion method.Liposome,LIPO was used as the nanomaterial carrier and Folic Acid（FA）as the targeting agent.Indocyanine Green（ICG）as Photothermal Agent（PTA）,CpG ODN（synthetic containing Unmethylated cytosine-phosphate-guanine oligodeoxynucleotide,CpG ODN）as an immune adjuvant,The immune photothermal nanomaterial FLIC NPs with targeting and immune activation function was formed.（2）The absorbance and fluorescence spectra of FLIC NPs were detected.The drug loading and encapsulation efficiency of ICG and CpG ODN in FLIC NPS were detected by Quant-i T Picogreen ds DNA quantitative kit,and the encapsulation rate and drug loading were determined.（3）Morphological characterization of FLIC NPs was observed by scanning electron microscopy（SEM）and transmission electron microscopy（TEM）,and photothermal conversion efficiency of FLIC NPs at different concentrations was detected by thermal imager.（4）HeLa cells were used to perform CCK-8 cytotoxicity assay,laser confocal assay and flow cytometry assay to evaluate the toxicity,cell uptake and photothermal therapeutic effect of FLIC NPs.（5）FLIC NPs was injected into cervical cancer tumor bearing mice through the tail vein,and the tumor targeting and metabolic distribution of FLIC NPs were recorded by thermal imager and small animal live imager.（6）The survival of mice was observed,and H&E staining was performed on important tissues and organs to evaluate the biotoxicity of FLIC NPs in vivo.Part Ⅲ:（1）1×10⁷/200μL U14 cervical cancer cells were injected subcutaneously into the right upper limb of female C57BL/6 mice.（2）The tumor growth was observed for 2weeks.The animals’ weight and tumor size were measured the next day.According to the volume calculation formula（volume = length × width 2/2）,the size of the tumor is determined once the tumor volume reaches 50mm³.（3）U14 tumor-bearing mice were divided into four groups（n=4 mice per group）: Group A and control group（PBS group）;Group B,CpG group（200μg/mL）;Group C,FA@LIPO@ICG+laser group(200μg/mL,1.5mg kg^-1 calculated by ICG content);Group C,FLIC NPs+laser group(200μg/mL,1.5mg kg^-1 calculated by ICG content).Different therapeutic interventions were performed on each group of mice,respectively on the 1st,4th and 7th day（three times in total）,and the survival and tumor growth of mice at the 14 th day were observed.（5）Mouse tumor infiltrating lymphocytes,spleen lymphocytes and lymph node lymphocytes were extracted,and the changes in the number and function of immune cells in lymph nodes,tumor tissues and spleen tissues of cervical cancer mice after immunophotothermal treatment were detected by cytometry flow analysis.（6）The distribution of CD8⁺ T cells in tumor tissues was analyzed by immunohistochemistry.And the proliferation and apoptosis of tumor tissue.Results:In the first part,（1）x Cell algorithm and EPIC algorithm analysis showed that there were significant differences between effector CD8⁺ T cells in cervical cancer tumor microenvironment and normal tissues（P < 0.05）.（2）analysis of The differential gene CD8⁺ T cell in cervical cancer samples and normal cervical samples showed that a total of 190 CD8⁺ T cell genes were annotated in cervical cancer samples and 40 in normal cervical samples.After combining the expression data of CD8⁺ T cells of the two samples,the difference analysis showed that there were 167 differentially expressed genes in cervical cancer and normal cervical samples,of which 126 genes were down-regulated and 41 genes were up-regulated.（3）By KEGG pathway enrichment analysis,CD8⁺ T cells were mainly enriched in immune response,T cell receptor signaling,IFN-γ signaling,apoptosis and cell cycle regulation pathways.（4）Immunohistochemical results showed that the proportion of CD8⁺ T cells infiltrated in cervical cancer tissues was significantly higher than that in paracancer tissues（P<0.05）.In the second part,（1）the drug loading capacity（DLC）of ICG in FLIC NPs is 4.5%,（1）The drug loading capacity（DLC）of ICG in Flic NPS is 4.5%,and the encapsulation rate（EE）is 90%.The drug loading capacity（DLC）of CpG ODN was 3.4%,and the encapsulation rate（EE）was 85.7 percent.（2）FLIC NPs solution（200μg/mL dissolved in deionized water）is a yellowish-green emulsion with an average particle size of 150 nm.（3）FLIC NPs has good photothermal stability,and the photothermal conversion rate is as high as 39.05%,Flic NPS has good photothermal stability,and the photothermal conversion rate is as high as 39.05%,which is higher than the 15.28% of ICG NPs.（4）In vitro photothermal experiments show that the photothermal properties of FLIC NPs are positively correlated with solution concentration and laser power density.（5）FLIC NPs and He La cells were incubated for 2 hours.（6）The results of CCK8 cytotoxicity test indicated that the survival rate of He La cells was greater than 80%,indicating that HELA cells had low cytotoxicity.（7）The results of laser confocal experiments suggested that FLIC NPs could be effectively taken up by He La cells and showed obvious tumor killing effect.（8）Flow cytometry showed that FLIC NPs had good photothermal treatment effect on tumor cells.（9）After FLIC NPs is injected into mice through the tail vein,FLIC NPs can be targeted to the tumor site,and its fluorescence intensity reaches a peak after 24 hours and continues within 48 hours,and the temperature of the tumor site can reach 51℃ after laser irradiation.（10）Evaluation of in vivo toxicity and biocompatibility of FLIC NPs showed no significant damage to the tissue structure and cell morphology of major organs in mice.In the third part,（1）In the immunophotothermal treatment group,the proportion of m DC cells in mouse tumor tissues increased significantly,while the total proportion of CD8⁺ T cells did not change significantly,and the number of activated CD8⁺ T cells increased significantly.The subsets of CD8⁺ T cells in the immunophotothermal treatment group showed that the proportion of CD8⁺ TN decreased,the proportion of CD8⁺ Tcm increased,and the proportion of CD8⁺ Tem increased.The secretion of TNF-α and IFN-γ by tumor-infiltrating CD8⁺ T cells was.And the proportion of CD8⁺ tem increased.The secretion of TNF-α and IFN-γ by tumor-infiltrating CD8⁺ T cells was significantly increased,while the secretion of IL-6 and IL-10 was significantly decreased in the immunophotothermal treatment group.（2）In the immunophotothermal treatment group,the proportion of m DC cells in spleen and lymph node tissues of mice was significantly increased,while the total proportion of CD8⁺ T cells was not significantly changed,and the number of activated CD8⁺ T cells was significantly increased.（3）In the immunophotothermal treatment group,the proportion of activated CD4⁺ T cells increased significantly,the proportion of CD4⁺ TN decreased,and the proportion of CD4⁺ Tem increased,while the levels of TNF-α and IFN-γ secreted by CD4⁺ T cells increased,and the proportion of CD4⁺ tem increased,while the levels of TNF-α and IFN-γ secreted by CD4⁺ T cells increased,and the levels of IL-6 and IL-10 decreased.（4）In the immunophotothermal treatment group,the total proportion of CD4⁺ T cells increased significantly,the proportion of activated CD4⁺ T cell subsets increased,and the cytokines secreted by CD4⁺ T cells also changed.（5）Immunohistochemical results showed that the expression of Ki67 in tumor tissues of the immunophotothermal treatment group was significantly decreased,which further indicated that the treatment had an inhibitory effect on tumor proliferation.Conclusion:（1）Bioinformatics analysis revealed significant differences in CD8⁺ T cells in cervical cancer tissues and normal tissues,and CD8⁺ T cells play an important role in the occurrence and development of cervical cancer.（2）Immunohistochemical results indicated that CD8⁺ T cell infiltration in cervical cancer tissue was high,but its anti-tumor immune function was decreased.（3）CD8⁺ T cells played a key role in inhibiting the growth of cervical cancer tumors,and were an important immunomodulatory component in the occurrence and development of cervical cancer,with potential therapeutic value.（4）Using liposome as a carrier,ICG and CpG ODN were successfully coated to form FLIC NPs with good stability and biocompatibility.（5）Photothermal experiments verified the good photothermal conversion rate of FLIC NPs,and confirmed its ability to target accumulation at tumor sites.（6）Flow cytometry analysis showed that after immunophotothermal treatment,the proportion of m DC cells infiltrated in tumor tissues was significantly increased,and the activation state of CD8⁺ T cells was also significantly improved.（7）The results suggest that immunophotothermal therapy may play a therapeutic role by regulating immune cell activity and inhibiting tumor proliferation.This study provides an important experimental basis for immunophotothermal therapy of cervical cancer.