| Chapter 1:Exploring the efficacy and prognostic factors associated with salvage endoscopic surgery for recurrent nasopharyngeal carcinomaObjective:The purpose of this chapter is to investigate the efficacy of nasal endoscopic surgery for recurrent nasopharyngeal carcinoma and the correlation between factors such as age,gender,T-stage,time frame of recurrence,internal carotid artery management,lymph node metastasis,bone or soft tissue necrosis status,and known important molecular indicators and prognosis in patients with recurrent nasopharyngeal carcinoma.Methods:(1)A clinical database of recurrent nasopharyngeal carcinoma treated with nasal endoscopic surgery was constructed,and relevant clinical data and samples of all patients who underwent nasal endoscopic surgery for recurrent nasopharyngeal carcinoma in our department from January 2016 to June 2022 were included.(2)To analyze the 3-year overall survival(OS)and 3-year progression-free survival(PFS)associated with the enrolled patients,and to analyze the correlation between patient age,gender,T classification,time frame of recurrence,internal carotid artery management,lymph node metastasis,and bone or soft tissue necrosis status and prognosis.(3)Immunohistochemical techniques were used to detect 25 pairs of patients with nasopharyngeal carcinoma,the expression of Ki-67 and EGFR at the time of initial presentation and at the time of recurrence,and correlation analysis was performed.(4)Flow cytometry technique was used to detect CD34~+EPCAM~+cells in the tumor tissue suspension of nasopharyngeal carcinoma,and the density of the above cells was compared between initial and recurrent nasopharyngeal carcinoma.Results:(1)Compared with the initial nasopharyngeal carcinoma,the overall survival rate and progression-free survival rate of patients with recurrent nasopharyngeal carcinoma were poorer,and the 3-year overall survival rate and progression-free survival rate were 56.2%and 43.9%,respectively.(2)Age,concomitant bone or soft tissue necrosis and T-stage of patients with recurrent nasopharyngeal carcinoma treated by nasal endoscopic surgery were significantly correlated with overall survival(P<0.05);while gender and whether the internal carotid artery was interfered or not were significantly correlated with progression-free survival(P<0.05).(3)Ki-67 protein expression was significantly lower in recurrent nasopharyngeal carcinoma tissues compared with initial nasopharyngeal carcinoma tissues(P<0.001).EGFR protein expression was also significantly lower(P<0.01).(4)The density of CD34~+EPCAM~+cells in the tumor microenvironment of recurrent nasopharyngeal carcinoma tended to be reduced compared with that of primary nasopharyngeal carcinoma,but more studies are needed to confirm this(P>0.05).Conclusion:The overall survival and progression-free survival rates of recurrent nasopharyngeal carcinoma were poor compared with those of primary nasopharyngeal carcinoma.However,the expression of classical prognostic-related markers was lower in the tissues of recurrent nasopharyngeal carcinoma compared with that of primary nasopharyngeal carcinoma.This suggests that the prognosis of recurrent nasopharyngeal carcinoma and the related indicators affecting prognosis differ from those of the initial nasopharyngeal carcinoma.Chapter 2: Single-cell transcriptome sequencing and spatial transcriptome sequencing to map the tumor microenvironment of recurrent and incipient nasopharyngeal carcinomaObjective: Based on the study in Chapter 1,this chapter analyzes the differences between the tumor microenvironment of recurrent and incipient nasopharyngeal carcinoma by spatial transcriptome sequencing combined with high-throughput single-cell transcriptome sequencing,aiming to explore the spatial heterogeneity of gene expression during the evolution of the tumor microenvironment in the development of recurrent nasopharyngeal carcinoma and the changes in cell-to-cell interactions,to provide a theoretical basis for finding new immunotherapeutic strategies and to design new insights into the design of precision medicine.Methods:(1)Fresh tumor samples from 12 patients with different disease stages of nasopharyngeal carcinoma(6 in the spatial transcriptome and 6 in the single cell transcriptome)were collected and prepared into two sample types required for sequencing experiments.(2)10×Genomics 3’ single-cell transcriptome sequencing library construction and sequencing(3)10×Genomics spatial transcriptome library construction and sequencing(4)Bioinformatics analysis of single-cell transcriptome and spatial transcriptome sequencing data(5)Flow cytometry validation of clinical tissue samples.(6)In vitro cytology experiments to explore the possible biological functions of cell subpopulations.Results:(1)The improved spatial transcriptome sample preparation method can better guarantee the quality of sample data and avoid the waste of precious clinical samples.(2)The tumor microenvironment of nasopharyngeal carcinoma consists of CD4+ T cells,CD8+ T cells,NK cells,B cells,plasma cells,dendritic cells,giant eosinophils,monocytes,fibroblasts,endothelial cells,and malignant epithelial cells + epithelial cells.(3)Mapping of the spatial transcriptome to each pathological region showed that the microenvironmental diversity of recurrent nasopharyngeal carcinoma was reduced compared with that of the initial nasopharyngeal carcinoma.(4)Nasopharyngeal carcinoma epithelial cells could be divided into six cell subpopulations based on specific highly expressed genes,including three normal epithelial cell populations and three malignant epithelial cell populations.The malignant transitional subpopulation of normal epithelial cells,h E0,highly expresses Myc pathway-related genes such as CDKN1 A,RPS2A1,VIM,RUT1 and PRKDC.The malignant cell population,on the other hand,highly expresses genes related to mitotic instability.(5)Combined spatial transcriptome and single cell transcriptome Cell Trek analysis showed three subclonal populations of malignant epithelial cells,of which subclone 2 was in the malignant transition phase,which was characterized by epithelial mesenchymalization and enrichment of immune response pathways.Copy KAT analysis of malignant epithelial cells showed a specific increase in the copy number of genes such as TYMP and FAM210 B,which was consistent with the results of single cell analysis.(6)SOX2 may be a driver gene for malignant transformation of normal epithelial cells.Its high expression during malignant epithelial cell development is confirmed by the spatial transcriptome.(7)Malignant epithelial cell subpopulations h M0 and h M2 are significantly higher than normal cell subpopulations in metabolic pathways such as citric acid cycle,glycolysis/glucose survival and fatty acid metabolism.h E0 is similar to malignant epithelial cells as a transitional subpopulation of benign and malignant epithelial cells.The high expression of malignant cells was shown to be inhibited by pathways such as keratinocyte differentiation,humoral immune response and immune response.(8)T cells are also heterogeneous in the tumor microenvironment of nasopharyngeal carcinoma.Both cytotoxic T cells and regulatory T cells were less dense in recurrent tissues than in primary nasopharyngeal carcinoma.As shown by the analysis of the GSE102349 dataset,the responsiveness of nasopharyngeal carcinoma to immunotherapy was proportional to both the T-cell toxicity fraction and the depletion fraction.(9)The responsiveness of recurrent nasopharyngeal carcinoma to immunotherapy may be worse than that of the initial nasopharyngeal carcinoma.And CXCL13 high CD4+ T-cell fraction was proportional to the responsiveness to immunotherapy.(10)Myeloid cells in the nasopharyngeal carcinoma tumor microenvironment can be divided into 15 small subpopulations,which can be further categorized into four large myeloid cell populations by clustering their highly expressed genes,namely the giant myeloid cell population,the plasmacytoid dendritic cell population(p DC),the conventional dendritic cell population(c DC),and the monocyte population.SPP1+ macrophages are specifically highly distributed in recurrent nasopharyngeal carcinoma,and the results of in vitro experiments suggest that they may promote tumor invasion and may also affect the tumor response to immunotherapy.(11)The presence of M1-M2 coupled polarization of megalophils in the nasopharyngeal carcinoma microenvironment may have a role in immune regulation.(12)A population of proliferative fibroblasts with specific high expression of proliferation-related genes exists in the nasopharyngeal carcinoma microenvironment,and the proportion of such fibroblasts is higher in recurrent nasopharyngeal carcinoma than in primary nasopharyngeal carcinoma.(13)In the results of Cell Chat analysis,fibroblasts were shown to have the highest communication with other cells both in terms of number and weight.Conclusion: This chapter provides a preliminary description of the gene expression changes during the development of tumor cells in the nasopharyngeal carcinoma tumor microenvironment by spatial transcription and single-cell sequencing.The developmental process of recurrent nasopharyngeal carcinoma tumor cells was initially revealed by combined analysis of single-cell data and spatial transcriptome data.Cellular profiles of other cell subpopulations in the nasopharyngeal carcinoma microenvironment were mapped and validated with in vitro experiments and clinical samples to provide a basis for further interpretation of the heterogeneity of the nasopharyngeal carcinoma tumor microenvironment.46 figures,6 tables,3 schedules,88 reference... |
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