Multi-Omics-Based Mechanism And Prognostic Biomarkers For Lymph Node Metastasis In Papillary Thyroid Microcarcinoma

Part I Comprehensive analysis of tissue proteomics and function study in patients with papillary thyroid microcarcinomaBackgroundThe development of ultrasonography and fine-needle aspiration biopsy has increased the detection rate of newly diagnosed papillary thyroid microcarcinoma(PTMC).Lymph node metastasis(LNM)is a common mode of metastasis of PTMC,leading to an increased risk of recurrence and poor prognosis.In PTMC,the known risk factors associated with LNM include patient age,sex,multifocality,calcification,and so on.Sex is an important factor in the pathogenic mechanism,diagnosis,and prognosis of various cancers,particularly sex-specific organs.Sex disparities in cancers of some organs shared by males and females,such as the thyroid,liver,and lung,have also been reported.Sex disparities play promotional roles in the development and progression of thyroid cancer.Multiple previous studies have suggested that the male is a risk factor for LNM in papillary thyroid carcinoma or PTMC.Nevertheless,there is no reliable basic research to elucidate the potential mechanism of sex disparities of LNM at the molecular level,which is difficult to provide the scientific basis for clinical decision-making.This present study aimed to investigate tissue proteomics and function in PTMC patients to unveil the molecular mechanisms underlying LNM and its significant sex disparities in PTMC development.MethodsA direct data-independent acquisition(DIA)proteomics approach was used to identify differentially expressed proteins(DEPs)in tumorous tissues from PTMC with LNM or without LNM(NLNM)and from male and female patients with LNM to reveal the potential mechanisms at the protein level.The functional annotation of DEPs was performed using bioinformatics methods.Protein-protein interaction(PPI)analysis was conducted and visualized using Cytoscape.Furthermore,The Cancer Genome Atlas Thyroid Carcinoma(TCGA-THCA)dataset and immunohistochemistry(IHC)were used to validate selected DEPs.ResultsThe proteomics profile in PTMC with LNM differed from that of PTMC without LNM.The metastasis-related DEPs were primarily enriched in categories associated with mitochondrial dysfunction and may promote tumor progression by activating oxidative phosphorylation and PI3K/AKT signaling pathways.Comparative analyses of these DEPs revealed downregulated expression of specific proteins with well-established links to tumor metastasis,such as SLC25A15,DIRAS2,PLA2R1,and MTARC1.Additionally,the proteomics profiles of male and female PTMC patients with LNM were dramatically distinguishable.An elevated level of ECM-associated proteins might be related to more LNM in male PTMC than in female PTMC patients.The upregulated expression levels of MMRN2 and NID2 were correlated with sex disparities and showed a positive relationship with unfavorable variables,such as LNMs and poor prognosis.ConclusionsProteomic profiles were significantly different in tumorous tissues of PTMC with LNM and PTMC without LNM.SLC25A15,DIRAS2,PLA2R1,and MTARC1 expression was downregulated in thyroid tumorous tissues with LNM compared to thyroid tumorous tissues with NLNM,which was associated with poor prognosis.This is the first application of a direct DIA proteomics approach to explore significant sex differences in LNM in patients with PTMC.Sex disparities may be due to the upregulated expression of extracellular matrix protein in male PTMC with LNM,especially MMRN2 and NID2 upregulation,which can provide basic theories for male as a risk factor of LNM.This present study can further our understanding of the functional networks and signaling pathways related to PTMC.Part II Research on serum protein N-glycosylation changes in papillary thyroid microcarcinoma and establishment of nomogramsBackgroundPapillary thyroid cancer(PTC)is one of the most common head and neck cancers,with the incidence rising steadily over the past three decades.Papillary thyroid microcarcinoma(PTMC)as the subtype of PTC accounts for approximately 35%-70%of all PTC cases.Although the system of diagnosis and treatment of PTMC has kept on completing,there are still some shortcomings in the preoperative diagnosis and risk stratification.The American Thyroid Association guidelines recommend against the use of fine-needle aspiration biopsy(FNAB)for thyroid nodules smaller than 10 mm,even those with highly suspicious ultrasound patterns.FNAB cannot obtain sufficient tissue for cytological analysis due to its small size and tends to result in a high false-negative rate.Therefore,effective and novel liquid biopsies for the diagnosis of PTMC are urgently needed.The rates of misdiagnosis and missed diagnosis are high in the identification of lymph node metastasis(LNM)due to atypical clinical manifestations of PTMC and the low sensitivity of ultrasound detection of LNM.Approaches for the preoperative prediction of LNM are still needed,thereby contributing to accurate prediction.Serological glycomic profiling is a rising research field for the discovery of potential biomarkers for cancer diagnosis,disease surveillance,and prognosis assessment in recent years.This study aimed to investigate serum protein N-glycomic features of PTMC patients,explore specific glycan biomarkers,and establish nomograms based on N-glycomic biomarkers for diagnosis of PTMC and prediction of LNM.MethodsSerum protein N-glycans were enzymatically released and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after linkage-specific derivatization of sialic acids.A robust N-glycome quantitative strategy based on mass spectrometry detection was used to obtain N-glycomic features of PTMC consisting of LNM and non-LNM(NLNM)patients and matched healthy controls(HC).Furthermore,univariate and multivariate analyses were carried out to identify specific serum N-glycomic biomarkers.The logistic regression models were created and presented as nomograms.Additionally,we conduct preliminary research on the N-glycomic profiles between PTMC with capsular invasion(CI)and with non-CI(NCI)at the level of serum glycomics.ResultsIn this study,96 directly detected N-glycans were identified,and 91 derived N-glycan traits were calculated from the 96 directly detected N-glycan traits based on their common structural characteristics.Serum N-glycan traits were found to differ between PTMC and HC in high-mannose,complexity,fucosylation,and bisection,of which four derived glycan traits(TM,CA1,CA4,and A2Fa)were significantly associated with PTMC.The nomogram that integrated with the four glycans achieved good performance for the auxiliary diagnosis of PTMC(area under the curve[AUC]=0.876),Serum N-glycan traits were found to differ between LNM and NLNM in high-mannose,complexity,galactosylation,and sialylation.In addition,two derived glycan traits(CA4 and A2F0S0G)exhibited an obvious correlation with LNM.The nomogram based on two traits showed relatively good performance(AUC=0.746)for the preoperative prediction of LNM.We also found differences in N-glycan traits between CI and NCI.ConclusionsThis study reported serum N-glycome signatures in PTMC for the first time.The predictive nomograms integrated with aberrant N-glycans could be useful tools for auxiliary diagnosis of PTMC and preoperative prediction of LNM in clinical practice.Moreover,the study also indicated that CI is not associated with LNM at the level of serum glycomics.Part Ⅲ Proteomics analysis and function study of serum exosome derived from papillary thyroid microcarcinomaBackgroundPapillary thyroid cancer(PTC)is a well-known malignancy of the endocrine system,with the rate of incidence increasing globally.The main reason for this is likely due to an increase in the incidence of papillary thyroid microcarcinoma(PTMC).Due to indolent biological behavior and favorable prognosis,the treatment scheme of PTMC is still controversial.Active surveillance was accepted as an alternative management for patients with low-risk PTMC by American Thyroid Association guidelines.Although most PTMC patients possess an indolent clinical course,PTMC within a proportion of patients has shown aggressive characteristics such as cervical lymph node metastasis(LNM),extrathyroidal extension,or distance metastasis which could lead to recurrence and reduced survival time,requiring a necessary operation to reduce the mortality rate.Cervical LNM is the main and common metastatic pathway of PTMC.However,there is no available tool to accurately identify LNM before operation.It is a major obstacle for surgeons to formulate treatments without accurate preoperative judgments of LNM in PTMC patients.Thus,it is necessary to fully elucidate the molecular mechanism triggering cancer progression and discover novel biomarkers for the preoperative prediction of LNM in PTMC.Exosomal proteins have been considered promising non-invasive tumor biomarkers for the abundant proteins from originating cells and availability in serum.This study aimed to explore the potential role of exosome protein profiling and to discover novel biomarkers to be used in the auxiliary diagnosis and risk stratification of PTMC.MethodsThe serum exosomes of PTMC patients with lymph node metastasis(LNM),PTMC patients without LNM,and benign thyroid nodules(BN)patients were isolated via size exclusion chromatography.Cell phenotypic experiments such as proliferation,migration,and tube information were carried out to explore the effects of exosomes from different sources on the malignant biological behavior of papillary thyroid cancer.The data-independent acquisition(DIA)proteomics technique combined with bioinformatic strategies was conducted to compare the protein profiles of serum exosomes purified from three groups and to discover the biomarkers.To further validate the performance of exosomal bone marrow stromal cell antigen 2(BST2)as a diagnostic and prognosis biomarker in PTMC,BST2 was quantitatively detected by ELISA.Cell counting kit-8(CCK8),Transwell,and tube formation assays were used to evaluate the effects of BST2 on the malignant biological behaviors of human papillary thyroid cancer(PTC)cell lines and HLEC cells line in vitro by knocking down or overexpressing BST2.The effects in vivo were explored by popliteal LNM mode.ResultsExosomes were successfully isolated from the serum of patients.Phenotypic experiments indicated that serum exosomes from PTMC patients could promote the proliferation and migration of PTC cells and lymphangiogenesis of HLEC cells.In addition,PTMC patients with LNM and PTMC with NLNM have different expression profiles of serum exosomal proteins.Proteomic analysis and ELISA indicated that the expression of serum exosomal BST2 was elevated during PTMC progression.The functional experiments in vitro demonstrated that exosomes with high expression of BST2 contributed to PTC cell proliferation,migration,and lymphangiogenesis in HLEC.The experiments in vivo showed that the swelling popliteal lymph node was obviously larger in the BST2-enriched exosome group.ConclusionsThe exosomal protein profiles between PTMC patients with LNM and without LNM were significantly different.Through both in vivo and in vitro experiments,we found that serum exosomal BST2 promoted LNM of PTMC and has the potential to be used as the diagnostic and prognostic biomarker in PTMC.