| Part 1Using single-cell RNA sequencing to explore the effect of tumor microenvironment in pituitary neuroendocrine tumorigenesisObjectivesPituitary neuroendocrine tumors(PitNETs)are neuroendocrine tumors derived from adenohypophyseal neuroendocrine cells.Functioning and non-functioning PitNETs severely affect life quality and lifespan of patients because of the abnormal hormone secretion,tumor mass effects and/or invasion of surrounding tissues.Recurrent and aggressive PitNETs remain to be challenges in clinical treatment.Several factors have been found to contribute to the pathogenesis of PitNETs,including single gene variants such as GNAS,AIP,USP8,MEN1,epigenetics,abnormal expression of cell cycle regulators.Recently,it has been found that the tumor microenvironment(TME)may also play an important role in the invasion and management of PitNETs.PitNETs have complex classifications and various tumor subtypes,and the mechanism of pathogenesis has not been fully elucidated.Single-cell RNA sequencing(scRNA-seq)is a new technology for transcriptomic sequencing at the single cell level,which can be used to explore the gene expression in individual cells and solve the cellular heterogeneity problem that occur in traditional tumor sample sequencing.It provides important evidence for understanding the behavior and mechanisms of individual cells in PitNETs.Therefore,this study aims to construct a single-cell transcriptomic landscape of PitNETs by scRNA-seq and explore the role of TME in the pathogenesis of PitNETs;further elucidate the molecular characteristics and dynamic changes of cell subpopulations associated with tumor development,as well as search for potential biological markers and novel therapeutic targets.MethodsFresh tissue samples were collected from 11 PitNETs patients who underwent surgery in our hospital.Clinical non-functioning PitNETs(NF-PitNETs)included 2 null cell tumors,2 non-functioning tumors with positive immunohistochemical staining for PIT-1 and PRL(PIT-1+NF),2 silent corticotroph adenomas(SCA).Functioning PitNETs included 2 growth hormone(GH),2 prolactin(PRL)and 1 thyrotropin(TSH)adenomas.ScRNA-seq was performed after single cell suspension preparation,and the sequenced raw data were subjected to information processing,unsupervised dimensional reduction,cell clustering and annotation,differential expression analysis,pathway enrichment analysis,cell communication analysis,pseudo-time trajectory analysis,and transcription factor module analysis for major cell populations.Infer-CNV was used to distinguish endocrine tumor cells from normal cells in the endocrine cell population,and function analysis of T cells and inflammatory response of monocytes/macrophages were performed respectively.Results1.Single-cell profile of PitNETsBased on the expression of known marker genes in cell populations,eight major cell clusters were annotated and identified,including endocrine cells,monocytes/macrophages,T lymphocytes,NK cells,endothelial cells,neutrophils,B cells,epithelial and mesenchymal cells.Endocrine cells showed the highest proportion.The major cell compositions and proportions of PitNETs subtypes showed heterogeneity,with the highest proportion of endocrine cells in functioning PitNETs,which was consistent with the clinical presentation and immunohistochemical findings.In contrast,null cell and PIT1+NF tumors showed an enrichment of immune cells and a relatively low content of endocrine cells,with high proportions of monocytes/macrophages and T lymphocytes.Notably,SCA had no clinical manifestations of hormone hypersecretion but exhibited similar cellular compositions to that of functioning tumors.Cellular communication analysis showed that monocytes/macrophages and neutrophils had significant interactions with endocrine cells,and intercellular communication was stronger in GH,PRL,TSH adenomas and SCA,and weaker in null cell and PIT-1+NF tumors,but monocytes/macrophages still showed strong intercellular interactions in NF-PitNETs.2.Single-cell transcriptomic characteristics of endocrine cells in PitNETsThe 35,111 endocrine cells were re-clustered into seven cell subpopulations:lactotrophs(28%),thyrotroph(23%),somatotroph(21%)and corticotroph(18%),as well as gonadotroph,stem cells and proliferating Poulfl cells.There was heterogeneity in the endocrine cell composition of functioning and NF-PitNETs,with the maj ority of endocrine cells in functioning PitNETs and SCA being exclusively hormone-secreting cells,while null cell and PIT-1+NF tumors showed complex compositions and contained a proportion of stem cells.Stem cells were subclustered into three subpopulations,Stem1 and Stem2 cells were mainly present in NF-PitNETs,while Stem3 was mainly derived from GH and PRL adenomas.Pathway enrichment analysis revealed that Steml was mainly associated with extracellular matrix(ECM)organization and collagen.Stem2 cells were enriched in pathways of MHC complex and endoplasmic reticulum structure,while GO terms in Stem3 cells were associated with intercellular junctions,plasma membrane,and vesicle lumen.Tumor necrosis factor(TNF)-α,interferon(IFN),epithelial mesenchymal transition(EMT),angiogenesis,and Notch signaling were also significantly activated in stem cells.Functional heterogeneity exists in endocrine cells of PitNETs subtypes:pathways such as RNA catabolic processes,metabolic regulation,protein-targeted transportation,valine,leucine and isoleucine biosynthesis were activated in GH adenomas,while activation and binding of transcription factors,biotin metabolism were activated in TSH adenoma.In SCA,ligand binding domains,cortical actin cytoskeleton pathways were enriched.Several metabolic pathways,including oxidative phosphorylation and tricarboxylic acid cycle,was significantly activated in SCA.Enriched ECM,regulation of epithelial cell proliferation and migration,angiogenesis,EMT,and Wnt signaling pathways were observed in null cell tumors.PIT-1-NF tumors were associated with antigen presentation and vesicular transportation from endoplasmic reticulum-to-Golgi,and Golgi-associated vesicle membranes.3.Characterization of endocrine tumor cells in PitNETs subtypesThe 28,452 tumor cells and 6,659 non-tumor cells were distinguished in endocrine cells.Majority of differential expression genes(DEGs)were downregulated in null cell(66.5%,602/905),PRL(96.1%,171/178)and TSH adenomas(53.1%,587/1105),whereas in GH(59.3%,80/135)and PIT-1+NF tumors(59.7%,1239/2075)were mainly upregulated genes.Functional analysis of tumor cells in the six PitNETs subtypes showed significant heterogeneity,GO terms such as protein-targeted localization,protein transport to endoplasmic reticulum and ribosomes were enriched in GH adenomas.Conversely,pathways including cellular ribosomes and protein-targeted transport were downregulated in PRL tumors.A large number of downregulated genes in TSH PitNETs were highly associated immune activities such as MHC receptor activity,IFN-y,lymphocyte differentiation,and leukocyte activation.The downregulated genes in null cell tumors were enriched to multiple pathways associated with ECM,and the downregulated genes in PIT1+NF were associated with cell adhesion,junction,and actin-based regulation.To clarify common features and differences between functioning and NF-PitNETs,DEGs were further compared in functioning PitNETs(GH vs PRL vs TSH adenomas),NF-PitNETs(SCA vs null cell vs PIT-1+NF)and PRL immunohistochemically positive PitNETs(PRL adenomas vs PIT-1+NF),and intersected genes were identified.Among the functioning PitNETs,only CHGB was upregulated in three PIT-1 lineage tumors,and eight genes(IER2,PTMA,TMSB4X,BTG2,KLF6,NR4A1,SAT1,S100A6)were downregulated in these subtypes.In NF-PitNETs,only two RNA genes,SNHG14 and KCNQ1OT1,were consistently upregulated.In PRL adenomas and PIT-1+NF,CHGB,MEG3,MEG8,and VGF genes were significantly overexpressed in both tumors and 150 genes were downregulated.Developmental progression of seven endocrine cell subclusters was visualized by pseudotime trajectory analysis,the developmental trajectories of corticotroph and PIT-1 lineages(thyrotroph,lactotroph,somatotroph)showed a clear separation.Corticotroph and gonadotroph presented overlaps during developmental process,while stem cells and proliferating cells demonstrated a dynamic functional status in every trajectory,involving in differentiation of hormone-secreting cells.4.Exploring potential markers for PitNETsPotential markers for PitNETs were selected from three ways.(1)DEGs expression in seven endocrine cell subpopulations was investigated and ten candidate molecules were selected,and their expression levels were further evaluated in PitNETs subtypes and normal endocrine cells.ADH1A and ENPP2 were significantly upregulated in TSH adenoma and normal thyrotroph,but presented low expression in other PitNETs subtypes and cell types,which may serve as potential cell-specific markers.TMEM158,THSD7A,BCAT1,and NDRG1 expression highly expressed in lactotroph tumor cells,thus may serve as potential tumor-specific markers.PCDH9 and GLUL expression were both upregulated in null cell tumors and SCA,thus may serve as NFPitNETs-related markers.(2)Evaluation of DEGs in endocrine tumor cells.THSD7A and NDRG1 were significantly highly expressed in PRL adenomas,and JPH4 was highly expressed in null cell tumors and SC A.The expression of marker genes associated with PitNETs in the literature were also evaluated,including ADAMTS6,ADAMTS1,BIRC5,CEBPD,NOTCH3,PITX2,SSTR5 and SSTR2,but none of them were found to be specifically expressed in PitNETs or normal endocrine cells.(3)Intersected DEGs in functioning and NF-PitNETs.MEG3 and VGF were consistently upregulated in PRL and PIT-1+NF tumors,but were low expressed in normal lactotroph.TMSB4X was downregulated in all PitNETs subtypes,and IER2 showed consistent downregulation in three functioning PitNETs.Thirteen candidate markers were selected and validated in the 10 GEO datasets of PitNETs.The expression of THSD7A,VGF and IER2 in the GEO datasets were consistent with the results of sc-RNA seq.Transcription factors(TFs)analysis further supported the role of THSD7A,VGF and IER2 as potential markers of PitNETs.5.Single-cell landscape of T lymphocytes in PitNETs The 2,973 T cells were re-clustered into five subpopulations.Both T1 and T3 subpopulations contained naive T cells and exhausted T cells,with naive T cells being the major component of T1 and T3 and low level of exhausted T cells in T3.T2 and T4 subpopulations consisted of cytotoxic T cells and exhausted T cells,with a high content of cytotoxic T cells.The T5 subset contained mainly naive T cells and cytotoxic T cells.Heterogenous distribution and function of T cells were observed in PitNETs subtypes.Functioning PitNETs showed higher levels of T1 and T2 cells,while NF-PitNETs had higher levels of T1 and T3 cells.Naive T and cytotoxic T cells levels were similar in GH and TSH adenomas,but PRL adenomas exhibited higher levels of naive T lymphocytes.Naive T cells were higher than cytotoxic T cells in NF-PitNETs,while all PitNETs subtypes showed low exhausted T lymphocytes levels.T cells in PitNETs share some common functions,such as activation of inflammation and initiation of translation,but also exhibited subtype-specific features.T cells in GH adenomas were associated with ligand-binding activity and cytokine binding.Pathways such as ribosomes,translation initiation,reorganization of T cell receptors,and differentiation of T cells were significantly activated in PRL adenomas,while TSH adenomas were enriched for multiple pathways of IFN response and Fc receptor signaling.T cells in SCA were associated with transcriptional and translational processes,and pathways such as IFN response,ATPase regulation,helper T cell differentiation,and antigen processing and presentation were activated in null cell tumors and PIT-1+NF.The developmental trajectory of T cells started from T3(naive T cells),through T2(cytotoxic T cells),and terminated at T4(cytotoxic T cells)and T1(naive T cells).T3 and T2 formed the main trajectory backbone,while T4 and T1 cells were located at different directions,indicating a differentiation process from naive T cells to cytotoxic T lymphocytes,accompanied with a small number of exhausted T cells.Both proliferation and exhausted scores were low in PitNETs,while cytotoxic scores were higher than naive scores in most PitNETs.However,naive score was higher in PRL adenomas.6.Proinflammatory phenotype mediated by monocytes/macrophages in PitNETsThe 9,839 monocytes/macrophages were re-clustered into five subpopulations,and monocytes/macrophages were mainly derived from NF-PitNETs.A high proportion of Mo1 and Mo2 were found in NF-PitNETs,while Mo3 and Mo4 subpopulations were predominant in functioning PitNETs.Mol exhibited high levels of dendritic cells and macrophages marker genes,pathways including leukocyte and T cell differentiation,B cell activation were enriched in Mo1.Mo2 highly expressed transcription factors and chemokines,thus was associated with differentiation of myeloid and fat cells,and transcriptional regulation.Mo3 was mainly found in PRL adenomas,with upregulated ribosomal protein genes associated with RNA catabolic processes,protein transcription and intracellular targeted transport.Mo4 was widely distributed in PitNETs except null cell tumors,and cell chemotaxis,leukocyte migration,interleukin(IL)-production pathways were activated.Mo5 was almost exclusively found in null cell tumors,showing high expression of multiple inflammatory response-related genes and chemokines,thus inflammatory signaling,leukocyte and T cell activation,cell adhesion regulation,and TNFα-NFκB pathways were activated.Monocytes/macrophages in GH adenomas were associated with hormone responses,lipoprotein granules,and cell chemotaxis.Neutrophil activation and regulation as well as antigen presentation activity were increased in PRL adenomas.Significant activation of IFN-related signaling pathways was demonstrated in TSH adenoma,whereas IFN pathways were not active in GH and PRL adenomas.Compared to other PitNETs subtypes,SCA showed significant activation of the TNFα-NFκB pathway,and active lipopolysaccharide response,leukocyte migration and IL-2 production.Monocytes/macrophages in null cell tumors were involved in immune responses to toxic substance,lipopolysaccharides,and reactive oxygen species,but the IFN pathway was not activated.Monocytes/macrophages in PIT-1+NF were mainly associated with amide and peptide binding,cell growth,and immune system regulation.All monocyte/macrophage subpopulations in PitNETs exhibited a pro-inflammatory feature,and the polarization of M2 macrophages were more predominated than M1.Different subtypes showed heterogeneity in the expression and activation status of TFs.ConclusionThe composition and functions of endocrine cells,T lymphocytes and monocytes/macrophages of PitNETs subtypes are heterogeneous,and the differences in the functions of endocrine tumor cells suggest distinguishment in pathogenesis and therapeutic targets.This study elucidates the detailed characteristics,functions and developmental trajectories of T cells and monocytes/macrophages in TME of PitNETs at the single cell precision,providing novel insights for immune-related diagnosis and the selection of therapeutic targets.Part 2Using single-cell RNA sequencing to explore the tumorigenesis of TPIT lineage pituitary neuroendocrine tumorsObjectivesSC A is a specifical clinically non-functioning PitNETs.In contrast to pituitary ACTH adenomas,patients with SC A usually lack the clinical manifestations of hypercortisolism and show pathologically positive for ACTH and/or TPIT.In the past,SCA was clinically defined as a non-functioning adenoma,however,SCA is more aggressive,proliferative and recurrent than other non-functioning adenomas.SCA can transform into functioning ACTH adenoma,and the clinical outcome of patients vary greatly,so the mechanism of SCA needs to be explored.In contrast,pituitary ACTH adenomas are very rare,have lower remission rates than other functioning adenomas and are prone to recurrent,their treatment remain to be a worldwide challenge.The pathogenesis of SCA and pituitary ACTH adenoma have not been fully elucidated.The aim of this study was to construct a singlecell transcriptomic profile of TPIT lineage PitNETs using scRNA-seq and to explore the role of TME in tumorigenesis and progression,and provide evidence for potential markers and drug targets.MethodsFresh tissue samples were collected from 6 TPIT lineage PitNETs,including 2 SC As and 4 pituitary ACTH adenomas(Cushing’s disease,CD).ScRNA-seq was performed after single-cell suspension preparation,and the sequenced raw data were subjected to information processing,unsupervised dimensional reduction,cell clustering and annotation.Infer-CNV was used to distinguish endocrine tumor cells from normal cells in endocrine cells.Differential expression analysis,pathway enrichment analysis,cell communication analysis,pseudo-time trajectory analysis,and monocyte/macrophage characterization scoring were performed in endocrine cells and monocytes/macrophages.Results1.Single-cell profiles of TPIT lineage PitNETsThe major cell composition of SCA and pituitary ACTH adenomas were similar,with the highest proportion of endocrine cells followed by monocytes/macrophages.Differently,higher proportions of NK cells,neutrophils and T lymphocytes were observed in CD,and a certain proportion of epithelial and mesenchymal cells was also observed.Cell communication analysis showed that monocytes/macrophages had the most significant interactions with endocrine cells,followed by neutrophils.Intercellular communication was stronger in SCA compared to CD,existing between endocrine cells and other major cell populations.2.Single-cell transcriptomic characteristics of endocrine cells in SCA and CDThe endocrine cells in SCA and CD were heterogeneous in composition and function.Corticotroph was most abundant in CD,while a specific cell may be present in SCA that was in an intermediate state of differentiation from stem cells to corticotroph.These cells exhibited both characteristics of corticotroph and stem cells.SCA and CD showed intratumoral heterogeneity and exhibited differences with immunohistochemical findings.GO terms including neuronal cell body,sex determination and adrenal gland development were enriched in corticotroph.In addition,corticotroph was associated with cell membrane structure such as membrane rafts,membrane microdomain,membrane region,sarcoplasmic reticulum and sarcoplasm pathways.KEGG analysis revealed that corticotroph was associated with apoptosis and p53 signaling pathways.Multiple metabolic pathways were activated in corticotroph and TBX19+stem cells compared to other endocrine cell subpopulations,suggesting active biosynthetic metabolic activity.Pseudo-time trajectory analysis showed overlapped developmental trajectories of corticotroph and stem cells,suggesting that there may be different characteristic corticotroph subsets that appear at different periods during pituitary neuroendocrine cell differentiation.Functions of endocrine cells in SCA and CD were heterogeneous.GO analysis showed that CD was enriched to pathways of neuronal cell body,hormone activity,and neuropeptide hormone activity,while SCA was associated with ligand domain binding.SCA also exhibited active synthetic and metabolic activities,with multiple pathways including oxidative phosphorylation,tricarboxylic acid cycle,fatty acid,and amino acid metabolism highly activated,but these pathways were inactivated in CD.3.Endocrine tumor cell heterogeneity of SCA and CDThe 19,579 normal endocrine cells and 7,407 tumor cells were identified by InferCNV.73.3%of DEGs in CD were down-regulated genes(341/465),while the number of up-and down-regulated genes in SCA was similar(26/46,56.5%).POMC was upregulated in CD.Functional analysis found activation of pathways such as enzyme inhibitor activity,endopeptidase inhibitor and regulator activity,and peptidase inhibitor activity in CD tumor cells.Tumor cells in SCA showed downregulation of POMC and large numbers of ribosomal genes.Inactivation was observed in pathways related to transcriptional translation,such as cytoplasmic ribosomes,protein targeting to endoplasmic reticulum,and cell membrane.4.Monocyte/macrophage heterogeneity of SCA and CDThe monocyte/macrophage subpopulations of SCA and CD showed heterogeneous distribution.CD consisted of Mo1,Mo2,Mo4,and Mo6 subpopulations,with a high proportion of Mo1 and Mo2.In contrast,SCA was mainly composed of Mo3 and Mo5.The distribution of monocytes/macrophages also showed obvious intratumoral heterogeneity.Mo1 was associated with phosphatase,protein kinase,MAPK signaling pathway and transcriptional misregulation in cancer.Mo2 was enriched to multiple pathways related to neutrophil activation,regulation and angiogenesis.Mo4 was not only related to neutrophil and macrophage activation and regulation,but also involved in chemotaxis and migration of leukocytes and granulocytes.In Mo6,signaling pathways such as regulation of immune response,antigen processing and presentation,and MHC Ⅱcomplex were significantly activated.In SCA,inflammatory response pathways such as IFN,TNF,and NF-kappaB were significantly activated in Mo3,and Mo5 was closely related to regulation of lipid transport,lipid localization,and steroid esterification.The DEGs and functions of SCA and CD monocytes/macrophages also showed significant heterogeneity.Monocytes/macrophages in CD were associated with cytoskeleton,regulation of actin filaments,neutrophil activation,degranulation,and vesicle secretion.Monocytes/macrophages in SCA were enriched to activated pathways of immune responses such as lymphocyte migration,NK cell chemotaxis,and lipid transport.Pathways of IFN-a,y responses,TNF,and IL-6 signaling were significantly activated in SCA,while the IFN-a pathway was inactivated in CD.However,MYC,transforming Growth Factor(TGF)-β,WNT,Notch,and mTOR pathways,which are associated with cell proliferation,were highly activated in CD and inactivated in SCA.SCA and CD also showed heterogeneity in the inflammatory characteristics and differentiation trajectories of monocytes/macrophages.The differentiation trajectory of monocytes/macrophages trajectory started from Mo6(anti-inflammatory),passed through Mo4(anti-inflammatory),Mo2(intermediate),Mo3(pro-inflammatory),Mo5(proinflammatory),and terminated at Mo1(pro-inflammatory).More monocytes/macrophages in CD were found in early and late stages,and fewer in intermediate differentiation stage,consisted of pro-inflammatory,anti-inflammatory and intermediate cells.In contrast,monocytes/macrophages in SCA were mainly in the middle stage of differentiation and fewer cells in the late stage,all of which were pro-inflammatory.ConclusionThis study constructs single-cell atlas of TPIT lineage PitNETs,revealing heterogeneity in the composition and function of endocrine cells and monocytes/macrophages in SCA and pituitary ACTH adenomas.Distinguishment of endocrine cells in SC A and pituitary ACTH adenomas,downregulation of POMC as well as abnormalities in intracellular ribosomes and proteins transport in endocrine tumor cells of SCA,and different activation of pathways in monocytes/macrophages of pituitary ACTH adenomas and SCA may explain the functioning and silent status of these two tumors.Part 3Study of clinical characteristics,diagnosis and treatment of patients with ectopic ACTH syndromeObjective:Ectopic adrenocorticotropic hormone syndrome(EAS)is a rare cause of Cushing’s syndrome,because of complex etiological causes and heterogenic clinical features,the diagnosis and management of EAS remain crucially challenging.This study retrospectively summarized the clinical spectrum of EAS cases with clear etiological diagnosis in a single center,provide evidence for the standard diagnosis and treatment,and lay foundation to further explore the nature of pituitary ACTH adenomas and ectopic ACTH neuroendocrine tumors.Methods:A retrospective study was conducted to identify 88 confirmed EAS cases at Peking union medical college hospital from 1984 to 2019.The clinical,biochemical,imaging and pathological features were analyzed.The control group was 262 patients with pituitary ACTH adenomas.Results:1.Etiological distribution characteristicsOf the 88 eligible EAS patients,the most common cause was bronchial/pulmonary neuroendocrine neoplasms(NENs)(41/88,47%),followed by thymic/mediastinal NENs(29/88,33%).2.Clinical featuresPatients with EAS and pituitary ACTH adenoma both showed clinical manifestations and biochemical characteristics of ACTH-dependent hypercortisolism,but EAS patients have a higher prevalence of glucose metabolism(81.4%vs.29.6%,p<0.05),osteoporosis(76.6%vs.37.4%,p<0.05),urolithiasis(31.8%vs.10.1%,p<0.05).HPA axis hormonal abnormalities and electrolyte disturbances were more significant in EAS patients(ACTH:161 vs 63.2 pg/mL;UFC:946.28 vs 388.74 μg/24h;serum potassium:2.8 vs 3.86 mmol/L,all p<0.05).3.Sensitivity and specificity of localization testsInferior petrosal sinus sampling(IPSS,97.4%)and computed tomography(CT,85.4%)provided high positive diagnostic accuracy.CT is also a useful tool to identify tumors in chest cavity compared with non-chest lesions(91.2%vs 57.1%,p<0.05).18F-FDG and 68Ga-DOTATATE PET/CT presented positive rates in non-chest tumors,but no significant difference was found between these two groups.4.Comparison of clinical features of EAS due to pulmonary and thymic/mediastinal neuroendocrine neoplasmsCompared to pulmonary NENs,tumor volume was larger in thymic/mediastinal NENs(4.54 cm vs.1.44 cm,p<0.05).Baseline ACTH,blood and urinary cortisol levels were not significantly different between the two groups,but 83.3%of patients with thymic/mediastinal NENs were unsuppressed in the high-dose dexamethasone suppression test,significantly higher than patients with pulmonary NENs(51.5%,p<0.05).Conclusions:EAS had more severe clinical presentations than pituitary ACTH adenomas,and the most common causes of EAS are pulmonary and thymic/mediastinal NENs.IPSS combined with CT and other radiological tests can improve the localization diagnosis of EAS. |
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