| ObjectivePituitary somatotroph tumor is one of functional pituitary neuroendocrine tumors(PitNETs),accounting for 5.7%to 16.5%of all PitNETs.As one kind of rare diseases,the annual prevalence of pituitary somatotroph tumors in Europe is(2.8～13.7)/100,000 and the annual incidence is(0.2～1.1)/100,000.The excess of growth hormone(GH)and insulin-like growth factor-1(IGF-1)can cause complications in multiple organs and systems,seriously affecting the quality of life of patients,and even significantly increase the risk of malignant tumors as wells as mortality.Invasive somatotroph tumors are difficult to resected completely through surgery,which has been a major challenge in the treatment of disease and research hotspot.The association between cytokines and biological behavior of tumors has been a hotspot in recent years.A variety of inflammatory and anti-inflammatory cytokines play an important role in regulating tumor cell survival,proliferation and metastasis,including interleukin(IL),tumor necrosis factor(TNF),and interferon(IFN).Thymic stromal lymphocytes hormone(TSLP)can induce terminal breast cancer and lung cancer cell differentiation to block out early tumorigenesis.Chitinase-3-like protein 1(CHI3L1)can promote lung metastasis of melanoma,and is closely related to the progression of glioblastoma and rectal cancer.Pentraxin 3(PTX3)exerts both tumor-promoting and anti-tumor effects.PTX3 is the potential biomarker for glioblastoma,meningioma,pancreatic cancer,gastric cancer and liver cancer.Osteopontin(OPN)is a biomarker of ovarian cancer,and is involved in regulating the proliferation,invasion and metastasis of esophageal cancer,colorectal cancer,prostate cancer and melanoma.Osteocalcin(OC)may be the marker of bone metastasis in breast cancer and prostate cancer and a therapeutic target for the prevention of bone metastasis.Sella floor bone destruction exists in some invasive PitNETs cases,but there are still few studies on bone-derived factors in PitNETs.Previous studies have shown that cytokine levels are closely associated with the prognosis of neuroendocrine tumors(NETs).For example,serum levels of IL-1β,IL-6,IL-8,IL-10,IL-18 and TNF in gastrointestinal and pancreatic NETs patients were significantly different between NET patients and healthy controls.In addition,cytokine levels were significantly correlated with tumor stage,incidence of distant metastasis and disease progression,suggesting cytokines are related to the occurrence,development and prognosis of NETs.Several studies have found that matrix metalloproteinases(MMPs)are associated with the invasiveness of PitNETs,including MMP-1,MMP-2,MMP-9,MMP-3 and MMP-14.At present,although the above cytokines have been studied to varying degrees in other types of tumors,there is no study on serum cytokine in patients with PitNETs,especially pituitary somatotroph tumors.The pituitary gland is composed of a variety of hormone secreting cells,stromal cells and immune cells.However,the data obtained by traditional transcriptomic sequencing are the comprehensive results of all types of cells in the sample tissue,which is difficult to accurately reflect the changes of gene expression levels in different cells in PitNETs.As a basis of tumor cell survival during treatment,the intratumoral heterogeneity(ITH)becomes the main obstacle to improving the prognosis of patients.However,there are still few studies on ITH related mechanisms.Research on the cellular/molecular mechanisms of PitNETs related ITH may be of great significance in guiding the diagnosis and treatment of diseases,especially invasive PitNETs.Single cell RNA sequencing(scRNA-seq)is expected to further reveal the invasive mechanism at the single-cell level.However,there is few single-cell transcriptomic studies on underlying mechanism of invasive growth of somatotroph tumors so far.The aim of this study is to detect cytokines in the serum of patients with pituitary somatotroph tumors using Luminex technology to screen out peripheral biomarkers of invasive pituitary somatotroph tumors.Meanwhile,scRNA-seq is used to analyze the cellular components and gene expression in tumor tissues of invasive and non-invasive somatotroph tumors,in order to explore the relevant mechanisms of invasion and growth of pituitary somatotroph tumors.Methods1.Serum samples from 50 patients with invasive somatotroph tumors,48 patients with non-invasive somatotroph tumors,and 47 control individuals matched in age and gender 1:1 were collected.Luminex technology was used to detect 34 cytokines in samples.The correlation between serum cytokines and multiple invasive features of somatotroph tumors were analyzed.2.The fresh tumor tissue samples of 7 somatotroph tumors were collected.After preparation of single cell suspension,single cell RNA sequencing(scRNA-seq)was conducted.Based on the process of the sequencing data and quality control,the cells were further annotated.Cell composition analysis,differential gene expression analysis and pathway enrichment analysis were performed on invasive and noninvasive pituitary somatotroph tumors.Results1.The study of serum cytokines in patients with invasive and non-invasive pituitary somatotroph tumors1.1 The baseline characteristics of invasive and non-invasive pituitary somatotroph tumorsThere was no significant difference in baseline GH and IGF-1 to upper limit of normal(ULN)ratio(IGF-1/ULN)between the invasive and non-invasive tumor groups(PGH=0.164,PIGF-1/ULN=0.649).The nadir-GH during OGTT in invasion tumor group was significantly higher than that in non-invasion tumor group(14.9[6.5,34.3]ng/mL vs.8.0[3.7,17.4]ng/mL,P=0.038).The proportion of macroadenomas in the invasive group was significantly higher than that in the non-invasive tumor group(93.0%vs.72.9%,P=0.005).No significant differences were observed between the groups of different Ki-67 index,P53 staining and multiple complications(Ki-67 index≥3%:P=0.816;P53 positive:P=0.899;Hypertension:P=0.221;Abnormal glucose metabolism:P=0.230;Hyperlipidemia:P=0.458).The median postoperative follow-up period was 11 months in this study.At the last follow-up,the fasting GH value and nadir-GH in the invasive tumor group were significantly higher than those in the non-invasive tumor group(2.5[1.2,6.7]ng/mL vs.0.5[0.3,2.0]ng/mL,PGH fasting<0.001)(0.7[0.2,2.8]ng/mL vs.0.2[0.1,0.3]ng/mL,Pnadir-GH<0.001),while there was no significant difference of IGF-1/ULN between the groups.1.2 The overall levels of cytokines in serum of objects1.2.1 Serum cytokine levels of patients with somatotroph tumors and the association between cytokine levels and invasiveness of tumorsPTX3 levels in the invasive tumor group were significantly lower than those in the non-invasive tumor group(10.1[6.3,14.5]ng/mL vs.16.6[12.3,24.1]ng/mL,P=0.001).The levels of IL-12(p40),IL-29/IFN-λ1 and IL-11 in group of Knosp grade≥3 were lower than those in group of Knosp grade<3(16.9[11.3,23.6]pg/mL vs.23.6[14.7,38.9]pg/mL,PIL-12(p40)=0.017;2.1[1.0,3.2]pg/mL vs.3.2[1.9,3.9]pg/mL,PIL29/IFN-λ1=0.030;0.6[0.4,0.8]pg/mL vs.0.8[0.6,1.2]pg/mL,PIL-11=0.010).The serum MMP-1 level in the group of impaired sellar floor was significantly higher than that in group of intact sellar floor(453.8[252.6,706.2]pg/mL vs.295.2[202.7,448.1]pg/mL,P=0.039),while the level of PTX3 was lower in the group of impaired sellar floor(10.0[6.5,15.7]pg/mL vs.14.0[9.8,23.3]pg/mL,P=0.041).BAFF/TNFSF13B levels in the macroadenoma cases were significantly higher than those in the microadenoma cases(8.1[5.6,11.3]pg/mL vs.6.5[4.6,7.4]pg/mL,P=0.018).1.2.2 Comparison of serum cytokine levels between groups of relatively high or low hormone levelsThe included patients were divided as group of high IGF-1 levels and the group of lower IGF-1 levels according to median IGF-1/ULN of these patients.The levels of IL12(p40),APRIL/TNFSF13,PTX3 and IL-10 in high IGF-1 group were significantly lower than those in low IGF-1 group(16.9[11.3,23.6]pg/mL vs.23.6[16.9,38.9]pg/mL,PIL-12(p40)=0.033)(3.2[0.1,53.4]ng/mL vs.36.6[0.7,59.2]ng/mL,PAPRIL/TNFSF13=0.031)(11.5[7.4,16.1]ng/mL vs.16.6[9.0,24.4]ng/mL,PPTX3=0.013)(2.9[1.8,4.1]pg/mL vs.3.9[2.6,5.6]pg/mL,PIL-10=0.033).There was no statistical difference of all cytokines between the groups of different GH levels.1.2.3 Comparison of serum cytokine levels between groups of pathological features and complicationsThere were no significant differences in serum cytokine levels between groups of different Ki-67 index and P53 staining.Serum levels of gp130/sIL-6Rβ,IL-34,IL-35,APRIL/TNFSF13,sCD163,OC and sTNF-R1 were significantly higher in patients with hypertension than in those without hypertension(82.0[68.1,93.0]ng/mL vs.56.3[27.0,87.5]ng/mL,Pgp130/sIL-6Rβ=0.040)(94.5[60.8,118.1]pg/mL vs.65.0[30.1,94.5]pg/mL,PIL-34=0.037)(36.4[27.6,48.2]pg/mL vs.28.5[16.1,40.9]pg/mL,PIL-35=0.041)(49.9[0.8,63.4]ng/mL vs.9,3[0.2,47.7]ng/mL,PAPRIL/TNFSF13=0.039)(71.3[44.8,93.1]ng/mL vs.40.1[23.8,80.8]ng/mL,PsCD163=0.029)(6.5[3.7,10.3]ng/mL vs.4.2[1.9,6.8]ng/mL,POC=0.021)(1.2[0.7,1.6]ng/mL vs.0.8[0.4,1.4]ng/mL,PsTNF-R1=0.033).There was no statistical difference in cytokine levels between patients with or without abnormal glucose and lipid metabolism.1.2.4 Regression analysis of serum cytokine levels and invasive features of pituitary somatotroph tumorsAge,sex,hypertension and abnormal glucose or lipid metabolism were adjusted simultaneously in the adjusted model to explore the independent correlation between peripheral cytokine levels and several invasive features.MMP-3 and CHI3L1 were positively and independently correlated with invasiveness[OR=1.288,95%CI(1.0921.519),PMMP-3=0.003][OR=1.949,95%CI(1.085-3.501),PCHI3L1=0.025].Osteocalcin was negatively and independently associated with invasiveness[OR=0.880,95%CI(0.785～0.986),P=0.027].IL-12(p40)and IL-11 were negatively correlated with Knosp grade ≥3[OR=0.953,95%CI(0.911-0.997),PIL-12(p40)=0.037][OR=0.149,95%CI(0.026～0.840),PIL-11=0.031].Four cytokines were positively and independently correlated with impairment of sellar floor,including IFN-y[OR=1.912,95%CI(1.092～3.347),P=0.023],MMP-3[OR=1.275,95%CI(1.091～1.490),P=0.002],TWEAK/TNFSF12[OR=1.365,95%CI(1.058～1.762),P=0.017]and CHI3L1[OR=1.917,95%CI(1.079～3.404),P=0.026].2.Single cell RNA sequencing of pituitary somatotroph tumors2.1 Single-cell transcriptome characteristics of invasive and non-invasive pituitary somatotroph tumorsA total of 40,352 cells from 3 invasive and 4 non-invasive pituitary somatotroph tumors were clustered into 25 clusters and annotated as 5 major cell groups and 12 cell subgroups.These cells contain three kinds of endocrine cells(EPCAM+,POUIF1+,GH1+,GHRHR+),immune cells(PTPRC+,CD68+,CD79A+,GATA2+),stromal cells(PECAM+,COL1A2+),stem cells(SOX2+,SOX9+)and proliferating cells(CENPF+).Endocrine cells were found with the highest count,followed by immune cells,stromal cells and proliferating cells.Compared with non-invasive pituitary somatotroph tumors,invasive pituitary somatotroph tumors presented with a higher proportion of T/NK cells,macrophages,cDC,and stem cells.In two tumor simples,both PRL and GH1 were expressed in the single cell,suggesting the presence of mixed state cells.In invasive pituitary somatotroph tumors,TNFα-NFκB,IFN-α and IFN-γ,P53 signaling,folding protein response and other pathways were significantly activated.2.2 Characteristics of endocrine tumor cell in invasive and non-invasive pituitary somatotroph tumorsEndocrine cells were identified as 22,226 endocrine tumor cells and 10,487 normal endocrine cells by InferCNV method.Several genes of the Heat Shock Protein(HSP)family were up-regulated in the tumor cells from invasive pituitary somatotroph tumors,including HSPA6,HSPB1,HSPH1,HSP90AA1 and DNAJB1.In addition,ATF3(activating factor 3),IER2(immediate early response 2)and ARHGAP36(Rho GTPase activating protein 36)were also up-regulated in tumor cells from invasive pituitary somatotroph tumors.Based on GO analysis of the Top 50 DEGs in both invasive and non-invasive tumors,the invasive somatotroph tumors are enriched in protein folding,ubiquitin protein ligase binding and myofibrillar pathways,while non-invasive growth hormone cell tumors are associated with cytoplasm translation,ribosome,hormone activity as well as growth hormone secretion.ConclusionIn this study,we found the serum levels of multiple cytokines were associated with tumor invasiveness,which are expected to be new biomarkers of invasive pituitary somatotroph tumors,including MMP-3、CHI3L1、sIL-6Rα、OC、IL-12(p40)、IL-11、PTX3、gp130/sIL-6Rβ、IFN-γ and TWEAK/TNFSF12.A series of differentially expressed genes between invasive and non-invasive pituitary somatotroph tumors were identified based on scRNA-seq,such as HSPA6、HSPB1、HSPH1、HSP90AA1、DNAJB1 and ATF3,IER2 and ARHGAP36,providing theoretical support for future research on mechanisms of invasiveness and therapeutic targets of pituitary somatotroph tumors.ObjectivePitNETs,deriving from adenohypophysial cells,were relatively common among intracranial tumors and the incidence of PitNETs is only lower than those of meningioma and glioma.The treatment of refractory PitNETs has been a serious challenge.The rapid development of scRNA-seq technology provides an opportunity for a comprehensive understanding of the cellular components and complex regulatory networks within PitNETs.Through previous work on scRNA-seq of 11 PitNETs,we have screened out a series of potential biomarkers according to the analysis of subpopulation cell composition,differential gene expression and function of different subtypes of PitNETs.IER2(immediate early reactive 2)was uniformly down-regulated in three functional PitNETs compared with nonfunctional PitNETs.VGF(VGF nerve growth factor inducible)in lactotroph tumors and immature PIT-1+PitNETs were overexpressed.THSD7A(thrombospondin type 1 domain containing 7A)was overexpressed in lactotroph tumors.The expression level of ADH1A(alcohol dehydrogenase 1A,alpha polypeptide)in thyrotroph tumor cells and normal thyrotroph cells is higher than that in other subtypes of PitNETs and other types of endocrine cells.This study aimed to validate the potential markers screened through scRNA-seq.Furthermore,we explored the effects of pan-PitNETs subtype-associated IER2 and VGF on cell growth,migration,and invasion of tumor cells in cell experiments.Methods1.RNA was extracted from frozen tissue samples of 103 PitNETs and converted into cDNA by reverse transcription.The expression levels of IER2,VGF,THSD7A and ADH1A were detected by RT-qPCR.The expression levels of the above four genes were analyzed between functional and non-functional PitNETs,macroadenomas and microadenomas,invasive and non-invasive PitNETs,and different subtypes of PitNETs.2.The paraffin-embedded tissues of PitNETs were sliced,and the sections were stained with IER2(n=55),VGF(n=77),THSD7A(n=70),and ADH1A(n=61).The IHC results were reviewed and scored by experienced pathologist(IHC score).The IHC score of the above four molecules were compared between different groups.The grouping method was the same as that of RT-qPCR analysis.3.GH3 cells were transfected with IER2-and VGF-expression plasmids.The CCK8 assay,wound scratch assay and Transwell assay were conducted in the transfected GH3 cells to explore the effects of the overexpression of these two genes on cell proliferation,migration and invasion.Results1.The validation results in PitNETs tissue through RT-qPCRAccording to the RT-qPCR results,the expression levels of IER2 and VGF in different subtypes of PitNETs were heterogeneous.The expression level of THSD7A in lactotroph tumors was significantly higher than those of other PitNETs subtypes.The expression level of ADH1A was low in all subtypes of PitNETs.Based on the betweengroup comparisons,the macroadenomas expressed lower levels of IER2.The expression level of VGF in functional PitNETs was significantly lower than that in non-functional PitNETs.The expression level of THSD7A in lactotroph tumors was significantly higher than that of other subtypes of PitNETs.No significance was observed in levels of ADH1A between different groups.2.The validation results through IHCAccording to the IHC score,the expression levels of IER2,VGF and ADH1A in different subtypes of PitNETs were heterogeneous.The expression level of IER2 in macroadenomas was significantly lower than that of microadenomas.The expression level of VGF was lower in functional PitNETs than in non-functional PitNETs.Lactotroph tumors expressed higher levels of THSD7A and macroadenomas expressed lower levels of THSD7A.There was no statistical difference of ADH1A expression between different groups.3.Experimental results of cell function after transfection with IER2 and VGF overexpression plasmidThe 36-fold improvement of IER2 expression was determined after transfection with IER2 overexpression plasmid.At 72th h of wound scratch experiment,the scratch healing rate of IER2 overexpression group was higher than that of control(EGFP)group(P=0.004).In the CCK8 assay,there was no significant difference in OD values between the IER2 overexpression group and the control group.In the Transwell assay,there was no significant difference in the number of cells passing through the ventricle between groups.The 60-fold improvement of VGF expression was determined after transfection with VGF overexpression plasmid.In 72 h of scratch experiment,there was no difference in the scratch healing rate between VGF overexpression group and EGFP group.In the CCK8 assay,there was no significant difference in OD values between the VGF overexpression group and the control group.In the Transwell assay,there was no significant difference in the number of cells passing through the ventricle between groups.ConclusionIn this study,we validated the potential markers screened through sc-RNA seq of PitNETs at the tissue level and further cell function experiments.The major findings are listed as follows:①IER2 might be involved in the movement and migration of tumor cells of PitNETs;②VGF might be related to hormone secretion in PitNETs;③THSD7A might be a specific marker of lactotroph tumors.Our study provides some enlightenments as well as research basis for further research on biomarkers and mechanism of PitNETs.ObjectiveAs a rare type of PitNETs/pituitary adenomas(PAs),GH/TSH cosecreting PitNETs secrete GH and thyroid stimulating hormone(TSH)simultaneously.Due to the extraordinarily low occurrence of GH/TSH cosecreting PitNETs,most related papers were single-case reports.This study aimed to summarize the clinical characteristics and experience of diagnosis and treatment among patients with GH/TSH cosecreting PAs from Peking Union Medical College Hospital during 12 years.MethodsWe retrospectively reviewed GH/TSH cosecreting PitNETs cases admitted to Peking Union Medical College Hospital between January 1st,2010,and August 30th,2022 to investigate the clinical characteristics,hormone detection,imaging findings,treatment patterns and outcomes of follow-up.The age-and sex-matched cases of growth hormone mono-secreting adenomas(GHPAs)were screened in a ratio of 1:2 using the propensity score matching method(n=42)as control group.We further compared the clinical features of mixed adenomas with those of GHPAs.Results1.Demographic characteristicsTwenty-one patients with GH/TSH cosecreting PitNETs were included in this study.The average age of first visit to our hospital was 45.1±13.7 years old and the male to female ratio was approximately 3:2(13:8).The median age of onset was 41.6 years old and 90.5%(19/21)of the patients ranged from 20 to 60 years old.Delayed diagnosis occurred in 57.1%(12/21)of the patients and the median duration of delayed diagnosis was 21 months.2.Clinical featuresThe most common initial symptom of mixed GH/TSH pituitary adenoma was thyrotoxicosis(10/21,47.6%).The median GH,IGF-1/ULN,TSH and free thyroxine(FT4)before treatment were 4.0 ng/mL,1.9,3.4 μIU/mL and 2.6 ng/dL,respectively.All patients were large adenomas,of which 23.8%(5/21)were giant adenomas.In addition,33.3%(7/21)and 57.1%(12/21)of the patients presented sellar floor bone destruction and cavernous sinus involvement,respectively.3.Treatment and follow-upIn 90.5%(19/21)of patients,surgery was chosen as the primary treatment,and only 36.8%(7/19)of these adenomas were completely resected at the first surgery.In 66.7%(14/21)of patients,somatostatin analogues(SSAs)were applied as initial therapy.Four patients(19.0%)received comprehensive treatment combined with drugs,surgery and radiotherapy.The median follow-up duration was 40 months.Only 23.8%(5/21)of patients achieved long-term biochemical remission of GH/IGF-1.At the last follow-up,1/3(7/21)of patients achieved a complete remission(both GH/IGF-1 and TSH),1/3(7/21)of patients achieved a partial response(either GH/IGF-1 or TSH)and the remaining 1/3(7/21)of patients presented non-remission or relapse.Hormone levels decreased significantly in the first 3 months after surgery or preoperative SSAs.The combination of surgery and SSAs or comprehensive treatment combined with SSAs,surgery and radiotherapy is more conducive to the long-term stability of hormone levels.4.Comparative analysis of GH/TSH cosecreting PitNETs and GHPAsCompared with the age-and sex-matched GHPAs,the mixed GH/TSH pituitary adenoma showed larger tumor size(maximum tumor diameter:24.0 mm vs.14.7 mm,P=0.005)and a higher incidence of cavernous sinus involvement(57.1%vs.23.8%,P=0.009).The rate of complications was higher in the mixed PA group than that of GHPAs group,mainly including arrhythmia(28.6%vs.2.4%,P=0.004),cardiac enlargement(33.3%vs.4.8%,P=0.005),and osteopenia/osteoporosis(33.3%vs.2.4%,P=0.001).In terms of therapeutic response,the proportion of patients with long-term remission was lower in mixed adenoma group(28.6%vs.71.4%,P<0.001),and the proportion of cases who sustained nonremission or underwent relapse was higher in mixed adenoma group(61.9%vs.28.6%,P=0.019).ConclusionThe diagnosis and treatment of GH/TSH cosecreting PitNETs has been a great challenge.The comprehensive treatment program combined surgery with medication or radiation therapy is more effective than treatment of single modality. |
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