【作者】 孙毅；

【导师】 魏强；

【作者基本信息】 四川大学 ， 外科学（泌尿）， 2021， 博士

【摘要】 研究目的及背景:目前,手术治疗和雄激素剥夺治疗是前列腺癌的主要治疗手段。雄激素剥夺治疗主要依据前列腺癌的雄激素依赖性。虽然临床研究证明这两种治疗方法对大多数患者的治疗效果较好,但仍有相当比例患者会发展为去势抵抗性前列腺癌(Castration-Resistant Prostate Cancer,CRPC)。去势抵抗性前列腺癌在很大程度上不再依赖雄激素,因此针对去势抵抗性前列腺癌目前还没有非常有效的治疗方案。据报道,免疫治疗已在多种肿瘤上取得较好的治疗效果,且有临床研究表明针对某些靶点对去势抵抗性前列腺癌患者使用免疫治疗可能会取得不错的效果,因此免疫治疗成为一个治疗前列腺癌的热门研究方向。免疫治疗最为重要的基础是寻找有效的“治疗靶点”,以及确定调控“治疗靶点”的通路。当前国际上免疫治疗主要的“治疗靶点”是“程序性死亡受体-1(Programmed Cell Death Protein 1,PD-1)”及“程序性死亡配体-1(Programmed Cell Death Protein Ligand-1,PD-L1)”。针对PD-1/PD-L1的免疫治疗方案已经应用于多种肿瘤,并取得了令人欣慰的效果。与此同时,与PD-L1具有重要协同关系的吲哚胺-2,3-双加氧酶-1(Indoleamine 2,3-Dioxygenase-1,IDO-1)也引起了越来越多的关注,而且目前有报道表明,在一些肿瘤中针对IDO-1进行治疗效果良好。总之,针对PD-L1/IDO-1的免疫治疗手段在近年来得到了越来越多的关注。而且目前也有临床数据展现了免疫治疗在前列腺癌上的应用前景。尽管如此,前列腺癌中PD-L1/IDO-1的表达水平、调控方式和相应机制方面的报道相对其它肿瘤依然较少,基础研究也处于起步阶段。目前有临床证据表明,PD-L1/IDO-1的表达水平与前列腺癌患者预后有着紧密联系,且在晚期前列腺癌,尤其是在恶性程度最高的小细胞神经内分泌前列腺癌(Small Cell Neuroendocrine Prostate Cancer;SCNEPC)中,其表达量明显高于早期前列腺癌。EMT进程对PD-L1/IDO-1的表达具有重要影响,再联系EMT与神经内分泌转化之间的关系,我们有理由相信EMT有可能与前列腺癌PD-L1/IDO-1的表达有重要联系。但是关于这一现象以及相应机制的研究在前列腺癌中报道较少。因此我们的研究将从探索前列腺癌中PD-L1/IDO-1表达水平与前列腺组织类型(包括良性与恶性)之间的关系着手,重点关注PD-L1/IDO-1在不同EMT程度的前列腺恶性肿瘤中的表达差异,及相关调控分子机制。已有研究表明,干扰素Gamma(Interferon Gamma,IFN-g)-JAK-STAT(The Janus Kinase-Signal Transducer and Activator of Tranions)调控通路在PD-L1/IDO-1的表达方面具有重要作用,故我们的机制研究将重点针对此通路。材料和方法:1.PD-L1/IDO-1表达水平与前列腺不同组织类型之间的关系:1.1我们首先检测患者组织标本中PD-L1/IDO-1的表达水平。我们收集杜克医院泌尿外科收治的行“前列腺癌根治性切除术”或“前列腺穿刺活检术”的患者的病理标本,并将标本分为:“良性前列腺增生”、“早期前列腺腺癌”、“去势抵抗性前列腺癌”和“前列腺小细胞神经内分泌肿瘤”四个不同的组别。良性前列腺增生患者可以仅通过H&E染色确定;早期前列腺腺癌除参考H&E染色外,与去势抵抗性前列腺癌进行区分时,主要依据患者病史——使用过激素剥夺治疗,但是治疗无效或肿瘤有进展则为去势抵抗性前列腺癌;去势抵抗性前列腺癌患者其前列腺组织中小细胞神经内分泌肿瘤细胞占全部肿瘤细胞比例少于10%;而神经内分泌肿瘤除具备“使用过激素剥夺治疗,但是治疗无效或肿瘤有进展”这一特点外,其组织中小细胞神经内分泌肿瘤细胞占全部肿瘤细胞比例应高于10%。小细胞神经内分泌肿瘤细胞主要通过形态(细胞体积小,胞质少,核消失,分布不规律且无腺管形态)及三种标记物(Neuron specific enolase,NSE;Chromogranin A,Cg A;Synaptophysin,SYP)来确认。我们将患者标本固定在石蜡中,设计出几组不同类型的组织标本微阵列(Tissue Microarrays,TMA),切片后进行染色。然后通过免疫组织化学检测(Immunohistochemistry;IHC)来确定病理标本上面是否有超过1%的肿瘤细胞表达PD-L1,如果超过1%就统计为阳性标本;对于IDO-1的检测则采用超过90%视为阳性的标准;对于EMT的标记物N-cadherin则采用超过60%为阳性的标准,这些标准均依据相关文献报道制定。最后,统计每个样本的IHC评分并进行分析。另外,我们还分析了PD-L1/IDO-1与EMT水平之间的线性关系;1.2在细胞水平检测PD-L1/IDO-1的表达时,根据前列腺癌类型的不同将不同细胞系分成四组,与患者的组别相对应。即代表“良性前列腺组织”的“BPH-1”细胞系;代表“早期腺癌”的“LNCap”细胞系;代表“去势抵抗性前列腺癌”的“CWRR-1”和“C4-2”细胞系;代表“神经内分泌肿瘤”的“PC3”、“LASCPC”和“NCI-H660”细胞系。将不同组别的细胞系使用IFN-g进行处理,通过Western blot和PCR(Polymerase Chain Reaction)技术,检测不同组别之间PD-L1/IDO-1表达水平的不同。同时明确这些细胞系的EMT状态,区分EMT阳性与阴性细胞系,并比较其PD-L1/IDO-1的表达差异;1.3为进一步探索PD-L1/IDO-1的表达与前列腺癌发展不同阶段之间的关系,我们研究了PD-L1/IDO-1在动物模型中的诱导表达情况。为更好优化实验条件,排除干扰因素,我们选择使用NSG小鼠(Biocytogen-NOD-Prkdcscid Il2rgnull;免疫缺陷型小鼠)。我们将小鼠模型分为“早期腺癌组”和“神经内分泌肿瘤组”,分别采用皮下注射“LNCap”和“PC3”细胞系的方式构建小鼠肿瘤模型。在种植肿瘤后,我们将两种不同模型的小鼠再分为两个组用于重复之前细胞水平的实验:注射IFN-g的实验组和注射二甲基亚枫(Dimethyl sulfoxide,DMSO)的对照组。连续给药两天,观察48小时后将小鼠处死并将肿瘤取下。标本切割成两部分:一部分使用福尔马林固定并使用石蜡包埋,以便进行免疫组织化学检测;另一部分利用单细胞分离技术将组织分离成单细胞状态,然后进行流式细胞学检测或者提取RNA进行PCR。将流式细胞学,PCR与免疫组织化学染色检测的数据进行比较,判断两组中PD-L1/IDO-1表达情况的差异。再与之前在患者标本及细胞实验中获得的数据进行比较。2.IFNGR1-JAK-STAT通路调控PD-L1/IDO-1表达的研究:2.1我们首先阻断整个IFNGR1(IFN-g Receptor-1)-JAK-STAT通路,以便确定PD-L1/IDO-1受IFNGR1-JAK-STAT通路的调控。然后,为完整检测IFNGR1-JAK1-STAT1信号通路,我们在患者标本、细胞标本和动物标本中检测IFNGR1及p-IFNGR1(激活后的IFNGR1)的表达,并试图构建IFNGR1,p-IFNGR1同PD-L1/IDO-1表达水平及EMT状态之间的联系;2.2基于不是所有细胞系中都可以检测到JAK-STAT通路中的全部成员,我们推测不同细胞系是通过不同的JAK/STAT通路来调控的。因此,我们检测了细胞系中JAK1/STAT1和JAK2/STAT3两条途径的表达和磷酸化情况,再比较不同细胞系中PD-L1/IDO-1的表达来确定具体的调控通路。同时我们还分别阻断STAT1和STAT3通路,来进一步比较其对PD-L1/IDO-1表达的影响。3.EMT与PD-L1/IDO-1相互调节的研究:3.1为确定EMT状态与PD-L1/IDO-1表达之间的联系,我们首先利用免疫组化检测动物标本中不同级别肿瘤组织上面EMT状态的变化,并与PD-L1/IDO-1的表达水平进行比较;3.2随后,我们检测PC3/N-cadherin敲除细胞模型(PC3-N-cadherin Knock Out,PC3-N-cad KO)和EMT激活的LNCap细胞模型(LNCap C1 C2 C3)中PD-L1/IDO-1的表达水平。并将这一模型应用于动物实验,检测改变EMT状态后PD-L1/IDO-1的变化。随后,我们使用PD-L1阻断剂和IDO-1产物犬尿氨酸刺激细胞,以研究PD-L1/IDO-1对EMT进程的影响;3.3通过检测PC3-N-cad KO模型和LNCap C1 C2 C3模型上面JAK/STAT通路各成员的表达及激活情况,来阐述EMT对JAK/STAT通路的表达及激活水平的影响,进一步确定EMT调控PD-L1/IDO-1表达的机制。结果:1.PD-L1/IDO-1的表达水平在不同级别前列腺癌中的变化以及与EMT状态的联系:1.1组织标本检测数据表明,PD-L1/IDO-1在小细胞神经内分泌肿瘤(即EMT阳性肿瘤组)中的表达水平明显高于早期低级别前列腺癌,而且在EMT阳性的标本上面,其表达水平明显提高,更重要的是只有小细胞神经内分泌肿瘤才表现出EMT阳性;1.2细胞水平实验中,PD-L1/IDO-1在蛋白水平与RNA水平的表达趋势是一致的:1)施加IFN-g刺激后,只有神经内分泌肿瘤细胞系才可以诱导出PD-L1。2)尽管在去势抵抗性前列腺癌组也可以检测到IDO-1,但其表达水平远低于小细胞神经内分泌组。3)EMT阳性细胞系中PD-L1/IDO-1表达水平明显高于EMT阴性细胞系;1.3动物实验结果表明,只有在代表EMT阳性肿瘤、小细胞神经内分泌肿瘤的PC3小鼠模型中,PD-L1和IDO-1才可以被IFN-g诱导出来,而在LNCap小鼠模型(EMT阴性肿瘤、早期腺癌)中则不能。这一结果证明只有神经内分泌肿瘤/EMT阳性肿瘤才具有IFN-g反应性,并在其诱导下分泌PD-L1/IDO-1。2.IFNGR1-JAK-STAT通路调控PD-L1/IDO-1:2.1我们发现虽然IFNGR1在早期腺癌、去势抵抗性前列腺癌和小细胞神经内分泌肿瘤中均有表达,但其激活形式p-IFNGR1只存在于去势抵抗性前列腺癌和小细胞神经内分泌肿瘤中,而且在小细胞神经内分泌肿瘤组的表达水平最高。在动物实验中,IFNGR1只在PC3动物模型中被激活。另外,在EMT阳性组中IFNGR1/p-IFNGR1的表达水平明显高于EMT阴性组;2.2 STAT1在大多数肿瘤细胞上面都有表达,但是JAK1只在神经内分泌肿瘤中存在,而且STAT1也只在神经内分泌肿瘤中被IFN-g激活,但是STAT3在去势抵抗性前列腺癌和神经内分泌肿瘤中都可以被激活,且在PC3中STAT3不表达,这一系列结果侧面反映了STAT1可以调控PD-L1和IDO-1的表达,而STAT3只能调控IDO-1的表达。为详细阐述这一信号通路,我们使用JAK/STAT通路阻断剂处理细胞,处理后的PC3、LASCPC都无法表达PD-L1。进一步研究表明,STAT3阻断剂可以阻断IDO-1的表达,而STAT1阻断剂对PD-L1和IDO-1的表达都有影响。总之,我们初步证实了JAK1-STAT1可以调控PD-L1和IDO-1,而JAK2-STAT3只能调控IDO-1。3.只有小细胞神经内分泌肿瘤动物模型(种植PC3肿瘤的小鼠)中可以发现N-cadherin。PC3-N-cad KO细胞系构建的细胞模型和动物模型中PD-L1与IDO-1的表达明显受抑制。EMT激活的LNCap细胞系(LNCap C1 C2)里面,PD-L1/IDO-1可以被诱导出来,但是LNCap C3却不能。虽然这一细胞系也有少量的N-cadherin,但是表达水平相比LNCap C1 C2较低,而E-cadherin却较高,总体EMT水平相对比较低。LNCap C1肿瘤动物模型中可以诱导出PD-L1/IDO-1,与细胞水平结果一致。使用犬尿氨酸刺激细胞后发现EMT标记物的表达水平明显上调,说明IDO-1也可以正向促进EMT进程。阻断PD-L1之后,细胞水平的EMT标记物的表达明显受到抑制,间接证实了PD-L1可以影响EMT进程。结论:本课题是由临床研究、细胞研究和动物实验三部分组成。综合三部分的研究结果我们发现:1)PD-L1/IDO-1在EMT阳性前列腺癌中的表达水平明显升高;2)EMT进程伴随着前列腺癌神经内分泌转化过程而发生并不断强化;3)阻断EMT可以明显降低PD-L1/IDO-1的表达,PD-L1/IDO-1也可以反过来提高肿瘤的EMT激活水平。这说明针对前列腺神经内分泌肿瘤(即EMT阳性肿瘤)进行免疫治疗(尤其是PD-L1/IDO-1为靶点的治疗)或许效果要好于其它阶段的前列腺肿瘤。更为重要的一点在于EMT过程对PD-L1/IDO-1的表达至关重要,所以在选择针对PD-L1/IDO-1进行用药的同时,还可以尝试联合应用与EMT相关的药物来提高前列腺癌免疫治疗的效果。更多还原

【Abstract】 ObjectiveAt present,the main treatment of prostate cancer is surgical and Androgen Deprivation Treatment(ADT).Androgen deprivation therapy is according to that grow of prostate cancer based on the androgen.Although these two treatments can be very effective in most patients,a considerable proportion of patients will develop to Castration Resistant Prostate Cancer(CRPC).However,there is no effective treatment plan for those patients.Clinical studies have shown that immunotherapy is efficiency in some cancers,and also have well effect in CRPC,so immunotherapy has become a hot research direction.The most important foundation of immunotherapy is to find the effective target and the regulatory pathway.Immunotherapy,especially for programmed cell death protein-1(PD-1)and programmed cell death protein ligand-1(PD-L1),has been applied in many kinds of tumors,and has achieved very gratifying therapeutic effect.At the same time,indoleamine-2,3-dioxygenase-1(IDO-1)has also attracted more and more attention.Although immunotherapy for PD-L1/IDO-1 has attracted more and more attention in recent years.But there are not enough reports about the treatment of PD-L1/IDO-1on prostate cancer,and the basic research is rarely.At present,a considerable number of clinical data show that the expression level of PD-L1/IDO-1 is closely related to the prognosis of prostate cancer patients.Moreover,its expression level is significantly higher in the small cell neuroendocrine prostate cancer than that of other early tumors.Small cell neuroendocrine prostate cancer is usually accompanied by Epithelial Mesenchymal Transition(EMT).In some studies about other tumors,EMT process has an important impact on the expression of PD-L1/IDO-1,according with the relationship between EMT and Neuroendocrine transformation,we propose this hypothesis that EMT may be able to regulate the expression of PD-L1/IDO-1 in prostate cancer.However,there are few reports about this phenomenon and mechanism in prostate cancer.Our research will start from exploring the relationship between the expression level of PD-L1/IDO-1 on prostate cancer and prostate tissue type.In order to further exploration about the mechanism.According to the published reports,IFN gamma receptor and the Janus kinase/signal transducer and activator of tranions(IFNGR-JAK-STAT)pathway plays an important role in the expression of PD-L1/IDO-1.Therefore,we will conduct in-depth study on this path way.Materials and Methods1.1 In the clinical research part,we collected the pathological samples of the patients who had undergone radical prostatectomy or puncture biopsy in the Urology of Duke Hospital.According to the pathological identification,the patients were divided into four different groups: Benign Prostatic Hyperplasia,adenocarcinoma,CRPC and small cell neuroendocrine tumor of prostate.Then,the immunohistochemistry(IHC)was used to determine whether there was more than1% PD-L1 expression on the pathological samples.If there was more than 1%PD-L1 expression,it would be considered as positive.More than 90% of IDO-1 was positive.More than 60% of N-cadherin was positive.At the same time,the IHC scores of each sample were calculated and analyzed.And the liner relationship between PD-L1/IDO-1 and N-cadherin also studied;1.2 Different cell lines were divided into four groups,corresponding to the group of patients.BPH-1 cell line representing benign prostate tissue,LNCap cell line representing adenocarcinoma,CWRR-1 cell line and C4-2 cell line representing CRPC cell line,PC3 cell line,LASCPC cell line and NCI-H660 cell line representing neuroendocrine tumor.Different cell lines were treated with IFN-g.Western blot and PCR were used to detect whether the expression of PD-L1/IDO-1 could be induced and the different expression of PD-L1/IDO-1 among several groups.At the same time,EMT status of these cell lines was determined,EMT positive and EMT negative cell lines were distinguished,and the expression levels of PD-L1/IDO-1 were compared;1.3 In order to further improve the relationship between PD-L1/IDO-1 expression and different types of prostate cancer,we also studied the expression of PD-L1/IDO-1 in NSG mice.Animal models were made by subcutaneous implantation of human prostate cancer tumor cells.In order to make the animal model more representative,we also chose two different groups: in early prostate cancer group,we chose LNCap,while in neuroendocrine group,we chose PC3.After subcutaneous implantation of tumor,we cultured the mice under the same culture conditions.When the tumor size reached 1.5cm-2cm,IFN-g reagent was given by intraperitoneal injection.DMSO was used in the control group.The mice were killed and the tumor was removed after48 hours.Then the specimen was cut into two parts,one was fixed with formalin,and embedded in paraffin for immunohistochemistry detection,the other was separated into single cell state by single cell separation technology for RNA extraction and flow cytometry.Compare the data from mice model in two different groups with the data obtained in patients and cells.2.1 When we study the specific regulatory pathway of PD-L1/IDO-1,we blocked JAK/STAT pathway to detect whether PD-L1/IDO-1 expression could still be induced.In order to completely detect IFNGR1-JAK1/STAT1 signaling pathway,we detected the expression and activation of IFNGR1 at the patient level,cell level and animal experiment.Then we compared the expression levels of IFNGR1,p-IFNGR1 with PD-L1/IDO-1,and EMT status;2.2 In addition,as far as we know,not every factor in JAK/STAT pathway can be detected in all cell lines.Different cell lines representing different tumor types may be regulated by different JAK/STAT pathways.Therefore,we first need to detect the expression and phosphorylation of JAK1/STAT1 and JAK2/STAT3 in cell lines,and then compare the expression of PD-L1/IDO-1 in different cell lines to determine the specific regulatory pathway.At the same time,we also blocked STAT1 and STAT3 pathways to compare their effects on the expression of PD-L1/IDO-1.3.1 In order to determine the relationship between EMT and PD-L1/IDO-1,we first detected the relationship between the EMT status and tumor types in animal samples.And compared with the expression of PD-L1/IDO-1;3.2 We used PC3N-cadherin knockout cell model(PC3-N-cad KO)and EMT activated LNCa P cell model(LNCap C1,C2,C3)to detect the expression of PD-L1/IDO-1.Then use those models in animal to detect the expression of PD-L1/IDO-1.At the same time,we also applied PD-L1 blocking antibodies or stimulate cells with tyrosine to study its effect on EMT process.PC3-N-cad KO model and LNCap C1 C2 C3 models were used in studied the effect of EMT on the expression and activation of JAK/STAT pathway to determine the mechanism of EMT regulating PD-L1/IDO-1 expression.Results1.1 Our study showed that the expression of PD-L1/IDO-1 in small cell neuroendocrine tumor was significantly higher than that in other types of prostate cancer,moreover,the expression level of PD-L1/IDO-1 was significantly increased in EMT positive specimens;1.2 The expression of protein level is consistent with that of RNA level.Only on neuroendocrine tumor cell lines,PD-L1 can be induced under IFN-g stimulation.Although the expression of IDO-1 can also be found in CRPC group,the level is lower than that of small cell neuroendocrine group.This result is consistent with the clinical data;1.3 The results of animal experiments showed that PD-L1 and PD-L1/IDO-1 could be expressed only in PC3 model stimulated by IFN-g,but there was no expression in LNCap group.This result further proves that only neuroendocrine tumors have IFN-g reactivity and can express PD-L1/IDO-1.2.1 By detecting the expression of IFNGR1 and p-IFNGR1 in patients,we found that although IFNGR1 was expressed in both CRPC patients and small cell neuroendocrine tumor patients,p-IFNGR1 was only found in small cell neuroendocrine tumor patients.It is well known that p-IFNGR1 is the activation state of IFNGR1.Only IFNGR1 is activated can further activate the downstream JAK/STAT signal pathway.In addition,the expression level of IFNGR1/p-IFNGR1 in EMT positive group was significantly higher than that in EMT negative group.In vivo experiment,the results also prove that only neuroendocrine prostate cancer has IFN reactivity;2.2 Then we detected the activation of different JAK/STAT pathways.The results showed that STAT1 was expressed in most tumor cells,but JAK1 was only expressed in several neuroendocrine tumors.At the same time,STAT1 can only be activated in neuroendocrine tumors.However,STAT3 can be activated on CRPC,which provides the evidence that STAT1 can only induce the expression of IDO-1instead of PD-L1.While the PC3 do not express STAT3,so JAK1/STAT1 pathway can regulate both PD-L1 and IDO-1.Above all,we also clarified that JAK1-STAT1 regulates the expression of PD-L1 and IDO-1,while JAK2-STAT3 can only regulate the expression of IDO-1.The discovery was also reported for the first time.3.1 The effect of PD-L1/IDO-1 expression during EMT.Our study shows that EMT activation only occurs in the neuroendocrine prostate group(patients,cells and animals),and PD-L1 expression only be induced in EMT activated mice model;3.2The expression of N-cadherin was found only in the small cell neuroendocrine group(PC3).PC3-N-cad KO cell line has become EMT negative after N-cadherin knockout.After using these two models,we found that the expression levels of PD-L1 and IDO-1 significantly decreased after inhibited N-cadherin.Meanwhile,PD-L1/IDO-1 can be induced in EMT activated LNCap cell line(LNCa P C1 C2).However,PD-L1/IDO-1 could not be induced in primary LNCap cells.But in LNCap C3,PD-L1/IDO-1 could not be expressed.Although there was a small amount of N-cadherin expression in this cell line,the expression level was lower than that of LNCap C1 C2,but the expression level of E-cadherin was higher,and the overall EMT level was lower.The results of animal model experiments suggest that PD-L1 and IDO-1 can be induced in LNCap C1 tumor model after EMT activation.However,PD-L1 and IDO-1 could not be induced in PC3-N-cad KO model after N-cadherin knockout;3.3 We used tyrosine to stimulate the cells and detected the expression level of EMT markers.The results showed that the expression level of EMT markers was significantly up-regulated.The results proved IDO-1 can also regulate EMT process with positive feedback.After blocking PD-L1,we found that the expression level of EMT markers dramatically decreased;3.4Further study found that EMT can affect the activation of JAK-STAT pathway.This is the mechanism by which EMT regulates the expression of PD-L1/IDO-1.ConclusionThis project is composed of clinical research,cell level research and animal experiment.We found that the expression of PD-L1/IDO-1 significantly increased in EMT positive prostate cancer.The EMT process is accompanied by the neuroendocrine transformation.Blocking EMT can significantly reduce the expression of PD-L1/IDO-1,and PD-L1/IDO-1 can in turn increase the level of EMT activation.This suggests that immunotherapy(especially target to PD-L1/IDO-1)for prostate neuroendocrine tumors may be better than other stages of prostate tumors.More importantly,since EMT process is crucial to the expression of PD-L1/IDO-1,we can try to combine EMT related drugs with PD-L1/IDO-1 to improve the effect of immunotherapy of prostate cancer.更多还原