CN116515937A - eCAF在免疫检查点阻断治疗法治疗癌症的疗效预测中的应用 - Google Patents
eCAF在免疫检查点阻断治疗法治疗癌症的疗效预测中的应用 Download PDFInfo
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Abstract
本发明公开了eCAF在免疫检查点阻断治疗法治疗癌症的疗效预测中的应用,本发明深入研究eCAF亚群在癌症免疫检查点阻断治疗法治疗疗效上的影响,发现eCAF亚群及其特征基因POSTN和FAP在癌症中活跃程度与免疫检查点阻断治疗法治疗癌症效果相关,这表明eCAF亚群及其特征基因可以作为判断免疫检查点阻断治疗法治疗癌症疗效的潜在标记物,并且eCAF亚群及其特征基因的抑制剂可以作为免疫检查点阻断治疗法治疗癌症疗效的增强剂。
Description
技术领域
本发明属于生物医药领域,涉及eCAF在免疫检查点阻断疗法治疗癌症的疗效预测中的应用。
背景技术
免疫检查点阻断(ICB)治疗已经彻底改变了癌症治疗,免疫检查点阻断疗法的基本原理是基于免疫细胞T细胞的激活机制。程序性死亡受体表达在T细胞表面,其配体表达在肿瘤细胞和髓源性抑制细胞表面。程序性死亡受体与其配体结合可使T细胞衰竭而无法正常杀伤肿瘤细胞,肿瘤细胞便可逃脱宿主的免疫监视。因此,程序性死亡受体及其配体被称为“免疫检查点”。基于程序性死亡受体及其配体的免疫检查点阻断疗法通过抑制二者的结合,从而提高宿主免疫系统对肿瘤细胞的攻击性。
胃癌(GC)是全球第三大致死癌症,免疫检查点阻断(ICB)疗法已阐明了多种实体肿瘤的癌症治疗,但抗PD1单药治疗的益处为晚期GC患者提供的生存益处不到20%。因此,ICB耐药的潜在机制仍然是一个重大挑战。
肿瘤组织中大量的肿瘤相关成纤维细胞(cancer associated fibroblasts,CAFs)为肿瘤的发展构建了良好的环境(占基质细胞50%以上)。CAFs细胞分为肌成纤维细胞、周细胞、细胞外基质CAF(eCAF)和免疫调节CAF(iCAF),不同亚型CAFs表面特征基因表达情况不同,如成纤维细胞活化蛋白(FAP)、α-平滑肌动蛋白(α-SMA)、成纤维细胞特异性蛋白1(FSP1)、血小板衍生生长因子受体(PDGFR)等。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊,而这种简化或省略不能用于限制本发明的范围。鉴于现有技术存在的技术缺陷,本发明克服了现有技术存在的不足,并提供了以下的发明内容。
本发明提供了一种检测样本中eCAF细胞的试剂在制备免疫检查点阻断疗法治疗肿瘤疗效预测的产品中的应用。
进一步,所述检测样本选自器官或组织样本的固体组织,或活组织抽吸物。
进一步,所述检测样本为个体肿瘤组织。
术语“样本”是指大量生物材料,在一些实施例中,这些生物材料来自患者,并且包含细胞和/或无细胞DNA;也就是说,该术语可与术语“标本”或“组织样本”交换使用。术语“样本”有时也分别用在获得(例如)重组核酸的更大组或量的子集或部分的统计学意义上;具体地讲,术语“样本”的统计学用法也可被理解为意指“代表性样本”,那样,这类样本被理解为反映或者近似于(例如)组织中不同核酸的相对频率。本领域的技术人员能够从上下文中辨别该术语的正确用法。特别的,本发明中所使用的样本是指从患者组织获得的类似细胞的集合。组织或细胞样本的来源可以来自新鲜、冷冻和/或保存的器官或组织样本的固体组织,或者活组织检查或者抽吸物。组织样本可以是初级或体外培养的细胞或细胞株。可选地,组织或细胞样本从疾病组织/器官获取。组织样本可能包含不与所述组织自然混合的化合物,诸如防腐剂、抗凝血剂、缓冲剂、固定剂、营养剂、抗生素、或类似化合物。
术语“个体”、“患者”或“主体”在本文中可以互换使用,是指哺乳动物、鸟、鱼、爬行动物或任何其它动物。术语“个体”亦包括人类。术语“个体”亦包括家庭宠物。家庭宠物之非限制性实例包括:狗、猫、猪、兔、大鼠、小鼠、沙鼠、仓鼠、天竺鼠、雪貂、鸟、蛇、蜥蜴、鱼、龟及蛙。术语“个体”亦包括家畜动物。家畜动物之非限制性实例包括:羊驼、野牛、骆驼、牛、鹿、猪、马、美洲驼、骡、驴、绵羊、山羊、兔、驯鹿、牦牛、鸡、鹅及火鸡。
如本文所用之术语“哺乳动物”是指任何哺乳动物,诸如人类、小鼠、兔、非人类灵长类动物。在一优选实施方式中,所述哺乳动物为人类。
进一步,所述肿瘤为实体瘤。
进一步,所述实体瘤包括肺癌、脑癌、结肠癌、直肠癌、前列腺癌、乳腺癌、肝癌、肾癌、胃癌、子宫颈癌、头颈癌、卵巢癌、睾丸癌、垂体癌、食道癌、皮肤癌、胰腺癌或骨癌。
进一步,所述实体瘤为胃癌。
进一步,所述检测样本中eCAF细胞的试剂包括检测eCAF细胞活性的试剂、检测eCAF细胞分泌物的试剂、或检测eCAF细胞相关特征基因的试剂。
术语“试剂”意味着包含能够防止、改善、或治疗疾病或其它医疗状况的任何分子、化学实体、组合物、药物、治疗剂、化疗剂、或生物试剂。术语“试剂”包括小分子化合物、反义试剂、siRNA试剂、抗体、酶、有机或无机肽分子、天然或合成化合物等。试剂可在临床试验、预实验测试、或FDA-批准后的任何阶段根据本发明的方法分析。
进一步,所述检测eCAF细胞活性的试剂包括以下方法中所使用的试剂,所述方法包括化学染色法、荧光染色法、克隆-集落形成实验、MTT比色法、XTT比色法、CKK-8比色法、Alamar Blue法、乳酸脱氢酶释放法、SRB法、ATP含量测定法、液体闪烁计数仪法、或流式细胞术。
术语“细胞活性”是指活细胞浓度与细胞总浓度之间的比例,其中后者也包括死细胞。这个比例可以在任何一种方式下进行计算。其中一种方法是用染料标记这些细胞,而在活或死细胞上的染料会有差别。一个用于分辨活和死细胞的例子是使用台盼蓝染料。接下来,就可以数出所标记的细胞并确定活和死细胞的数量。然后计算出这个细胞活性,在其中就可以计算在活细胞数和细胞总数之间的比例。术语“细胞活性”也包括在细胞在增殖分裂过程的快慢,凋亡速度的快慢等。
术语“细胞分泌”是指细胞将在粗面内质网上合成而又非内质网组成部分的蛋白和脂通过小泡运输的方式经过高尔基体的进一步加工和分选运送到细胞内相应结构、细胞质膜以及细胞外的过程称为细胞的分泌。“细胞分泌”分为两种∶分泌的物质主要是供细胞内使用;另一种是要通过与细胞质膜的融合进入细胞质膜或运输到细胞外。术语“细胞分泌物”则包括各种酶类、激素、神经递质、局部介质、血清蛋白、抗体,以及细胞外基质成分。
进一步,所述检测eCAF细胞相关特征基因的试剂包括检测eCAF细胞相关特征基因POSTN、或FAP的试剂。
进一步,所述检测eCAF细胞相关特征基因POSTN、或FAP的试剂包括特异性识别POSTN或FAP基因的探针,或特异性扩增POSTN或FAP基因的引物,或特异性结合POSTN或FAP基因编码的蛋白的结合剂。
进一步,所述产品包括试剂盒、芯片、试纸、系统中的一种或多种。
如本文所使用,术语“试剂盒”是指用于递送物质的任何递送系统。包含性的术语“试剂盒”是用于研究和临床应用的试剂盒。此类递送系统包括容许从一个位置到另一位置反应试剂(例如适当的容器中的寡核苷酸、酶等)和/或支持物质(例如缓冲剂、用于进行分析的书面说明书等)的储存、转运、或递送的系统。例如,试剂盒包括含有相关反应试剂和/或支持物质的一个或多个壳体(例如盒子)。
本发明还提供了一种用于预测免疫检查点阻断疗法治疗肿瘤疗效的产品,所述产品包括检测样本中eCAF细胞的试剂。
进一步,所述检测样本选自器官或组织样本的固体组织,或活组织抽吸物。
进一步,所述检测样本为个体肿瘤组织。
进一步,所述产品包括试剂盒、芯片、试纸、系统中的一种或多种。
进一步,所述肿瘤为实体瘤。
进一步,所述实体瘤包括肺癌、脑癌、结肠癌、直肠癌、前列腺癌、乳腺癌、肝癌、肾癌、胃癌、子宫颈癌、头颈癌、卵巢癌、睾丸癌、垂体癌、食道癌、皮肤癌、胰腺癌或骨癌。
进一步,所述实体瘤为胃癌。
如本文所使用,与所定义的或描述的物件、组合物、装置、方法、过程、系统等元素相关的术语“包含”或“包括”及其变体,意思是包括一切的或开放式的,允许另外的元素,从而表示所定义的或描述的物件、组合物、装置、方法、过程、系统等包括那些指定的元素--或者,适当时,其等价物--并且其它元素可被包括并仍落入所定义的物件、组合物、装置、方法、过程、系统等的范围/定义内。
进一步,所述检测样本中eCAF细胞的试剂包括检测eCAF细胞活性的试剂、检测eCAF细胞分泌物的试剂、或检测eCAF细胞相关特征基因的试剂。
进一步,所述检测eCAF细胞活性的试剂包括以下方法中所使用的试剂,所述方法包括化学染色法、荧光染色法、克隆-集落形成实验、MTT比色法、XTT比色法、CKK-8比色法、Alamar Blue法、乳酸脱氢酶释放法、SRB法、ATP含量测定法、液体闪烁计数仪法、或流式细胞术。
进一步,所述检测eCAF细胞相关特征基因的试剂包括检测eCAF细胞相关特征基因POSTN、或FAP的试剂。
术语“标记”表示处于化学或生物实体形式的任何性状或特征。标记可以在性质上是形态的、功能的或生物化学的。在优选的实施例中,标记是差异性或优先地由特定细胞类型表达或不表达,或由细胞在某些条件(例如,在细胞周期的特定时点或在特定细胞外基质)下表达的细胞因子或表面抗原或膜蛋白或胞浆蛋白等。在本发明中更具体地指那些可借助其存在(阳性)或不存在(阴性)来指示细胞或细胞亚群的标记。
本发明还提供了eCAF细胞的抑制剂在制备增强ICB治疗肿瘤疗效的药物中的应用。
进一步,所述肿瘤为实体瘤。
进一步,所述实体瘤包括肺癌、脑癌、结肠癌、直肠癌、前列腺癌、乳腺癌、肝癌、肾癌、胃癌、子宫颈癌、头颈癌、卵巢癌、睾丸癌、垂体癌、食道癌、皮肤癌、胰腺癌或骨癌。
进一步,所述实体瘤为胃癌。
进一步,所述药物还包括任何药学可接受的辅料。
进一步,所述辅料包括表面活性剂、助流剂、润滑剂、增塑剂,包衣剂、胶囊材料、助溶剂、赋形剂、pH调节剂、抗氧剂、稳定剂、螯合剂、抑菌剂、等渗剂、防腐剂、乳化剂,但不限于此。所述其他药学辅料可以在制备过程中根据剂型制备需要选用。
用于制备其药物的有用的药物载体可以是固体,液体或气体;因此,所述组合物可以采用片剂、丸剂、胶囊、栓剂、粉末剂、肠包衣的或其它保护的制剂(例如结合于离子交换树脂或包装在脂蛋白泡囊中)、缓释制剂、溶液、混悬剂、酏剂、气溶胶等的形式。所述载体可以选自不同油,包括石油、动物油、植物油或合成来源的油,例如,花生油,大豆油,矿物油,芝麻油等。水,盐水,含水葡萄糖,和乙二醇是优选的液体载体,特别是(当与血液等渗时)用于可注射溶液。
根据本发明的治疗的术语“疗效”可以基于疾病在对根据本发明的应用或方法的反应的过程中的变化来测量。例如,治疗癌症的疗效可以通过肿瘤体积的减少,和/或发展自由存活时间的增加,和/或初级癌症切除后复发风险的降低来测量。更具体地,对于通过免疫疗法治疗的癌症来说,疗效的评估可以通过新颖的免疫相关反应标准(irRC)的免疫治疗剂的抗肿瘤反应的临床模式的范围来进行,其从实体瘤疗效评价标准(RECIST)和世界卫生组织(WHO)标准改编而来(J.Natl.Cancer Inst.2010,102(18):1388–1397)。感染性疾病的预防的疗效最终通过在人类群体中的流行病学研究来评估,其常与血清中的中和抗体的滴度和多功能病原体特异性T细胞反应的引发相关。临床前评估可包括用感染性病原体激发后对感染的抗性。感染性疾病的治疗可通过与病原体特异性抗体和/或T细胞免疫反应相关的病原体的生长的抑制或病原体的消除(并因此没有发现病原体)来测量。
术语“抑制剂”是指能够特异性的针对eCAF细胞的细胞骨架蛋白抑制剂、细胞凋亡诱导剂、细胞增殖抑制剂、或细胞杀伤剂,通过这些试剂使得eCAF细胞被抑制或特异性的杀伤并且死亡,或无法正常复制分裂。
术语“一个实施方案”、“一个实施例”、“实施方案”或“实施例”同义,是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”“在一个实施方案中”并非均指同一个实施例,也不是单独的或选择性的与其他实施例互相排斥的实施例。
附图说明
图1是eCAFs特征基因POSTN和FAP在正常组织和癌症组织中表达水平图,其中,A为POSTN在正常组织和癌症组织中表达水平图,B为FAP在正常组织和癌症组织中表达水平图;
图2是TCGA数据库中不同肿瘤中eCAF细胞特征基因POSTN表达与FAP表达关系图,其中,A为乳腺癌(BRCA)POSTN表达与FAP表达关系图,B为结肠腺癌(COAD)POSTN表达与FAP表达关系图,C为食道癌(ESCA)POSTN表达与FAP表达关系图,D为骨髓癌(LIHC)POSTN表达与FAP表达关系图,E为肺腺癌(LUAD)POSTN表达与FAP表达关系图,F为头颈部鳞状细胞癌(HNSC)POSTN表达与FAP表达关系图;
图3是TCGA数据库中eCAF细胞特征基因POSTN表达差异或FAP表达差异的肿瘤中的ICB治疗TIDE评分图,其中,A为结肠腺癌(COAD)POSTN表达差异的ICB治疗的TIDE评分图,B为食道癌(ESCA)POSTN表达差异的ICB治疗的TIDE评分图,C为肺腺癌(LUAD)POSTN表达差异的ICB治疗的TIDE评分图,D为结肠腺癌(COAD)FAP表达差异的ICB治疗的TIDE评分图,E为食道癌(ESCA)FAP表达差异的ICB治疗的TIDE评分图,TIDE评分越高代表ICB治疗效果越差;
图4是POSTN、FAP与CAFs特征性基因表达相关图,其中,A为IHC检测弥漫型、肠型和混合型胃癌患者间质区POSTN的位置图,比例尺:50μm,B为IHC检测弥漫型、肠型和混合型胃癌患者间质区FAP的位置图,比例尺:50μm,C为TCGA数据库中胃腺癌(STAD)中CAF特征基因表达水平的相关性图,D为TCGA数据库中胃腺癌(STAD)肿瘤组织和正常组织POSTN表达水平图,E为TCGA数据库中胃腺癌(STAD)肿瘤组织和正常组织FAP表达水平图;
图5是胃癌中POSTN和FAP双阳性CAF亚群IF检测图;
图6是TCGA数据库中eCAF特征基因POSTN、FAP差异表达与胃腺癌(STAD)预后情况生存曲线图,其中,A为POSTN差异表达与胃腺癌(STAD)预后情况生存曲线图,B为FAP差异表达与胃腺癌(STAD)预后情况生存曲线图,C为POSTN和FAP差异表达与胃腺癌(STAD)预后情况生存曲线图,高表达组:表达水平最高的50%,低表达组:表达水平最低的50%;
图7是收集样本中POSTN、FAP、CD163、CD68表达水平与ICB治疗胃癌疗效的IHC柱状分析图,其中,A为POSTN表达水平与ICB治疗胃癌疗效的IHC柱状图,B为FAP表达水平与ICB治疗胃癌疗效的IHC柱状图,C为CD163表达水平与ICB治疗胃癌疗效的IHC柱状图,D为CD68表达水平与ICB治疗胃癌疗效的IHC柱状图,PR:部分缓解,SD:疾病稳定,PD:进展性疾病;
图8是TCGA数据库中胃癌患者中POSTN和FAP的表达水平与TIDE评分的关系图,其中,A为POSTN的表达水平与TIDE评分的关系图,B为FAP的表达水平与TIDE评分的关系图;
图9是收集样本中POSTN和FAP表达水平在ICB治疗胃癌疗效的IHC结果图与三维化学染色图,其中,A为POSTN表达水平在ICB治疗胃癌疗效的IHC结果图,B为FAP表达水平在ICB治疗胃癌疗效的IHC结果图,C为胃癌组织标本中eCAF特征基因POSTN和FAP的免疫组织化学染色(×40)图,比例尺:50μm,PR:部分缓解,SD:疾病稳定,PD:进展性疾病;
注:***,p值<0.001;**,p值<0.01;*,p值<0.05;ns,无显著差异。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是本发明还可以采用其他不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似推广,因此本发明不受下面公开的具体实施例的限制。
实施例
一、材料和方法
1)患者样本
收集2018—2021年在北京协和医院接受抗PD1治疗的92例恶性肿瘤患者临床病理特征和总缓解率(ORR),具体信息如表1所示,其中包括22例胃癌患者,具体信息如表2所示,这些患者的数据用于分析ICB的ORR与eCAF丰度之间的关系。所有患者均经北京协和医院病理医师病理诊断为胃癌或其他恶性肿瘤。根据AJCC第8版分期标准进行TNM分期。采用RECIST 1.1评估所有患者的ORR。总生存期(OS)的计算从诊断日期至任何原因死亡日期。术后每3个月门诊或电话随访1次,随访时间截至2022年8月。本研究获得了中国医学科学院北京协和医学院伦理委员会批准。所有患者均签署了书面知情同意书。
表1 92例接受化疗联合免疫治疗的肿瘤患者的特征信息
表2 22例接受化疗联合免疫治疗的胃癌患者的特征信息
2)TCGA数据库的生物信息学分析
通过TCGA biolinks R包(https://portal.gdc.cancer.gov)下载TCGA患者的基因表达数据和临床信息。从XENA-TCGA_GTEx(https://xenabrowser.net/datapages/)下载癌症数据。将基因表达的TPM(transcriptper million reads)值转换为log2(TPM+1)进行进一步分析。以POSTN和FAP的中位值确定高表达组和低表达组。通过肿瘤免疫功能障碍和排斥(TIDE)算法预测对ICB的潜在反应。采用survminer和survival软件包分别进行Kaplan-Meier检验和log-rank检验。进一步评估POSTN和FAP表达水平及临床病理特征对OS的影响,采用单因素和多因素Cox回归分析。
3)人GC来源CAFs(hCAFs)获得
从患者胃癌组织中分离hCAFs,通过定量反转录-聚合酶链反应(qRT-PCR)和蛋白质印迹法(WB)进行鉴定。两种方法均获得了中国医学科学院和北京协和医学院伦理委员会的批准。所有供者均签署了知情同意文件。
4)RNA提取和qRT-PCR
TRIzol(Thermo Fisher Scientific;15596018),用氯仿纯化。然后,用异丙醇沉淀RNA,75%乙醇洗涤,重悬于30μl DEPC水中。RNA浓度通过Nanodrop分光光度法(ThermoScientific Nanodrop 2000/2000C)测定。随后,从每个样本中提取5000ng RNA逆转录为cDNA。将cDNA模板和引物与SYBR@Premix Ex TaqTM(TaKaRa,日本)混合在96孔板中进行qRT-PCR。采用QuantStudio Design&Analysis软件进行qRT-PCR检测。qRT-PCR扩增条件为:95℃初始变性10min,95℃10s循环40次,60℃退火40s。使用2-ΔΔCT方法对每个样本的数据进行半定量分析。所有引物序列见表3。
表3引物序列
5)Western blot
WB按照常规技术进行。WB使用的一抗包括GAPDH(CST#5174,Danvers,MA,USA)、PDGFRα(CST#3174)、MMP2(CST#40994)、HSP90(CST#4877)、HSP70(CST#4873)和β-Actin(CST;4970#),购自美国马萨诸塞州丹佛斯。Periostin(Abcam;ab79946)购自Abcam(Cambridge,Massachusetts,USA),外泌体抗cd63(Invitrogen;TS63)购自InvitrogenSuperScriptII(Carlsbad,CA,USA)。WB使用的二抗为羊抗兔IgG(H+L)HRP(MultiSciences,中国;70-GAR0072)和羊抗小鼠IgG(H+L)辣根过氧化物酶(中国;70-gam0072)。最后,采用化学发光ECL检测试剂(Millipore,USA)进行化学发光检测。
6)IHC和免疫荧光(IF)
采用常规技术进行IHC。一抗及用IHC染色的稀释度为抗periostin抗体(Abcam,ab79946;1:75)、抗fap抗体(Abmart;PA7036S;1:100)、CD163(Servicebio,GB113152;1:1000)、CD68(Abcam,ab201340;1:200)。二抗为HRP山羊抗兔IgG(Servicebio,GB23303;1:200)、HRP山羊抗小鼠IgG(Servicebio,GB23301;1:200),Alexa488偶联山羊抗兔IgG(Servicebio,GB25303;1∶400),以及cy3标记的山羊抗小鼠IgG(Servicebio,GB21301;1:300)。应用HALO图像分析软件(Indica Labs)分析IF染色情况。eCAF阳性率的计算方法为POSTN或CD163阳性细胞百分比除以每个切片的细胞总数,阳性密度的定义为POSTN或CD163阳性细胞数/mm2。
7)统计分析
对于连续变量,正态分布的计量资料采用独立样本t检验,非正态分布的计量资料采用Wilcoxon秩和检验。分类变量采用卡方检验。采用Spearman秩相关或Pearson相关系数检验变量间的关系。采用Kaplan-Meier法进行生存分析,组间比较采用log-rank检验。采用Cox比例风险回归模型计算风险比(HR)。所有统计分析均采用R(v.4.0.2)软件包进行,以p值<0.05为差异有统计学意义。所有数据均使用ggplot2 R软件包进行构建。
二、实验结果
1)eCAFs与跨癌症的ICB耐药性相关。
我们使用了来自TCGA数据库的数据,并进行了pancancer分析。结果显示,eCAF特征基因POSTN和FAP在乳腺癌(BRCA)、结肠腺癌(COAD)、食道癌(ESCA)、骨髓癌(LIHC)、肺腺癌(LUAD)、头颈部鳞状细胞癌(HNSC)等实体肿瘤中的表达水平明显高于正常组织(p值<0.001),结果如图1所示。eCAF特征基因POSTN和FAP在多种实体肿瘤中的表达也呈显著正相关(p-value<0.001),结果如图2所示。eCAF特征基因POSTN或FAP的高表达与高TIDE评分呈显著正相关,结果如图3所示。因此,eCAF亚群可能存在于多种实体肿瘤中。
2)eCAFs与GC中ICB反应不佳相关
我们首先在TCGA数据库中证实了eCAF亚群的存在,POSTN和FAP作为eCAF亚群的特征基因,在不同类型的胃癌中都存在于肿瘤间质区,在eCAF亚群中大量表达,结果如图4所示。在GC肿瘤组织切片中,有成纤维细胞样细胞,通过IF进行eCAF双特征基因阳性染色,结果如图5所示,结果显示POSTN表达与FAP呈显著正相关。此外,eCAF特征基因POSTN和FAP高表达的GC患者在TCGA-STAD的OS更短,结果如图6和表4所示。接着我们分析了表1在PUMCH接受免疫治疗的92例患者的ORR。POSTN(p-value<0.001)、FAP(p-value<0.05)和CD163的高表达与免疫治疗反应不佳呈正相关,结果如图7所示。这些结果表明,eCAF及其特征基因POSTN、FAP与不同癌症的ICB治疗疗效相关。
表4TCGA-STAD患者OS的单因素和多因素Cox分析
同时在TCGA-STAD研究了eCAF特征基因POSTN(FAP)和TIDE评分之间的关系。eCAF特征基因POSTN或FAP高表达与高TIDE评分呈正相关,表明对ICB的反应不佳,结果如图8所示。随后,我们分析了在表2中PUMCH接受免疫治疗的22例GC患者的ORR。eCAF特征基因POSTN(p值<0.001)或FAP(p值<0.05)高表达与ICB治疗反应不佳呈正相关,结果如图9所示。这些结果证实了eCAF亚群分布在间质区,并与胃癌患者ICB反应不良相关。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
Claims (10)
1.检测样本中eCAF细胞的试剂在制备免疫检查点阻断疗法治疗肿瘤疗效预测的产品中的应用。
2.根据权利要求1所述的应用,其特征在于,所述检测样本中eCAF细胞的试剂包括检测eCAF细胞活性的试剂、检测eCAF细胞分泌物的试剂、或检测eCAF细胞相关特征基因的试剂;
优选地,所述检测eCAF细胞活性的试剂包括以下方法中所使用的试剂,所述方法包括化学染色法、荧光染色法、克隆-集落形成实验、MTT比色法、XTT比色法、CKK-8比色法、Alamar Blue法、乳酸脱氢酶释放法、SRB法、ATP含量测定法、液体闪烁计数仪法、或流式细胞术;
优选地,所述检测eCAF细胞相关特征基因的试剂包括检测eCAF细胞相关特征基因POSTN、或FAP的试剂;
优选地,所述检测eCAF细胞相关特征基因POSTN、或FAP的试剂包括特异性识别POSTN或FAP基因的探针,或特异性扩增POSTN或FAP基因的引物,或特异性结合POSTN或FAP基因编码的蛋白的结合剂。
3.根据权利要求1所述的应用,其特征在于,所述检测样本选自器官或组织样本的固体组织,或活组织抽吸物;
优选地,所述检测样本为个体肿瘤组织。
4.根据权利要求1所述的应用,其特征在于,所述肿瘤为实体瘤;
优选地,所述实体瘤包括肺癌、脑癌、结肠癌、直肠癌、前列腺癌、乳腺癌、肝癌、肾癌、胃癌、子宫颈癌、头颈癌、卵巢癌、睾丸癌、垂体癌、食道癌、皮肤癌、胰腺癌或骨癌;
优选地,所述实体瘤为胃癌。
5.根据权利要求1所述的应用,其特征在于,所述产品包括试剂盒、芯片、试纸、系统中的一种或多种。
6.一种用于预测免疫检查点阻断疗法治疗肿瘤疗效的产品,其特征在于,所述产品包括检测样本中eCAF细胞的试剂;
优选地,所述检测样本选自器官或组织样本的固体组织,或活组织抽吸物;
优选地,所述检测样本为个体肿瘤组织;
优选地,所述产品包括试剂盒、芯片、试纸、系统中的一种或多种。
7.根据权利要求6所述的产品,其特征在于,所述肿瘤为实体瘤;
优选地,所述实体瘤包括肺癌、脑癌、结肠癌、直肠癌、前列腺癌、乳腺癌、肝癌、肾癌、胃癌、子宫颈癌、头颈癌、卵巢癌、睾丸癌、垂体癌、食道癌、皮肤癌、胰腺癌或骨癌;
优选地,所述实体瘤为胃癌。
8.根据权利要求6所述的产品,其特征在于,所述检测样本中eCAF细胞的试剂包括检测eCAF细胞活性的试剂、检测eCAF细胞分泌物的试剂、或检测eCAF细胞相关特征基因的试剂;
优选地,所述检测eCAF细胞活性的试剂包括以下方法中所使用的试剂,所述方法包括化学染色法、荧光染色法、克隆-集落形成实验、MTT比色法、XTT比色法、CKK-8比色法、Alamar Blue法、乳酸脱氢酶释放法、SRB法、ATP含量测定法、液体闪烁计数仪法、或流式细胞术;
优选地,所述检测eCAF细胞相关特征基因的试剂包括检测eCAF细胞相关特征基因POSTN、或FAP的试剂;
优选地,所述检测eCAF细胞相关特征基因POSTN、或FAP的试剂包括特异性识别POSTN或FAP基因的探针,或特异性扩增POSTN或FAP基因的引物,或特异性结合POSTN或FAP基因编码的蛋白的结合剂。
9.eCAF细胞的抑制剂在制备增强免疫检查点阻断疗法治疗肿瘤疗效的药物中的应用;
优选地,所述肿瘤为实体瘤;
优选地,所述实体瘤包括肺癌、脑癌、结肠癌、直肠癌、前列腺癌、乳腺癌、肝癌、肾癌、胃癌、子宫颈癌、头颈癌、卵巢癌、睾丸癌、垂体癌、食道癌、皮肤癌、胰腺癌或骨癌;
优选地,所述实体瘤为胃癌。
10.根据权利要求9所述的应用,其特征在于,所述抑制剂包括特异性的细胞骨架蛋白抑制剂、细胞凋亡诱导剂、细胞增殖抑制剂、或特异性杀伤剂。
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