CN116763808A - 地西他滨联合RRx-001在治疗胶质瘤中的应用 - Google Patents
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Abstract
本发明公开了地西他滨联合RRx‑001在治疗胶质瘤中的应用。本发明首先在体外细胞水平上采用了RRx‑001联合地西他滨用药方案，在胶质瘤细胞系应用后检测其CD47表达水平，证明活化TAM的通路。进而在原位立体注射胶质瘤的小鼠模型中联合用药，并在抑制胶质瘤的生长，改善小鼠生存期，改善巨噬细胞活性等方面与单药组和溶剂对照组小鼠相对比，发现联合用药可活化免疫微环境中的巨噬细胞，显著抑制胶质瘤的生长，改善胶质瘤小鼠生存期，且二者联用体现出良好的协同增效作用，具有巨大的临床转化潜力和良好的临床应用前景。
Description
技术领域
本发明涉及地西他滨联合RRx-001在治疗胶质瘤中的应用，属于生物医药技术领域。
背景技术
在国际癌症研究机构(International Agency for Research on cancer)2018年发布的全球癌症发病率和死亡率GLOBOCAN估计值中，脑胶质瘤年发病率为3-6.4/10万，约占所有中枢神经系统肿瘤的23.3％，占中枢神经系统恶性肿瘤的78.3％。
胶质瘤恶性程度高，虽然手术、放射治疗和化学治疗等手段近年来不断更新，国内外治疗指南中也纳入了免疫治疗及靶向治疗方面具有应用前景的临床试验结果，但是胶质瘤的综合治疗效果仍然不佳。胶质瘤高度抑制的免疫微环境或许是导致疗效不佳的原因之一。此外，由于胶质瘤免疫微环境中主要以TAM(肿瘤相关巨噬细胞)为主，而T细胞浸润少，使得在其他肿瘤的治疗中有确切疗效的CTLA-4、PD-1及PD-L1靶向免疫抑制剂在胶质瘤患者中应答率低。
肿瘤表面的CD47作为一种免疫检查点，与巨噬细胞表面的SIPR1结合后使其胞内段酪氨酸磷酸化，进而激活SHP-1/2(酪氨酸磷酸酶)，引起巨噬细胞肌球蛋白中磷酸化的酪氨酸去磷酸化，经级联反应抑制巨噬细胞变形和吞噬。RRx-001最初在航空航天工业中被发现，并由EpicentRx公司开发成药物，作为CD47/SIRPα抑制剂，该小分子药物能够激活TAM，使肿瘤微环境正常化，并增强实体瘤对标准疗法的敏感性。目前已经进入了胶质瘤治疗的II期临床试验。
近年来，RNA甲基化对基因表达的影响以及其在肿瘤的发生发展过程中起到的作用不断被揭示。m5C是RNA中较常见的一类甲基化修饰形式，可以由NSUN家族和DNMT2催化。其中NUSN家族包含6个具有NOL1/NOP2/Sun结构域的成员，各自对不同类型RNA进行5mC修饰，既往认为NUSN5的催化底物主要是rRNA，在胶质瘤中主要与细胞抵抗应激的功能有关——由于CpG岛的高甲基化，NSUN5在胶质瘤中低表达，引起rRNA上特定位点的甲基化不足，导致胶质瘤细胞中蛋白的总表达量降低，而一部分应激基因如NQO1的表达上调。有生信分析提示，NSUN5的缺失会增强肾透明细胞癌中巨噬细胞的浸润，但NSUN5是否影响胶质瘤免疫微环境中浸润的巨噬细胞功能尚不明确。
地西他滨(Decitabine，C8H12N4O4)通过抑制DNMT1降低细胞的DNA甲基化水平，恢复肿瘤细胞中抑癌基因的表达，目前在临床上用于治疗骨髓异常增生综合征，但其对胶质瘤是否也有治疗效果尚不清楚。
RRx-001是一款具有First-in-Class(FIC)潜质的小分子药物，具有多种作用机制，包括CD47-SIRPα靶向、RONS生成以及表观遗传调节。RRx-001可以对多种肿瘤微环境异常起到调节作用，包括：下调CD47-SIRPα，使肿瘤相关巨噬细胞(TAM)复极化，由抗炎症M2表型转为促炎症M1表型；通过表观遗传抑制活性激活抑癌基因，逆转化疗耐药；使肿瘤血管正常化以增加化疗药物渗透，产生代谢产物RONS导致肿瘤细胞坏死。临床II期研究数据显示，RRx-001在小细胞肺癌等实体肿瘤的治疗中具有良好的疗效和安全性，但其在胶质瘤中是否也有治疗效果尚不清楚。
发明内容
本发明的目的是：为了解决如何提高胶质瘤的疗效的技术问题，本发明提供了地西他滨联合RRx-001在治疗胶质瘤中的应用，本发明的结果表明，地西他滨联合RRx-001在胶质瘤中具有协同抗肿瘤的作用。
为达到解决上述问题的目的，本发明所采取的技术方案是提供地西他滨联合RRx-001在制备治疗胶质瘤的药物中的应用。
优选地，所述胶质瘤包括胶质母细胞瘤。
优选地，所述药物的剂型包括注射剂、片剂、粉剂、混悬剂、胶囊剂、丸剂或糖浆。
与现有技术相比，本发明的有益效果在于：
NSUN5是RNA m5C甲基化酶NSUN家族的成员，本发明通过RRx-001联合地西他滨用药能够以直接和间接两种途径下调胶质瘤CD47表达，进而活化免疫微环境中的巨噬细胞，抑制胶质瘤的生长，促进胶质瘤的清除，并经过动物体内实验表明可显著改善胶质瘤小鼠生存期，且二者联用体现出良好的协同增效作用，具有巨大的临床转化潜力和良好的临床应用前景。
附图说明
图1展示了NSUN5在胶质瘤中低表达，影响巨噬细胞吞噬活性；其中，a-c为从TISCH2公共数据库中提取的Glioma_GSE89567和Glioma_GSE141982两组数据集中分析了胶质瘤和癌旁组织中NSUN5的表达水平；“NS”：not significant，*p<0.05，**p<0.01，***p<0.001，****p<0.0001；d为用pHrodo染色的T98G、U937共培养细胞图像，具有代表性的phrodo阳性细胞用红色箭头标记；e为phrodo染色共培养细胞图像中phrodo阳性细胞的定量和统计分析；
图2展示了NUSN5通过下调胶质瘤β-catenin-CD47信号轴，促进巨噬细胞吞噬；其中，a为利用CGGA公共数据库中的mRNA-seq对不同级别胶质瘤中NSUN5与CTNNB1和CD47的相关性进行分析的结果展示；b-e分别为在胶质瘤细胞系U87MG，U251MG，T98G和GL261中过表达NSUN5后，使用Western blot检测β-catenin和CD47表达水平的结果展示；f为在胶质瘤细胞系T98G中使用两种shRNA敲低NSUN5后，使用Western blot检测β-catenin和CD47表达水平的结果展示；
图3展示了流式细胞术检测NSUN5对巨噬细胞的吞噬作用；其中，a-f分别为在胶质瘤细胞系U87MG，U251MG，T98G中过表达NSUN5后，使用RT-qPCR检测CTNNB1和CD47 mRNA水平的结果展示；g-h为共培养实验后，使用流式细胞仪检测巨噬细胞CFSE荧光强度，反映肿瘤过表达NSUN5前后巨噬细胞吞噬活性的变化；“NS”：not significant，*p<0.05，**p<0.01，***p<0.001，****p<0.0001；
图4展示了体外实验证明抑制DNMT1可上调NSUN5并引起下游改变；其中，a为在胶质瘤细胞系T98G细胞系中敲低DNMT1(使用两种shRNA)，使用Western Blot检测NSUN5、β-catenin、CD47表达水平的结果展示；b为使用不同浓度的5-氮杂胞嘧啶核苷(5-Azacytidine)处理胶质瘤细胞系U87MG，使用Western blot检测NSUN5、β-catenin、CD47表达水平的结果展示；
图5展示了联合使用RRx-001和地西他滨能够对小鼠胶质瘤具有治疗作用；其中，a为联合使用地西他滨和RRx-001的体外实验，对小鼠胶质瘤细胞系GL261分组处理，第一组为溶剂对照，第二组单用地西他滨，第三组单用RRx-001，第四组联合使用地西他滨(10μM)和RRx-001(10μM)；使用Western Blot检测NSUN5、β-catenin、CD47表达水平的结果；b为C57Bl/6小鼠原位成瘤后第7天起进行分组，第一组为单纯溶剂对照，第二组单用地西他滨(1mg/kg,i.p.)，第三组单用RRx-001(1mg/kg,i.p.)，第四组联合使用地西他滨(1mg/kg,i.p.)和RRx-001(1mg/kg,i.p.)，每三天给药一次；分别于成瘤后第7，14，17天进行luciferase成像；c为上述四组小鼠的Kaplan-Meier生存分析；d为上述四组小鼠的luciferase成像荧光强度统计，反映肿瘤体积；e为从上述四个小组中各取一只小鼠的脑组织制片并进行HE染色，虚线圈出部分为肿瘤；f为对上述小鼠的脑组织切片进行免疫荧光染色，统计F4/80和CD11b双阳性细胞，反映肿瘤中浸润的巨噬细胞数量；*P<0.05，**P<0.01，***P<0.001。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例
本实施例提供了地西他滨联合RRx-001在治疗胶质瘤中的应用：
(一)实验方法：
1、细胞培养及处理
人胶质瘤细胞系T98G、U87MG、U251MG、小鼠胶质母细胞瘤细胞系GL261均来自中国科学院细胞库(中国，上海)。人巨噬细胞细胞系U937由复旦大学附属妇产科医院慷慨提供。上述细胞的培养基为DMEM添加10％胎牛血清(黄)、100U/mL青霉素和100μg/mL链霉素。培养条件为5％CO2，37℃。DNMT1抑制剂地西他滨(1μM)、RRx-001(1μM)用于细胞处理。
2、公共数据库分析
The Tumor Immune Single Cell Hub 2(TISCH2，http://tisch.comp-genomics.org/)是一个关注肿瘤微环境(TME)的在线数据库，收集了27种癌症的76个肿瘤数据集，包含近200万个细胞的单细胞转录组谱。使用TISCH2数据库系统地研究了胶质瘤中TME的异质性。
3、RT-qPCR
使用SparkZol试剂(SparkJade，中国)提取细胞中的总RNA，然后取2μg总RNA使用III 1st Strand cDNA Synthesis Kit(Yeason，中国)进行逆转录。qPCR采用qPCR SYBR Green Master Mix(Yeason，中国)进行。使用2-ΔΔCt的计算方法对靶基因的相对表达水平进行三次重复的定量，β-Actin作为内源性对照用于标准化基因表达水平。基因的qPCR引物如下：
Human NSUN5(1128nt)-forward(SEQ ID NO：1)：
CGCTACCATGAGGTCCACTAC；
Human NSUN5(1341nt)-reverse(SEQ ID NO：2)：
GCATCTCGCACCACGTCTT；
Humanβ-actin(1128nt)-forward(SEQ ID NO：3)：
CTCCATCCTGGCCTCGCTGT；
Humanβ-actin(1341nt)-reverse(SEQ ID NO：4)：
GCTGTCACCTTCACCGTTCC；
Human CTNNB1(1128nt)-forward(SEQ ID NO：5)：
AAAGCGGCTGTTAGTCACTGG；
Human CTNNB1(1341nt)-reverse(SEQ ID NO：6)：
CGAGTCATTGCATACTGTCCAT；
Human CD47(1128nt)-forward(SEQ ID NO：7)：
CTCATCCATACCACCGGATCT；
Human CD47(1341nt)-reverse(SEQ ID NO：8)：
ACTTAAAGAAACAAGAGTGTGATGTG；
Mouseβ-actin(1128nt)-forward(SEQ ID NO：9)：
GATGTGGATCAGCAAGCAGGA；
Mouseβ-actin(1341nt)-reverse(SEQ ID NO：10)：
AGCTCAGTAACAGTCCGCCTA；
Mouse nsun5(1128nt)-forward(SEQ ID NO：11)：
CTGAAGCAGTTGTACGCTCTG；
Mouse nsun5(1341nt)-reverse(SEQ ID NO：12)：
CCCTTCCCCAGCAATAATTCAT；
Mouse ctnnb1(1128nt)-forward(SEQ ID NO：13)：
ATGGAGCCGGACAGAAAAGC；
Mouse ctnnb1(1341nt)-reverse(SEQ ID NO：14)：
TGGGAGGTGTCAACATCTTCTT；
Mouse cd47(1128nt)-forward(SEQ ID NO：15)：
TGGTGGGAAACTACACTTGCG；
Mouse cd47(1341nt)-reverse(SEQ ID NO：16)：
CGTGCGGTTTTTCAGCTCTAT。
4、蛋白质印迹实验(Western blot)
为了检测目标蛋白在细胞中的表达含量，Western blot实验被用于蛋白的验证。收集细胞并使用含1％cocktail的Western及IP细胞裂解液(碧云天，中国)裂解细胞、收集蛋白质，每组总蛋白30μg进行电泳。转膜后用5％的脱脂牛奶在室温下封闭膜1小时，并与抗靶蛋白的一抗在4℃孵育过夜。洗涤一抗后在室温下用抗兔(CST，7074S)或抗小鼠(CST，7076S)二抗孵育膜1h。洗涤二抗，使用化学发光ECL试剂盒(Tanon，中国)观察蛋白条带。主要抗体及其稀释度如下：
Beta actin(β-Actin内参抗体)：Proteintech，货号：66009-1-Ig，WB稀释比：1:5000；
NSUN5抗体：Proteintech，货号：15449-1-AP，WB稀释比：1:1000；
Beta catenin(β-catenin抗体)：Proteintech，货号：80488-1-RR，WB稀释比：1:5000；
CD47抗体：Proteintech，货号：66304-1-Ig，WB稀释比：1:2000；
DNMT1抗体：Proteintech，货号：24206-1-AP，WB稀释比1:1000；
5、小鼠原位成瘤实验
4～5周龄的雌性C57Bl/6小鼠购自上海SLAC实验动物有限公司(上海，中国)，并在生物安全2级(BL2)条件下饲养。将1x 106个小鼠胶质母细胞瘤GL261细胞立体定向植入小鼠纹状体(前囟外侧2.2mm，深2.5mm)原位成瘤。
6、体外吞噬分析
为了进行吞噬实验，U937细胞用100ng/ml phorbol myristate acetate(PMA)培养24小时，24孔细胞培养板中以每孔接种80000个细胞。计数T98G，以每孔40,000个细胞的密度与U937依照前述细胞培养条件共培养1.5小时。而后用PBS洗涤，在含有100mM pHrodo-红色琥珀酰亚胺酯(Thermo Fisher)和10μg/ml Hoechst33342(碧云天)的RPMI1640中孵育20分钟。PBS洗涤后用4％多聚甲醛在室温下固定15分钟。将固定细胞置于酸性缓冲液(8g/L氯化钠，0.2g/L氯化钾，0.254g/L醋酸钠和1.01g/L冰醋酸，pH＝4.0)中，倒置显微镜低倍镜下放大三个随机视野，计数具有红色荧光的巨噬细胞数量。
7、流式细胞术的吞噬作用分析
对胶质瘤细胞用羧基荧光素琥珀酰亚胺酯(CFSE)进行荧光标记。20分钟后，将CFSE标记的胶质瘤细胞(U87、U251、GL261)加入到先前接种到6孔板中的U937或腹部单核细胞中，在前述培养条件下共孵育1.5小时后收集细胞，并用1％ PFA染色。在流式细胞仪上通过FITC通道检测CFSE阳性巨噬细胞的相对量。
8、HE染色和免疫组化
组织在PBS稀释的4％多聚甲醛中固定过夜，并用石蜡包埋。将石蜡包埋的组织切成5μm厚的切片，脱蜡，再水合。使用0.01M柠檬酸盐(pH 6.0)进行热诱导抗原恢复。内源性过氧化物酶用3％过氧化氢在室温黑暗中封闭15min。非特异性结合用5％的血清在室温下阻断1小时。然后将载玻片在4℃稀释的一抗中孵育过夜。将载玻片与加入亲和素-生物素过氧化物酶复合物的二抗在室温下孵育1小时，用DAB试剂、苏木精染色。最后，使用直立显微镜系统(尼康)获得图像。免疫组化所用抗体如下：
CD11b抗体：Abcam，货号：133357，IHC稀释比：1:4000；
F4/80抗体：Cell Signaling Technology(CST)，货号：70076s，IHC稀释比：1:250。
(二)实验结果：
1、从TISCH2公共数据库中提取了Glioma_GSE89567和Glioma_GSE141982两组数据集，对两组数据集的胶质瘤和癌旁组织中NSUN5的表达水平进行了分析及对比，如图1a～c所示；体外吞噬分析结果如图1d～e所示，以上结果表明NSUN5在胶质瘤中低表达，影响巨噬细胞吞噬活性。
2、利用CGGA公共数据库中的mRNA-seq对不同级别胶质瘤中NSUN5与CTNNB1和CD47的相关性进行了分析，结果如图2a所示；在胶质瘤细胞系U87MG，U251MG，T98G和GL261中过表达NSUN5后，使用Western blot检测β-catenin和CD47表达水平，结果如图2b～e所示；在胶质瘤细胞系T98G中分别运用两种shRNA(自NSUN5的第214和741位起与原序列互补，分别记为shNSUN5#214、shNSUN5#741)敲低NSUN5后，使用Western blot检测β-catenin和CD47表达水平，结果如图2f所示；以上结果表明，NSUN5能够下调胶质瘤β-catenin-CD47信号轴，进而活化TAM。
3、分别在胶质瘤细胞系U87MG，U251MG，T98G中过表达NSUN5后，使用RT-qPCR检测CTNNB1和CD47 mRNA水平，结果如图3a-f所示；共培养实验后，使用流式细胞仪检测巨噬细胞CFSE荧光强度，反映肿瘤过表达NSUN5前后巨噬细胞吞噬活性的变化，结果如图3g-h所示；
4、在胶质瘤细胞系T98G细胞系中敲低DNMT1(运用两种shRNA，分别为自DNMT1的第2919和3072位起与原序列互补，分别记为shDNMR1#2919、shDNMT1#3072)，使用WesternBlot检测NSUN5、β-catenin、CD47表达水平，结果如图4a展示；使用不同浓度的5-氮杂胞嘧啶核苷(5-Azacytidine)处理胶质瘤细胞系U87MG，使用Western blot检测NSUN5、β-catenin、CD47表达水平，结果如图4b所示；以上结果表明，在体外细胞水平抑制DNMT1可上调NSUN5并引起下游改变。
5、联合使用地西他滨和RRx-001的体外实验，对小鼠胶质瘤细胞系GL261分组处理，第一组为溶剂对照，第二组单用地西他滨，第三组单用RRx-001，第四组联合使用地西他滨(10μM)和RRx-001(10μM)；使用Western Blot检测NSUN5、β-catenin、CD47表达水平，结果如图5a所示；C57Bl/6原位成瘤小鼠进行给药处理，成瘤后第7天起进行分组，每隔3天给药一次，第一组为单纯溶剂对照，第二组单用地西他滨(1mg/kg,i.p.)，第三组单用RRx-001(1mg/kg,i.p.)，第四组联合使用地西他滨(1mg/kg,i.p.)和RRx-001(1mg/kg,i.p.)，分别于第7，14，17天进行luciferase成像，结果如图5b所示；四组小鼠分别进行Kaplan-Meier生存分析，绘制的生存曲线如图5c所示；四组小鼠的luciferase成像进行荧光强度统计，用于反映肿瘤体积，结果如图5d所示；从上述四个小组中各取一只小鼠的脑组织制片并进行HE染色，虚线圈出部分为肿瘤，结果如图5e所示；对上述小鼠的脑组织切片进行免疫荧光染色，统计F4/80和CD11b双阳性细胞，用于反映肿瘤中浸润的巨噬细胞数量，结果如图5f所示；以上结果表明，地西他滨和RRx-001联合使用比单独使用地西他滨或RRx-001组在抑制胶质瘤的生长、延长小鼠生存期、改善巨噬细胞活性、改善胶质瘤免疫微环境等方面具有更好的效果。
上述实施例仅为本发明的优选实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (3)
1.地西他滨联合RRx-001在制备治疗胶质瘤的药物中的应用。
2.如权利要求1所述的应用，其特征在于，所述胶质瘤包括胶质母细胞瘤。
3.如权利要求1或2所述的应用，其特征在于，所述药物的剂型包括注射剂、片剂、粉剂、混悬剂、胶囊剂、丸剂或糖浆。
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