MSI-H/S结直肠癌差异表达分子的筛选及其在免疫治疗中的意义
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摘要
结直肠癌是常见的恶性肿瘤之一，全世界范围内结直肠癌的发病率，在男性及女性中分别处于恶性实体肿瘤的第3位和第2位[1]。最新研究表明2012年美国结直肠癌新发展病例数和死亡率均居第3位[2]。由于结直肠癌发病隐匿，初次就诊的患者近30%发现肿瘤转移，目前对于转移性结直肠癌的患者，化疗仍然是这部分患者的主要治疗手段，但由于肿瘤患者的个体差异性及不同患者的肿瘤异质性，尤其是近年来肿瘤细胞耐药现象的不断出现，使得结直肠癌的预后疗效并不显著。因此，如何提高结直肠癌患者的临床疗效一直是肿瘤治疗领域的核心问题之一。分析肿瘤患者预后的相关生物学标志物，探索对某种治疗方式敏感患者的共同特征，对不同患者采用不同的治疗方式，即肿瘤的个性化治疗，对提高结直肠癌患者的临床疗效，具有重大意义。目前治疗的个性化已成为目前肿瘤治疗学的一个新的特征与发展方向[3]。
微卫星不稳定性（Microsatellite instability, MSI）是结直肠癌重要的发病机制之一[4]，MSI作为结直肠癌的预后标志的研究，日益受到关注[5]。研究发现高频微卫星不稳定（High-frequency microsatellite instability, MSI-H）的结直肠癌患者，其与低频微卫星不稳定（Low-frequency microsatellite instability, MSI-L）及稳定型（Microsatellite stability, MSS）的相比，病理特点主要是肿瘤多位于近端结肠、黏液腺癌多见、分化较差，但却有相对较好的预后[6‐9]。对于这类MSI-H特殊的结直肠癌患者深入研究后发现，其体内由于处于基因外显子区域的微卫星序列反复突变，进而引起蛋白质的读框突变，最终导致机体产生异常的多肽片段，这些异常多肽片段更易激发机体的抗肿瘤免疫应答反应[10‐13]。研究还发现，其肿瘤微环境中免疫相关的调控因素更有利激发免疫系统，进而促进了免疫系统功能发挥[14‐20]。我们在以树突状细胞瘤苗（APDC）联合化疗治疗转移性结直肠癌的II期临床研究中，对部分临床样本进行了微卫星状态的分析，在初步筛选出的18例MSI-H患者，其中APDC联合化疗组中10例MSI-H，其中8例的临床疗效为部分缓解(Partial response，PR)，2例为轻微缓解(Minimal response，MR)， PFS为6.0月，而对照组（单独化疗）8例MSI-H的患者仅为2PR，2NC，4PD，PFS为2.7月，结果也提示：接受树突状细胞治疗性疫苗组中的MSI-H的结直肠癌患者可能获得高于单独接受化疗的临床疗效。这些都提示了MSI-H这类特殊的肿瘤患者可能由于其肿瘤本身的免疫原性更强，其体内的免疫系统中各种调节因素的共同作用，最终改善了其预后。
但现阶段由于缺乏有效的MSI结直肠癌细胞及动物模型，使得目前国际上大部分对于MSI-H结直肠癌的免疫学特性及其治疗的研究只是局限在临床样本的研究，不能对其生物学特性、与治疗之间的关系及其机理进行深入研究。
本课题旨在通过建立微卫星稳定及不稳定的结直肠癌细胞模型，筛选重要的差异分子，探讨其与免疫治疗的内在机理及关系，为以DC为基础的免疫治疗晚期结直肠癌的Ⅲ期临床的开展提供科学的数据。
本研究共分四个部分进行：
第一部分：MSI-H/S结直肠癌细胞模型的建立
肿瘤异质性是恶性肿瘤重要特征之一，其可以表现在肿瘤分化水平及肿瘤功能水平上的差异，也可以表现在具有异质性的抗原表达或出现不同生物特性细胞亚群。我们根据肿瘤的异质性特征，利用单克隆化技术，对文献已报道过的MSI-H的结直肠癌细胞进行单克隆化，通过荧光PCR反应及GENE SCAN技术对PCR产物进行扫描后，确定每个单克隆细胞株的微卫星状态，最终筛选出MSS的细胞群体，从而在体外建立同一细胞来源的MSI-H/S细胞模型。这部分研究对5株文献已经报道为MSI-H的结直肠癌细胞（分别是LS174t、LS180、HCT15、HCT116、SW48）进行了单克隆化，最终长成集落的单克隆细胞株数分别是LS174t为71株、LS180为81株、HCT15为25株、HCT116为65株、SW48为37株。通过荧光PCR技术，对最终筛选出的单克隆细胞株进行MSI分型后，确定为MSS的细胞株数并进行标记，标记方法为：不同批次单克隆化+序号，如LTE-1表示某批次单克隆化的第1号单克隆细胞株，其中LS174t为21株，标记为LTE-1，2，4，5，6，7，8，9，10，11，12; LTT-1，2，3，4，5，6，7，8，11，12，最终将这21株细胞群体合命名为LS174t-MSS，将其余50株细胞群体合命名为LS174t-MSI-H；LS180为11株，标记为HSO-1，2，10，12；HSW-1，2，3，6，8，9，11，最终将这11株细胞群体合命名为LS180-MSS，其余70株细胞群体合命名为LS180-MSI-H；HCT15为12株，标记为LSF-1，2，3，4，5，6，7，8，14，15，17，18，最终将这12株细胞群体合命名为HCT15-MSS，其余13株细胞群体合命名为HCT15-MSI-H；HCT116为10株，标记为HF-2，3，4，5，13，14，16，22，23，25，最终将这10株细胞群体合命名HCT116-MSS，其余55株细胞群体合命名为HCT116-MSI-H；SW48为10株，标记为SWS-2，6，10，12，14，15；SWT-1，2，3，4，最终将这10株细胞群体合命名为SW48-MSS，其余27株细胞群体合命名为SW48-MSI-H，从而在体外建立了同一细胞来源的MSI-H/S细胞模型，通过细胞形态学的研究发现MSI-H/S的细胞株存在差异，特别是LS180模型中MSI-H与MSS细胞形态差异明显。同时通过药敏实验研究，其结果与相关文献报道一致[24‐27]，即两种不同微卫星状态的细胞株其对于常用化疗药物5-氟尿嘧啶（5-Fu）及伊立替康（CPT）的敏感性存在差异，其中模型中MSI-H细胞对于5-Fu更加耐受，而对于CPT却更加敏感。
通过第一部分的研究我们从体外建立同一细胞来源的MSI-H/S的细胞模型，通过微卫星分析及药敏实验验证了模型中两类细胞的差异性，为下一步的研究奠定了基础。
第二部分：MSI-H/S结直肠癌细胞模型中差异表达分子的筛选及验证
肿瘤的发生、发展和转移与其本身的肿瘤生物学特性有着密切的联系[28]。研究MSI-H结直肠癌的发生除了与许多重要抑癌基因的突变相关，如KRAS[29]，BRAF[30]等，还与细胞生物学功能相关基因的改变有关，如DNA修复基因MRE11[31]、hRAD50[32]及TGF-β[33]。这部分研究中我们在体外建立MSI-H/S的结直肠癌细胞模型的基础上，通过基因、蛋白芯片筛选其中重要的差异分子，并最终通过qPCR，流式技术及Western方法验证差异分子的表达情况。1、MSI-H/S结直肠癌细胞模型中差异表达基因的分析：我们首先对三组MSI-H/S结直肠癌细胞模型（HCT116，LS180，LS174t）进行基因表达谱芯片（Whole-Genome gene array, Affymetrix）的分析，所有样品中每个基因（probeset）的信号值均采用Robust Multichip Analysis（RMA）归一化方法，采用SAM（Significance Analysis of Microarrays）软件进行差异表达基因的筛选。上调大于2倍的基因，筛选标准为：q-value≤5%；Fold Change≥2；下调大于2倍的基因，筛选标准为：q-value≤5%； Fold Change≤0.5；挑选出的差异表达基因在MAS系统中进行了Pathway统计分析。差异表达基因所涉及到具有显著性意义的pathway分类，P value反映此pathway在实验结果中的重要性，P value越低提示与Pathway更相关。结果显示在LS180-MSI-H与LS180-MSS模型中差异表达的基因有2216个，其中LS180-MSI-H细胞中表达上调的基因1456个，对这些上调的基因进行通路分析后发现，其最为相关的通路是细胞因子与细胞因子受体作用通路，共33个基因，包括IL1A、IL1B、 IL7、IL8、 CXCL1、 CXCL2、CXCL3、CXCR4、 CXCL5、 CXCL11、 CCL20、 VEGFA等；在LS174t-MSI-H与LS174t-MSS模型中有4206个，其中LS174t-MSI-H细胞中表达上调的基因3446个，对这些上调的基因进行通路分析后发现，其最为相关的通路是mRNA的多聚腺苷酸化通路，共5个基因，包括CPSF3、PAPOLA、CSTF3、PABPN1、CSTF2；在HCT116-MSI-H与HCT116-MSS模型中有989个，其中HCT116-MSI-H细胞中表达上调的基因721个，对这些上调的基因进行通路分析后发现，其最为相关的通路是细胞周期相关通路，共5个基因，包括CDK6、CCND1、SMAD4、CDKN2A、CCNE1。对三组芯片的结果进行聚类分析后发现，其共同表达上调的基因有7个，分别是Fas, Versican（多能聚糖），Ezrin（一种骨架蛋白），Translocated promoterregion（TPR），Chromosome14open reading frame139(C14orf139)，Similar toankyrin repeat domain20family, member A1(LOC727770)，Chromosome5openreading frame24(C5orf24)。
2、MSI-H/S结直肠癌细胞模型中细胞因子蛋白差异表达的分析：肿瘤微环境中的细胞因子在肿瘤发生、发展及转移过程中发挥了重要的作用[34‐37]。我们通过基因表达谱芯片的结果分析发现许多重要的细胞因子在MSI细胞中mRNA水平的表达上调现象，这些细胞因子在蛋白水平上的表达是否也有差异？我们通过三组MSI-H/S结直肠癌细胞模型（HCT116，LS180，LS174t）进行Human Cytokine Array细胞因子的蛋白芯片分析（Raybiotech），通过加样，孵育，Cy3-链霉亲和等过程，最后采用激光扫描仪Axon GenePix扫描信号，采用Cy3或者绿色通道（激发频率=532nm），将所有样品的数据取平均值，做归一化处理，根据各因子的标准曲线计算出各样品中各因子的浓度值。经3次重复性验证实验来确定最终的细胞浓度。结果及说明如下：上调因子：取比值大于1.5的因子；下调因子：取比值小于0.66的因子。结果分析后发现，LS180-MSI-H与LS180-MSS模型，LS180-MSI-H细胞中许多细胞因子表达上调，如炎性因子IL-8、TIMP-2，生长因子BMP-4、BMP-5、AR、GH、GDF-15、IGFBP-4、OPG等，趋化因子CCL28、CCL20、CXCL10、CXCL5，其中五种细胞因子的表达与基因表达谱的分析结果一致，分别是IL-8、TIMP-2、GDF-15、CXCL5、CCL20；LS174t-MSI-H与LS174t-MSS模型，LS174t-MSI-H细胞中也有许多细胞因子表达上调，如炎性因子TIMP-1，生长因子IGFBP-2、IGFBP-3，趋化因子GCP-2、TECK，其中两种细胞因子的表达与基因表达谱的分析结果一致，分别IGFBP-2、IGFBP-3；在HCT116-MSI-H和HCT116-MSS模型，HCT116-MSI-H细胞中，炎性因子TIMP-1，生长因子IGFBP-6、BMP-4、AR，趋化因子GCP-2、6Ckine；都有不同程度的表达上调。
3、多能聚糖（Versican）在MSI-H/S结直肠癌细胞模型中的表达分析验证：我们已经通过基因表达谱芯片分析，发现许多重要的免疫相关的因素在MSI-H细胞中mRNA水平的表达上调现象，这些因素mRNA水平是否在与其蛋白表达水平相一致呢？首先我们关注一种多能聚糖Versican，其是胞外基质重要的组成部分，参与体内的炎症反应，其受多种细胞因子的调控，与肿瘤的增殖及转移相关[38‐41]。其在三组模型中基因表达谱芯片结果分析发现，其mRNA水平均表达上调，通过qPCR技术进一步发现了其在LS180，LS174t，HCT116模型中，MSI-H与MSS相比，分别增长了59倍、4.5倍、7.4倍。这些胞外基质（如多能聚糖）受到多种细胞因子的调控，与肿瘤的转移关系密切[42]。
4、Fas表达在MSI-H/S结直肠癌细胞模型中的分析验证：我们通过对三组模型进行基因表达谱芯片结果还发现，在MSI-H细胞中Fas分子的mRNA表达水平均有上调的现象。Fas-FasL是免疫系统发挥效应的一个重要的杀伤机制，通过效应细胞表面的FasL与靶细胞的Fas结合，可以启动靶细胞的caspase细胞转导途径，诱导靶细胞的凋亡[43]。通过流式细胞仪检测三组模型中Fas表达水平，在LS180-MSI/MSS，LS174t-MSI/MSS，HCT116-MSI/MSS中分别为48.3%/29.9%；80.4%/70.2%；68.7%/48.6%。凋亡相关基因Fas在MSI-H中的表达上调，及其对于效应细胞敏感性的深入研究，对MSI-H患者预后机制的探讨提供了重要的研究方向。
5、肿瘤抗原表达在MSI-H/S结直肠癌细胞模型中的分析验证：我们通过对三组模型进行基因表达谱芯片结果还发现，MSI-H细胞中许多肿瘤抗原相关基因表达上调的现象。如癌睾丸抗原（CTAG）、前列腺癌相关抗原（PAGE）、黑色素瘤相关抗原（MAGE），其中LS180-MSI-H与LS180-MSS相比，CTAG（1NY-ESO-1）、CTAG2（NY-ESO-2）、MAGE-A4的mRNA水平分别上调219倍、137倍、36倍；LS174t-MSI-H与LS174t-MSS相比，PAGE1、MAGE-A12的mRNA水平分别上调26倍、4倍；HCT116-MSI-H与HCT116-MSS相比，MAGE-B1的mRNA水平上调5倍。肿瘤的免疫原性是免疫系统能否发挥效应作用的关键所在，其已经成为肿瘤免疫领域的研究热点之一。我们通过qPCR技术及流式分析也证实了LS180模型中，MSI-H细胞中NY-ESO-1、NY-ESO-2、MAGE-A4的表达上调现象，同时通过Western分析也证实了MSI-H细胞中NY-ESO-2的表达上调现象；同时利用qPCR技术证实了LS174t模型中MAGE-3、MAGE-12的表达上调现象。我们还通过流式技术进一步分析了10株结直肠细胞（7株MSI-H，3株MSS）NY-ESO-1、NY-ESO-2、MAGE-A4的表达情况，结果也显示MSI-H结直肠细胞其表达的NY-ESO-1、NY-ESO-2、MAGE-A4平均水平高于MSS结直肠癌细胞。第三部分：MSI-H/S结直肠癌免疫治疗预后相关机制探讨
通过前两部分的研究，我们已经发现在MSI-H细胞中Fas表达上调及肿瘤抗原表达上调的现象，但由于MSI缺乏有效的模型，其预后机制并不明确。这部分研究主要从这两方面着手研究其预后的相关机制。
1、Fas在MSI-H/S结直肠癌细胞模型中的表达：我们首先通过Fas激动剂介导模型中MSI-H与MSS发生凋亡，检测两者发生凋亡的差异情况。对LS180-MSI-H/MSS、LS174t-MSI-H/MSS、HCT116-MSI-H/MSS三组模型的细胞加入Fas激动剂后，通过流式技术分析凋亡细胞的比例结果显示，MSI-H中凋亡细胞的比例高于MSS，同时还通过检测细胞活性的方法也发现，MSI-H中活性细胞比例低于MSS。这一结果证实了MSI-H的细胞由于其表面Fas分子表达的水平更高，使其可能对于机体免疫系统中效应细胞更为敏感，增强MSI-H细胞对杀伤的敏感性。
2、MSI-H细胞表达肿瘤抗原体外诱导人外周血淋巴细胞的特异性免疫应答：我们通过NY-ESO-1、NY-ESO-2、MAGE-A4的多肽（均为HLA-A2限制性）分别致敏的HLA-A2阳性的健康志愿者的DC，体外刺激其同体的淋巴细胞，分析其产生特异性免疫应答的情况，结果发现NY-ESO-1、NY-ESO-2、MAGE-A4的多肽三组的淋巴细胞经丝裂霉素处理后的LS180-MSI-H、LS180-MSS细胞及OVA多肽在体外再刺激后，IFN-γ ELISPOT结果显示三组经LS180-MSI-H刺激后所产生的表达IFN-γ的淋巴细胞数目均显著高于LS180-MSS刺激组及OVA刺激组（P＜0.05），同时体外的杀伤实验结果也显示NY-ESO-1、NY-ESO-2、MAGE-A4的多肽致敏的DC刺激后的淋巴细胞其对于靶细胞LS180-MSI-H的杀伤能力显著高于LS180-MSS及对照组T2细胞(P＜0.05)，而OVA致敏的DC及未致敏DC刺激的淋巴细胞杀伤三种靶细胞的能力没有显著差异，以上实验结果提示LS180-MSI-H细胞所表达的肿瘤抗原多肽致敏的DC诱导出多肽特异性的CTL，更有利于CTL对特异性表达这些肿瘤抗原的LS180-MSI-H细胞的杀伤作用。
3、MSI-H细胞表达肿瘤抗原诱导小鼠脾淋巴细胞特异性免疫应答：同时我们还用NY-ESO-1、NY-ESO-2、MAGE-A4的多肽致敏的HLA-A2.1/Kb转基因小鼠的BMDC去免疫同系小鼠，发现此免疫能诱导HLA-A2.1/Kb转基因小鼠产生抗原特异性免疫应答，我们通过NY-ESO-1、NY-ESO-2、MAGE-A4的多肽分别致敏HLA-A2.1小鼠DC后免疫HLA-A2.1小鼠，获得其脾淋巴细胞，三组的小鼠脾细胞经丝裂霉素处理后的LS180-MSI-H、LS180-MSS细胞及OVA多肽体外再刺激后，IFN-γ ELISPOT结果显示三组经LS180-MSI-H刺激后所产生的表达IFN-γ的淋巴细胞数目均显著高于LS180-MSS刺激组及OVA刺激组（P＜0.05）同时体外的杀伤实验结果也显示NY-ESO-1、NY-ESO-2、MAGE-A4的多肽致敏的DC刺激后的脾淋巴细胞其对于靶细胞LS180-MSI-H的杀伤能力显著高于的LS180-MSS及对照组T2细胞(P＜0.05)，而OVA致敏的DC及未致敏DC刺激的淋巴细胞杀伤三种靶细胞的能力没有显著差异。以上结果提示LS180-MSI-H细胞所表达的肿瘤抗原多肽致敏的DC诱导出多肽特异性的CTL，更有利于CTL对特异性表达这些肿瘤抗原的LS180-MSI-H细胞的杀伤作用。
4、MSI-H细胞肿瘤抗原致敏DC体内抗肿瘤效应：6-8周龄的雌性裸鼠，每组8只，接种LS180-MSI-H肿瘤细胞，剂量为2×106细胞/鼠，5天后进行脾细胞尾静脉回输至荷瘤裸鼠体内，数量为1×108细胞/200μl·鼠，观察并记录肿瘤生长情况及裸鼠存活情况。实验结果显示NY-ESO-1和NY-ESO-2多肽抗原致敏的DC诱导的脾细胞抑瘤效果明显强于对照OVA组（P＜0.05），观察至90天，NY-ESO-1组还有1只仍未长出肿瘤， NY-ESO-2组还有2只仍未长出肿瘤，而对照组的荷瘤小鼠在37-50天内均已死亡。这一实验结果提示，LS180-MSI-H肿瘤细胞表达的肿瘤抗原可能在诱导机体产生特异性的免疫应答过程中，发挥了重要的作用。
第四部分：不同微卫星状态下肿瘤抗原表达差异的验证及对于结直肠癌患者预后的影响
前面部分的研究均证实了在体外建立的模型，其中在LS180-MSI-H/MSS、LS174t-MSI-H/MSS、HCT116-MSI-H/MSS中，MSI-H细胞表达的肿瘤相关抗原NY-ESO-1、NY-ESO-2、MAGE-A4其mRNA及蛋白水平均表达上调的现象，在这部分研究中，我们通过研究临床患者的石蜡组织样本肿瘤相关抗原的表达情况，进一步验证其在MSI-H中的表达，及与生存时间的关系。
在本部分的研究中我们收集了2005-2009年186例临床样本，其中104例来源于江苏省南京中医院，55例来源于上海市新华医院，22例来源于上海长海医院，5例来源于上海长征医院，通过微卫星分型分析，确定其微卫星的状态，其中123例MSS，63例MSI-H患者，采用免疫组化的方法分析了其肿瘤组织中NY-ESO-1、NY-ESO-2的表达情况，结果发现其中NY-ESO-1表达阳性的比例在MSI-H中为57.14%，MSS中的43.09%；NY-ESO-2表达阳性的比例在MSI-H中为61.90%，显著高于MSS中的38.21%（P=0.0022）。
采用回顾性分析不同微卫星状态下NY-ESO-1的表达差异对于患者生存时间的影响。在MSI-H组中NY-ESO-1表达阳性的患者三年存活率为74.18%，而NY-ESO-1表达阴性的患者三年存活率为55.71%（P=0.2133）。
同时回顾性分析不同微卫星状态下NY-ESO-2的表达差异对于患者生存时间的影响，在MSI-H组中NY-ESO-2表达阳性的患者三年存活率为80.96%，显著高于NY-ESO-2表达阴性的患者三年存活率46.05%（95%CI,0.08-0.62；P=0.0109），生存分析结果显示在MSI-H组中NY-ESO-2表达阳性的患者其三年生存期显著高于NY-ESO-2表达阴性的患者（P=0.0044）。
这部分结果发现MSI-H组中NY-ESO-2肿瘤抗原的表达显著高于MSS组，与细胞模型中的现象一致，MSI-H患者表达NY-ESO-2肿瘤抗原其三年生存期显著高于不表达此肿瘤抗原的患者，以上实验结果提示：MSI-H的患者可能由于其表达NY-ESO-2的肿瘤抗原更易激发体内免疫系统对其进行杀伤作用，从而改善了其预后。
结论：
1、根据肿瘤的异质性的特征，利用单克隆化技术，从文献已报道为MSI-H的细胞（LS174t、LS180、HCT15、HCT116、SW48）中筛选出MSS的细胞群体，通过微卫星分型及药敏实验证实了两者的差异，从而建立了同一细胞来源的MSI-H/S结直肠癌细胞模型。
2、利用基因、蛋白芯片技术对三组MSI-H/S模型（LS174t、LS180、HCT116）进行了分析，筛选出Fas，多能聚糖分子及肿瘤抗原分子（NY-ESO-1、NY-ESO-2、MAGE-A4）重要分子的表达差异，mRNA水平证实了多能聚糖表达的上调；同时mRNA，蛋白质水平进一步证实了在模型中的MSI-H细胞表面Fas、NY-ESO-1、NY-ESO-2、MAGE-A4分子表达上调的现象。
3、MSI-H细胞表面肿瘤抗原（NY-ESO-1、NY-ESO-2、MAGE-A4）显著高于MSS细胞，其能诱导机体产生更强的免疫应答， MSI-H的细胞由于其表面Fas分子表达的水平更高，使其可能对于机体免疫系统中效应细胞的杀伤更为敏感。
4、通过免疫组化方法分析186例（123MSS,63MSI-H）结直肠癌患者的肿瘤组织样本的肿瘤抗原表达情况，发现肿瘤抗原NY-ESO-2表达阳性的比例在MSI-H的表达显著高于MSS，在MSI-H组中NY-ESO-2表达阳性的患者三年存活率显著高于NY-ESO-2表达阴性的患者存活率，生存分析结果显示在MSI-H组中NY-ESO-2表达阳性的患者其三年生存期显著高于NY-ESO-2表达阴性的患者。
Colorectal cancer (CRC) is one of the most common solid tumors in the worldand becomes the one of major cause of cancer-related death. Though higher responserate have been achieved with the improvement in pharmaceutical strategies over thepast decades, the relatively high resistance to systemic chemotherapy and the geneticheterogeneity of colorectal cancer have lead to poor prognosis and unfavorableclinical outcomes. Almost50%of the patients died as the disease progresses.Therefore, it is necessary to develop novel strategies for the treatment of metastaticcolorectal cancer. Personalized medicine, which refers to the tailoring of medicaltreatment to the individual characteristics of each patient, has been verified to beeffective. Examples provided by Herceptin in breast cancer and Gleevec in chronicmyeloid leukaemia (CML) have long carried the mantle for individualized therapy.
Microsatellite instability (MSI) status is a potential prognostic factor in CRC,patients with high frequency MSI (MSI-H) have unique characteristics compared withmicrosatellite stability (MSS) cancers, such as proximal anatomic location and severeinflammatory cell infiltration. Several studies have also indicated that patients withMSI-H achieve better prognosis compared with MSS CRCs. In our previous study，tumor samples obtained at baseline from101patients, who were selected from arandom phase II clinical trial of sequential administration of OXA+5-Fu and dendriticcell (DC) vaccine in treatment of metastatic colorectal cancer, were suitable for MSIstatus analysis. Of them60patients received sequential administration ofOXA+5-Fu/LV and DC vaccine (Group A),41received OXA+5-Fu/LV (Group B)only. There were18MSI-H patients, of them,10MSI-H in group A and8MSI-H ingroup B. The results indicated that patients with MSI-H in Group A achieved80%(8/10) objective clinical response, while2patients achieved25%(2/8) objectiveclinical response in Group B.
However, the underlying mechanisms remain to be unidentified. Our researchwas intended to identify the biological features of CRCs with MSI-H, so as to elucidate the underlying mechanisms for the improved prognosis in CRCs withMSI-H. Our major findings are summarized in four parts as followed:
PartⅠ: Establishment of the colorectal cancer cell models with MSI-H and MSSin vitro.
Five colorectal cancer cells with MSI-H（LS174t, LS180, HCT15, HCT116,SW48) were obtained for isolating their corresponding MSS cells according to tumorcell heterogeneity via cloning. The multiple cell clones of each colorectal cancer cellline were obtained by serial dilutions and plating in96-microwell plates. The numberof cell clones of LS174t, LS180, HCT15, HCT16, SW48was71,81,25,65,37,respectively. Genomic DNA was extracted from all separate cell clones. MSI at agiven microsatellite locus was detected by comparison of the allele patterns ofmultiple separate cell clones of each cell line. The microsatellite markers included theBAT25, BAT26, D2S123, D17S250, D5S346. The numbers of different allele patternsfor each microsatellite marker in a given cell line was scored. If different alleles of agiven microsatellite marker were detected in the separate cell clones of a given cellline, the locus was then scored as MSI-positive, and if no different alleles weredetected, then it was MSS. When two or more of the five microsatellite markersshowed MSI, based on the NCI criteria, MSI-high was scored. We isolated multiplecolorectal cancer cells with MSS status, the number of MSS cells in LS174t, LS180,HCT15, HCT16, SW48was21,11,12,10,10and named them LS174t-MSS,LS180-MSS, HCT15-MSS, HCT16-MSS, SW48-MSS, respectively. The rest of celllines were named LS174t-MSI-H, LS180-MSI-H, HCT15-MSI-H, HCT16-MSI-H,SW48-MSI-H, respectively. Finally we established the colorectal cancer cell modelswith MSI-H and MSS in vitro. We detected the different morphological features inMSI-H/S cell models, especially in LS180cell model. We also detected the differentsensitivity of the established colorectal cancer cell models to5-Fu and CPT, andfound that MSI-H cells were more resistant to5-Fu and more sensitive to CPT, inconsistence with previous reports.
PartⅡ: Identification of differentially expressed molecules in MSI-H/S colorectalcancer cell models.
We profiled gene expression in3pairs of MSI-H/S colorectal cancer cell models,consisting of LS180-MSI-H, LS174t-MSI-H, HCT116-MSI-H, and LS180-MSS,LS174t-MSS, HCT116-MSS, and explored the molecular variation in MSI-H andMSS cells through Whole-Genome gene array (Affymetrix). We identified2216,3446,989genes in LS180, LS174t, HCT116cell model, respectively, the expressionof which were significantly different between MSI-H and MSS groups. Mostly relatedpathway about the1456up-regulated genes in LS180-MSI-H is cytokine-cytokinereceptor interaction, in LS174t-MSI-H is polyadenylation of mRNA pathway, and inHCT116MSI-H is cell cycle pathway. Cluster analysis of the up-regulated genes inthree MSI-H cells showed that as few as7genes was up-regulated in MSI-H,including Fas, Versican, Ezrin, translocated promoter region (TPR), chromosome14open reading frame139(C14orf139), similar to ankyrin repeat domain20family,member A1(LOC727770) and chromosome5open reading frame24(C5orf24).
We investigated the protein variation in MSI-H and MSS cells via HumanCytokine Antibody Array (RayBiotech). We found many up-regulated cytokines inMSI-H cells compared with MSS, such as elevated IL-8, TIMP-1, TIMP-2, BMP-4,BMP-5, AR, GH, GDF-15, IGFBP-4, CCL28, CCL20, CXCL10, CXCL5and so on inLS180-MSI-H cells. The mRNA levels of IL-8, TIMP-2, GDF-15, CXCL5andCCL20were consistently upregualated in LS180-MSI-H cells with gene array; withIGFBP-2and IGFBP-3upregulated in LS174t-MSI-H cells.
FACS analysis of Fas expression in three MSI-H/S models indicated48.3%Faspositive cells in MSI-H as compared with29.9%in MSS in LS180cell model;80.4%in MSI-H as compared with70.2%MSS in LS174t cell model, and68.7%MSI-H ascompared with48.6%MSS in HCT116cell model.
We also found up-regulated tumor associated antigen genes in MSI-H cellswithin three MSI-H/S models, including cancer/testis antigen (CTAG), melanomaantigen family A (MAGE-A) and P antigen family. CTAG-1(NY-ESO-1)，CTAG2(NY-ESO-2) and MAGE-A4were increased in LS180-MSI-H cells by qPCR and FACS analysis. QPCR analysis also revealed up-regulated MAGE-3and MAGE-12in LS174t-MSI-H cells, as well as up-regulated Versican in all three MSI-H/S cellmodels.
FACS analysis of the expression of NY-ESO-1，NY-ESO-2，MAGE-A4in10colorectal cancer cell lines (7MSI-H,3MSS), in which MSI status have been perviousascertained, showed that the average expression of NY-ESO-1， NY-ESO-2，MAGE-A4is higher in MSI-H than MSS cells.
Part Ⅲ: Potential mechannism analysis for better prognosis of MSI-H CRC
We measured Fas-agonist induced apoptosis in MSI-H cells by FACS analysis,and found that the percentage of Annexin and PI double positive cells is higher inMSI-H than MSS cells. The cell viability is lower in MSI-H than MSS cells via cellcounting CCK8-assay. Thus, the elevated expression of Fas in MSI-H may enhancethe sensitivity to CTLs.
To explore whether DCs pulsed with antigen specific peptides were capable ofinducing peptide-specific CTLs in vitro. PBMCs from HLA-A2positive volunteerwere separated by density gradient centrifugation using Ficoll-Hypaque. IsolatedPBMCs were plated (1×107cells/ml per well) into a6-well plate in RPMI1640medium supplemented10%fetal calf serum (FCS). After2hours of incubation,non-adherent cells as T-cell-enriched fractions were removed and placed in a new6-well plate. Adherent cells were cultured in RPMI1640medium supplemented with10%FCS,500U/ml GM-CSF and40ng/ml IL-4. On day6, DCs were collected,stimulated with10μg/ml synthesized peptides, NY-ESO-1(HLA-A2restricted),NY-ESO-2(HLA-A2restricted) and MAGE-A4(HLA-A2restricted) for48hoursand then washed twice.2×105peptide-pulsed DCs were co-cultured with2×106autologous T-cell-enriched non-adherent cells in1ml RPMI1640mediumsupplemented with10%FCS. The cells were further re-stimulated with freshpeptide-pulsed DCs every7days. On day3after second stimulation,20U/ml rhIL-2was supplemented. Media were changed every3days with half fresh media inpresence of rhIL-2and expanded as necessary. On day7after the last stimulation,cytotoxicity assays were performed using a standard CFSE-PI assay. The results showed that CTLs induced by NY-ESO-1, NY-ESO-2and MAGE-A4pulsed DCsexhibited a stronger peptide-specific response against LS180-MSI-H as comparedwith LS180-MSS and T2cells. On the contrary, no specific lysis was observed in Tcells co-cultured with OVA peptide pulsed DCs or untreated DC. In ELISPOT assay,LS180-MSI-H cells，LS180-MSS cells were treated with mitomycin and used asstimulators，with T lymphocytes as responders. The results showed that T cellsinduced by DC pulsed with NY-ESO-1, NY-ESO-2and MAGE-A4elicited moreIFN-γ positive blots upon in vitro restimulation of LS180-MSI-H than LS180-MSScells or OVA.
To investigate whether DCs pulsed with antigen specific peptides were capableof inducing peptide-specific CTLs in vivo. HLA-A2/Kbtransgenic mice,6–8weeksold, were subcutaneously immunized three times at a week’s interval with1×106BMDCs pulsed with10μg/ml NY-ESO-1, NY-ESO-2and MAGE-A4or untreatedBMDCs. Splenocytes from immunized mice were re-stimulated withMitomycin-treated LS180-MSI-H, Mitomycin-treated LS180-MSS or OVA peptide.Elispot results showed that more IFN-γ secreting cells were induced in splenocytesgenerated from mice immunized with NY-ESO-1, NY-ESO-2and MAGE-A4pulsedBMDCs restimulated with LS180-MSI-H, than LS180-MSS cells or OVA. Weinvestigated whether peptide-specific CTL could be induced by the immunization ofBMDCs pulsed with NY-ESO-1, NY-ESO-2and MAGE-A4. More potentpeptide-specific lysis of LS180-MSI-H were observed in splenocytes from miceimmunized with DCs pulsed with NY-ESO-1, NY-ESO-2and MAGEA4peptide thanlysis of LS180-MSS or T2cells, while effector cells from mice immunized with OVApulsed BMDCs or untreated BMDCs showed almost no killing of target cells. Overall,these results indicated that immunization of NY-ESO-1, NY-ESO-2and MAGE-A4pulsed BMDCs could induce peptide specific CTLs in vivo.
In order to analyze whether the splenocytes from immunized mice indeed havethe ability of tumor rejection, we created an in vivo model of adoptive transfer inC57BL/6nu/numice bearing human tumors. The human colorectal cancer cell linesLS180-MSI-H (HLA-A2positive) were tested for progressive tumor growth in nude mice. C57BL/6nu/numice were challenged subcutaneously with2×106LS180-MSI-Htumor cells in the flank area. The mice were injected intravenously with1×108splenocytes per animal on day5after tumor bearing. This adoptive transfer wasperformed one time, splenocytes induced by NY-ESO-1or NY-ESO-2peptide pulsedBMDCs in HLA-A2/Kbtransgenic mice were able to suppress LS180-MSI-H growthin nude mice. All the control mice developed palpable tumor11days after tumorchallenge. All mice in control groups died between day37and day50after tumorchallenge. In this two groups,1,2of the eight animals were tumor free ever since,respectively, while no significant protection or improvement in survival was observedin control groups. The experiments demonstrated that immunization of mice with theNY-ESO-1or NY-ESO-2pulsed DCs induces potent protective immune responseagainst LS180-MSI-H tumor cells.Part Ⅳ: Identification of cancer associated antigen expression on CRC
Tumor samples obtained from186patients with colorectal cancer regardless oftreatments, who were selected from four hospitals, from2005to2009.63MSI-H and123MSS were identified. In this study, the primary tumor lesion was resected bysurgery and IHC was performed using NY-ESO-1or NY-ESO-2antibodies.4-μmsections of paraffin-embedded tissue were used for immunostaining NY-ESO-1(1:250,Bioss), NY-ESO-2(1:700, Bioss) and slides were then deparaffinized and rehydrated.Endogenous peroxidase was blocked with3%hydrogen peroxide in methanol for20min. Antigen retrieval was achieved with heating in a pressure cooker using10mmol/L of sodium-citrate buffer (pH6.0). Sections were incubated with20%goatblood serum at room temperature for blocking nonspecific binding. After washingwith PBS, sections were incubated with primary antibody at37℃for two hours, andthen incubated with secondary antibody (HRP-R/M, EnVision, Dako) at37℃for30mins. All antibodies were visualized using3,3-diaminobenzidine as chromagen(DAB). Negative controls omitted the primary antibody.
NY-ESO-1+expression were detected as positive in36from63MSI-H patients(57.14%), as compared with53negative from123MSS patients (43.09%).NY-ESO-2+expression were detected as positive in39from63MSI-H patients (61.90%), significantly higher than MSS patients who achieved38.21%(47/123)(P=0.0022).
We analyzed the3-year survival rate of patients with NY-ESO-1and NY-ESO-2protein expression, according to MSI status. The results showed that3-year survivalrate of MSI-H patients with NY-ESO-1positive was74.18%, compared with55.71%survival in patients with NY-ESO-1negative (P=0.2133).
The results also indicated that3-year survival rate of MSI-H patients withNY-ESO-2positive was80.96%, significantly higher than in patients with NY-ESO-2negative46.05%(95%CI,0.08-0.62; P=0.0109). Kaplan-Meier Analysis also showedthat3-year survival rate of MSI-H patients with NY-ESO-2positive was significantlyhigher than the patients with NY-ESO-2negative (P=0.0044).
These results showed that the expression of NY-ESO-2in MSI-H patients wassignificantly higher than MSS, and3-year survival rate of MSI-H patients withNY-ESO-2positive was also significantly higher than that of patients with NY-ESO-2negative. The results suggested that MSI-H CRCs had a better prognosis, mayresulting from the expression of NY-ESO-2protein, which promotes theimmunogenicity of MSI-H colorectal cancer cells.
In conclusion, we successfully established the colorectal cancer cell models withMSI-H and MSS in vitro, and identified the differences in drug sensitivity and MSIstatus between MSI-H/S cell types. We also identified and characterized thedifferentially expressed molecules in MSI-H/S colorectal cancer cell models throughWhole-Genome gene array, showing that Fas as well as some tumor antigen associategenes are up-regulated in MSI-H cells within the MSI-H/S cell models.
MSI-H cells with tumor antigen expression (NY-ESO-1, NY-ESO-2, MAGE-A4)could elite more effective immune response than MSS. Fas protein up-regulated inMSI-H tumor cells may enhance the sensitivity to effector cells. The expression ofNY-ESO-2in MSI-H colorectal cancer was significantly higher than in MSS, and3-year survival rate of MSI-H patients with NY-ESO-2positive was also significantlyhigher than NY-ESO-2negative patients. The results indicated patients with MSI-H colorectal cancer could promote its immunogenicity through the expression ofNY-ESO-2protein, enhance the sensitivity to effector cells by upregulation of Fasprotein, all these effects would contribute to its improved prognosis.
微卫星不稳定性（Microsatellite instability, MSI）是结直肠癌重要的发病机制之一[4]，MSI作为结直肠癌的预后标志的研究，日益受到关注[5]。研究发现高频微卫星不稳定（High-frequency microsatellite instability, MSI-H）的结直肠癌患者，其与低频微卫星不稳定（Low-frequency microsatellite instability, MSI-L）及稳定型（Microsatellite stability, MSS）的相比，病理特点主要是肿瘤多位于近端结肠、黏液腺癌多见、分化较差，但却有相对较好的预后[6‐9]。对于这类MSI-H特殊的结直肠癌患者深入研究后发现，其体内由于处于基因外显子区域的微卫星序列反复突变，进而引起蛋白质的读框突变，最终导致机体产生异常的多肽片段，这些异常多肽片段更易激发机体的抗肿瘤免疫应答反应[10‐13]。研究还发现，其肿瘤微环境中免疫相关的调控因素更有利激发免疫系统，进而促进了免疫系统功能发挥[14‐20]。我们在以树突状细胞瘤苗（APDC）联合化疗治疗转移性结直肠癌的II期临床研究中，对部分临床样本进行了微卫星状态的分析，在初步筛选出的18例MSI-H患者，其中APDC联合化疗组中10例MSI-H，其中8例的临床疗效为部分缓解(Partial response，PR)，2例为轻微缓解(Minimal response，MR)， PFS为6.0月，而对照组（单独化疗）8例MSI-H的患者仅为2PR，2NC，4PD，PFS为2.7月，结果也提示：接受树突状细胞治疗性疫苗组中的MSI-H的结直肠癌患者可能获得高于单独接受化疗的临床疗效。这些都提示了MSI-H这类特殊的肿瘤患者可能由于其肿瘤本身的免疫原性更强，其体内的免疫系统中各种调节因素的共同作用，最终改善了其预后。
但现阶段由于缺乏有效的MSI结直肠癌细胞及动物模型，使得目前国际上大部分对于MSI-H结直肠癌的免疫学特性及其治疗的研究只是局限在临床样本的研究，不能对其生物学特性、与治疗之间的关系及其机理进行深入研究。
本课题旨在通过建立微卫星稳定及不稳定的结直肠癌细胞模型，筛选重要的差异分子，探讨其与免疫治疗的内在机理及关系，为以DC为基础的免疫治疗晚期结直肠癌的Ⅲ期临床的开展提供科学的数据。
本研究共分四个部分进行：
第一部分：MSI-H/S结直肠癌细胞模型的建立
肿瘤异质性是恶性肿瘤重要特征之一，其可以表现在肿瘤分化水平及肿瘤功能水平上的差异，也可以表现在具有异质性的抗原表达或出现不同生物特性细胞亚群。我们根据肿瘤的异质性特征，利用单克隆化技术，对文献已报道过的MSI-H的结直肠癌细胞进行单克隆化，通过荧光PCR反应及GENE SCAN技术对PCR产物进行扫描后，确定每个单克隆细胞株的微卫星状态，最终筛选出MSS的细胞群体，从而在体外建立同一细胞来源的MSI-H/S细胞模型。这部分研究对5株文献已经报道为MSI-H的结直肠癌细胞（分别是LS174t、LS180、HCT15、HCT116、SW48）进行了单克隆化，最终长成集落的单克隆细胞株数分别是LS174t为71株、LS180为81株、HCT15为25株、HCT116为65株、SW48为37株。通过荧光PCR技术，对最终筛选出的单克隆细胞株进行MSI分型后，确定为MSS的细胞株数并进行标记，标记方法为：不同批次单克隆化+序号，如LTE-1表示某批次单克隆化的第1号单克隆细胞株，其中LS174t为21株，标记为LTE-1，2，4，5，6，7，8，9，10，11，12; LTT-1，2，3，4，5，6，7，8，11，12，最终将这21株细胞群体合命名为LS174t-MSS，将其余50株细胞群体合命名为LS174t-MSI-H；LS180为11株，标记为HSO-1，2，10，12；HSW-1，2，3，6，8，9，11，最终将这11株细胞群体合命名为LS180-MSS，其余70株细胞群体合命名为LS180-MSI-H；HCT15为12株，标记为LSF-1，2，3，4，5，6，7，8，14，15，17，18，最终将这12株细胞群体合命名为HCT15-MSS，其余13株细胞群体合命名为HCT15-MSI-H；HCT116为10株，标记为HF-2，3，4，5，13，14，16，22，23，25，最终将这10株细胞群体合命名HCT116-MSS，其余55株细胞群体合命名为HCT116-MSI-H；SW48为10株，标记为SWS-2，6，10，12，14，15；SWT-1，2，3，4，最终将这10株细胞群体合命名为SW48-MSS，其余27株细胞群体合命名为SW48-MSI-H，从而在体外建立了同一细胞来源的MSI-H/S细胞模型，通过细胞形态学的研究发现MSI-H/S的细胞株存在差异，特别是LS180模型中MSI-H与MSS细胞形态差异明显。同时通过药敏实验研究，其结果与相关文献报道一致[24‐27]，即两种不同微卫星状态的细胞株其对于常用化疗药物5-氟尿嘧啶（5-Fu）及伊立替康（CPT）的敏感性存在差异，其中模型中MSI-H细胞对于5-Fu更加耐受，而对于CPT却更加敏感。
通过第一部分的研究我们从体外建立同一细胞来源的MSI-H/S的细胞模型，通过微卫星分析及药敏实验验证了模型中两类细胞的差异性，为下一步的研究奠定了基础。
第二部分：MSI-H/S结直肠癌细胞模型中差异表达分子的筛选及验证
肿瘤的发生、发展和转移与其本身的肿瘤生物学特性有着密切的联系[28]。研究MSI-H结直肠癌的发生除了与许多重要抑癌基因的突变相关，如KRAS[29]，BRAF[30]等，还与细胞生物学功能相关基因的改变有关，如DNA修复基因MRE11[31]、hRAD50[32]及TGF-β[33]。这部分研究中我们在体外建立MSI-H/S的结直肠癌细胞模型的基础上，通过基因、蛋白芯片筛选其中重要的差异分子，并最终通过qPCR，流式技术及Western方法验证差异分子的表达情况。1、MSI-H/S结直肠癌细胞模型中差异表达基因的分析：我们首先对三组MSI-H/S结直肠癌细胞模型（HCT116，LS180，LS174t）进行基因表达谱芯片（Whole-Genome gene array, Affymetrix）的分析，所有样品中每个基因（probeset）的信号值均采用Robust Multichip Analysis（RMA）归一化方法，采用SAM（Significance Analysis of Microarrays）软件进行差异表达基因的筛选。上调大于2倍的基因，筛选标准为：q-value≤5%；Fold Change≥2；下调大于2倍的基因，筛选标准为：q-value≤5%； Fold Change≤0.5；挑选出的差异表达基因在MAS系统中进行了Pathway统计分析。差异表达基因所涉及到具有显著性意义的pathway分类，P value反映此pathway在实验结果中的重要性，P value越低提示与Pathway更相关。结果显示在LS180-MSI-H与LS180-MSS模型中差异表达的基因有2216个，其中LS180-MSI-H细胞中表达上调的基因1456个，对这些上调的基因进行通路分析后发现，其最为相关的通路是细胞因子与细胞因子受体作用通路，共33个基因，包括IL1A、IL1B、 IL7、IL8、 CXCL1、 CXCL2、CXCL3、CXCR4、 CXCL5、 CXCL11、 CCL20、 VEGFA等；在LS174t-MSI-H与LS174t-MSS模型中有4206个，其中LS174t-MSI-H细胞中表达上调的基因3446个，对这些上调的基因进行通路分析后发现，其最为相关的通路是mRNA的多聚腺苷酸化通路，共5个基因，包括CPSF3、PAPOLA、CSTF3、PABPN1、CSTF2；在HCT116-MSI-H与HCT116-MSS模型中有989个，其中HCT116-MSI-H细胞中表达上调的基因721个，对这些上调的基因进行通路分析后发现，其最为相关的通路是细胞周期相关通路，共5个基因，包括CDK6、CCND1、SMAD4、CDKN2A、CCNE1。对三组芯片的结果进行聚类分析后发现，其共同表达上调的基因有7个，分别是Fas, Versican（多能聚糖），Ezrin（一种骨架蛋白），Translocated promoterregion（TPR），Chromosome14open reading frame139(C14orf139)，Similar toankyrin repeat domain20family, member A1(LOC727770)，Chromosome5openreading frame24(C5orf24)。
2、MSI-H/S结直肠癌细胞模型中细胞因子蛋白差异表达的分析：肿瘤微环境中的细胞因子在肿瘤发生、发展及转移过程中发挥了重要的作用[34‐37]。我们通过基因表达谱芯片的结果分析发现许多重要的细胞因子在MSI细胞中mRNA水平的表达上调现象，这些细胞因子在蛋白水平上的表达是否也有差异？我们通过三组MSI-H/S结直肠癌细胞模型（HCT116，LS180，LS174t）进行Human Cytokine Array细胞因子的蛋白芯片分析（Raybiotech），通过加样，孵育，Cy3-链霉亲和等过程，最后采用激光扫描仪Axon GenePix扫描信号，采用Cy3或者绿色通道（激发频率=532nm），将所有样品的数据取平均值，做归一化处理，根据各因子的标准曲线计算出各样品中各因子的浓度值。经3次重复性验证实验来确定最终的细胞浓度。结果及说明如下：上调因子：取比值大于1.5的因子；下调因子：取比值小于0.66的因子。结果分析后发现，LS180-MSI-H与LS180-MSS模型，LS180-MSI-H细胞中许多细胞因子表达上调，如炎性因子IL-8、TIMP-2，生长因子BMP-4、BMP-5、AR、GH、GDF-15、IGFBP-4、OPG等，趋化因子CCL28、CCL20、CXCL10、CXCL5，其中五种细胞因子的表达与基因表达谱的分析结果一致，分别是IL-8、TIMP-2、GDF-15、CXCL5、CCL20；LS174t-MSI-H与LS174t-MSS模型，LS174t-MSI-H细胞中也有许多细胞因子表达上调，如炎性因子TIMP-1，生长因子IGFBP-2、IGFBP-3，趋化因子GCP-2、TECK，其中两种细胞因子的表达与基因表达谱的分析结果一致，分别IGFBP-2、IGFBP-3；在HCT116-MSI-H和HCT116-MSS模型，HCT116-MSI-H细胞中，炎性因子TIMP-1，生长因子IGFBP-6、BMP-4、AR，趋化因子GCP-2、6Ckine；都有不同程度的表达上调。
3、多能聚糖（Versican）在MSI-H/S结直肠癌细胞模型中的表达分析验证：我们已经通过基因表达谱芯片分析，发现许多重要的免疫相关的因素在MSI-H细胞中mRNA水平的表达上调现象，这些因素mRNA水平是否在与其蛋白表达水平相一致呢？首先我们关注一种多能聚糖Versican，其是胞外基质重要的组成部分，参与体内的炎症反应，其受多种细胞因子的调控，与肿瘤的增殖及转移相关[38‐41]。其在三组模型中基因表达谱芯片结果分析发现，其mRNA水平均表达上调，通过qPCR技术进一步发现了其在LS180，LS174t，HCT116模型中，MSI-H与MSS相比，分别增长了59倍、4.5倍、7.4倍。这些胞外基质（如多能聚糖）受到多种细胞因子的调控，与肿瘤的转移关系密切[42]。
4、Fas表达在MSI-H/S结直肠癌细胞模型中的分析验证：我们通过对三组模型进行基因表达谱芯片结果还发现，在MSI-H细胞中Fas分子的mRNA表达水平均有上调的现象。Fas-FasL是免疫系统发挥效应的一个重要的杀伤机制，通过效应细胞表面的FasL与靶细胞的Fas结合，可以启动靶细胞的caspase细胞转导途径，诱导靶细胞的凋亡[43]。通过流式细胞仪检测三组模型中Fas表达水平，在LS180-MSI/MSS，LS174t-MSI/MSS，HCT116-MSI/MSS中分别为48.3%/29.9%；80.4%/70.2%；68.7%/48.6%。凋亡相关基因Fas在MSI-H中的表达上调，及其对于效应细胞敏感性的深入研究，对MSI-H患者预后机制的探讨提供了重要的研究方向。
5、肿瘤抗原表达在MSI-H/S结直肠癌细胞模型中的分析验证：我们通过对三组模型进行基因表达谱芯片结果还发现，MSI-H细胞中许多肿瘤抗原相关基因表达上调的现象。如癌睾丸抗原（CTAG）、前列腺癌相关抗原（PAGE）、黑色素瘤相关抗原（MAGE），其中LS180-MSI-H与LS180-MSS相比，CTAG（1NY-ESO-1）、CTAG2（NY-ESO-2）、MAGE-A4的mRNA水平分别上调219倍、137倍、36倍；LS174t-MSI-H与LS174t-MSS相比，PAGE1、MAGE-A12的mRNA水平分别上调26倍、4倍；HCT116-MSI-H与HCT116-MSS相比，MAGE-B1的mRNA水平上调5倍。肿瘤的免疫原性是免疫系统能否发挥效应作用的关键所在，其已经成为肿瘤免疫领域的研究热点之一。我们通过qPCR技术及流式分析也证实了LS180模型中，MSI-H细胞中NY-ESO-1、NY-ESO-2、MAGE-A4的表达上调现象，同时通过Western分析也证实了MSI-H细胞中NY-ESO-2的表达上调现象；同时利用qPCR技术证实了LS174t模型中MAGE-3、MAGE-12的表达上调现象。我们还通过流式技术进一步分析了10株结直肠细胞（7株MSI-H，3株MSS）NY-ESO-1、NY-ESO-2、MAGE-A4的表达情况，结果也显示MSI-H结直肠细胞其表达的NY-ESO-1、NY-ESO-2、MAGE-A4平均水平高于MSS结直肠癌细胞。第三部分：MSI-H/S结直肠癌免疫治疗预后相关机制探讨
通过前两部分的研究，我们已经发现在MSI-H细胞中Fas表达上调及肿瘤抗原表达上调的现象，但由于MSI缺乏有效的模型，其预后机制并不明确。这部分研究主要从这两方面着手研究其预后的相关机制。
1、Fas在MSI-H/S结直肠癌细胞模型中的表达：我们首先通过Fas激动剂介导模型中MSI-H与MSS发生凋亡，检测两者发生凋亡的差异情况。对LS180-MSI-H/MSS、LS174t-MSI-H/MSS、HCT116-MSI-H/MSS三组模型的细胞加入Fas激动剂后，通过流式技术分析凋亡细胞的比例结果显示，MSI-H中凋亡细胞的比例高于MSS，同时还通过检测细胞活性的方法也发现，MSI-H中活性细胞比例低于MSS。这一结果证实了MSI-H的细胞由于其表面Fas分子表达的水平更高，使其可能对于机体免疫系统中效应细胞更为敏感，增强MSI-H细胞对杀伤的敏感性。
2、MSI-H细胞表达肿瘤抗原体外诱导人外周血淋巴细胞的特异性免疫应答：我们通过NY-ESO-1、NY-ESO-2、MAGE-A4的多肽（均为HLA-A2限制性）分别致敏的HLA-A2阳性的健康志愿者的DC，体外刺激其同体的淋巴细胞，分析其产生特异性免疫应答的情况，结果发现NY-ESO-1、NY-ESO-2、MAGE-A4的多肽三组的淋巴细胞经丝裂霉素处理后的LS180-MSI-H、LS180-MSS细胞及OVA多肽在体外再刺激后，IFN-γ ELISPOT结果显示三组经LS180-MSI-H刺激后所产生的表达IFN-γ的淋巴细胞数目均显著高于LS180-MSS刺激组及OVA刺激组（P＜0.05），同时体外的杀伤实验结果也显示NY-ESO-1、NY-ESO-2、MAGE-A4的多肽致敏的DC刺激后的淋巴细胞其对于靶细胞LS180-MSI-H的杀伤能力显著高于LS180-MSS及对照组T2细胞(P＜0.05)，而OVA致敏的DC及未致敏DC刺激的淋巴细胞杀伤三种靶细胞的能力没有显著差异，以上实验结果提示LS180-MSI-H细胞所表达的肿瘤抗原多肽致敏的DC诱导出多肽特异性的CTL，更有利于CTL对特异性表达这些肿瘤抗原的LS180-MSI-H细胞的杀伤作用。
3、MSI-H细胞表达肿瘤抗原诱导小鼠脾淋巴细胞特异性免疫应答：同时我们还用NY-ESO-1、NY-ESO-2、MAGE-A4的多肽致敏的HLA-A2.1/Kb转基因小鼠的BMDC去免疫同系小鼠，发现此免疫能诱导HLA-A2.1/Kb转基因小鼠产生抗原特异性免疫应答，我们通过NY-ESO-1、NY-ESO-2、MAGE-A4的多肽分别致敏HLA-A2.1小鼠DC后免疫HLA-A2.1小鼠，获得其脾淋巴细胞，三组的小鼠脾细胞经丝裂霉素处理后的LS180-MSI-H、LS180-MSS细胞及OVA多肽体外再刺激后，IFN-γ ELISPOT结果显示三组经LS180-MSI-H刺激后所产生的表达IFN-γ的淋巴细胞数目均显著高于LS180-MSS刺激组及OVA刺激组（P＜0.05）同时体外的杀伤实验结果也显示NY-ESO-1、NY-ESO-2、MAGE-A4的多肽致敏的DC刺激后的脾淋巴细胞其对于靶细胞LS180-MSI-H的杀伤能力显著高于的LS180-MSS及对照组T2细胞(P＜0.05)，而OVA致敏的DC及未致敏DC刺激的淋巴细胞杀伤三种靶细胞的能力没有显著差异。以上结果提示LS180-MSI-H细胞所表达的肿瘤抗原多肽致敏的DC诱导出多肽特异性的CTL，更有利于CTL对特异性表达这些肿瘤抗原的LS180-MSI-H细胞的杀伤作用。
4、MSI-H细胞肿瘤抗原致敏DC体内抗肿瘤效应：6-8周龄的雌性裸鼠，每组8只，接种LS180-MSI-H肿瘤细胞，剂量为2×106细胞/鼠，5天后进行脾细胞尾静脉回输至荷瘤裸鼠体内，数量为1×108细胞/200μl·鼠，观察并记录肿瘤生长情况及裸鼠存活情况。实验结果显示NY-ESO-1和NY-ESO-2多肽抗原致敏的DC诱导的脾细胞抑瘤效果明显强于对照OVA组（P＜0.05），观察至90天，NY-ESO-1组还有1只仍未长出肿瘤， NY-ESO-2组还有2只仍未长出肿瘤，而对照组的荷瘤小鼠在37-50天内均已死亡。这一实验结果提示，LS180-MSI-H肿瘤细胞表达的肿瘤抗原可能在诱导机体产生特异性的免疫应答过程中，发挥了重要的作用。
第四部分：不同微卫星状态下肿瘤抗原表达差异的验证及对于结直肠癌患者预后的影响
前面部分的研究均证实了在体外建立的模型，其中在LS180-MSI-H/MSS、LS174t-MSI-H/MSS、HCT116-MSI-H/MSS中，MSI-H细胞表达的肿瘤相关抗原NY-ESO-1、NY-ESO-2、MAGE-A4其mRNA及蛋白水平均表达上调的现象，在这部分研究中，我们通过研究临床患者的石蜡组织样本肿瘤相关抗原的表达情况，进一步验证其在MSI-H中的表达，及与生存时间的关系。
在本部分的研究中我们收集了2005-2009年186例临床样本，其中104例来源于江苏省南京中医院，55例来源于上海市新华医院，22例来源于上海长海医院，5例来源于上海长征医院，通过微卫星分型分析，确定其微卫星的状态，其中123例MSS，63例MSI-H患者，采用免疫组化的方法分析了其肿瘤组织中NY-ESO-1、NY-ESO-2的表达情况，结果发现其中NY-ESO-1表达阳性的比例在MSI-H中为57.14%，MSS中的43.09%；NY-ESO-2表达阳性的比例在MSI-H中为61.90%，显著高于MSS中的38.21%（P=0.0022）。
采用回顾性分析不同微卫星状态下NY-ESO-1的表达差异对于患者生存时间的影响。在MSI-H组中NY-ESO-1表达阳性的患者三年存活率为74.18%，而NY-ESO-1表达阴性的患者三年存活率为55.71%（P=0.2133）。
同时回顾性分析不同微卫星状态下NY-ESO-2的表达差异对于患者生存时间的影响，在MSI-H组中NY-ESO-2表达阳性的患者三年存活率为80.96%，显著高于NY-ESO-2表达阴性的患者三年存活率46.05%（95%CI,0.08-0.62；P=0.0109），生存分析结果显示在MSI-H组中NY-ESO-2表达阳性的患者其三年生存期显著高于NY-ESO-2表达阴性的患者（P=0.0044）。
这部分结果发现MSI-H组中NY-ESO-2肿瘤抗原的表达显著高于MSS组，与细胞模型中的现象一致，MSI-H患者表达NY-ESO-2肿瘤抗原其三年生存期显著高于不表达此肿瘤抗原的患者，以上实验结果提示：MSI-H的患者可能由于其表达NY-ESO-2的肿瘤抗原更易激发体内免疫系统对其进行杀伤作用，从而改善了其预后。
结论：
1、根据肿瘤的异质性的特征，利用单克隆化技术，从文献已报道为MSI-H的细胞（LS174t、LS180、HCT15、HCT116、SW48）中筛选出MSS的细胞群体，通过微卫星分型及药敏实验证实了两者的差异，从而建立了同一细胞来源的MSI-H/S结直肠癌细胞模型。
2、利用基因、蛋白芯片技术对三组MSI-H/S模型（LS174t、LS180、HCT116）进行了分析，筛选出Fas，多能聚糖分子及肿瘤抗原分子（NY-ESO-1、NY-ESO-2、MAGE-A4）重要分子的表达差异，mRNA水平证实了多能聚糖表达的上调；同时mRNA，蛋白质水平进一步证实了在模型中的MSI-H细胞表面Fas、NY-ESO-1、NY-ESO-2、MAGE-A4分子表达上调的现象。
3、MSI-H细胞表面肿瘤抗原（NY-ESO-1、NY-ESO-2、MAGE-A4）显著高于MSS细胞，其能诱导机体产生更强的免疫应答， MSI-H的细胞由于其表面Fas分子表达的水平更高，使其可能对于机体免疫系统中效应细胞的杀伤更为敏感。
4、通过免疫组化方法分析186例（123MSS,63MSI-H）结直肠癌患者的肿瘤组织样本的肿瘤抗原表达情况，发现肿瘤抗原NY-ESO-2表达阳性的比例在MSI-H的表达显著高于MSS，在MSI-H组中NY-ESO-2表达阳性的患者三年存活率显著高于NY-ESO-2表达阴性的患者存活率，生存分析结果显示在MSI-H组中NY-ESO-2表达阳性的患者其三年生存期显著高于NY-ESO-2表达阴性的患者。
Colorectal cancer (CRC) is one of the most common solid tumors in the worldand becomes the one of major cause of cancer-related death. Though higher responserate have been achieved with the improvement in pharmaceutical strategies over thepast decades, the relatively high resistance to systemic chemotherapy and the geneticheterogeneity of colorectal cancer have lead to poor prognosis and unfavorableclinical outcomes. Almost50%of the patients died as the disease progresses.Therefore, it is necessary to develop novel strategies for the treatment of metastaticcolorectal cancer. Personalized medicine, which refers to the tailoring of medicaltreatment to the individual characteristics of each patient, has been verified to beeffective. Examples provided by Herceptin in breast cancer and Gleevec in chronicmyeloid leukaemia (CML) have long carried the mantle for individualized therapy.
Microsatellite instability (MSI) status is a potential prognostic factor in CRC,patients with high frequency MSI (MSI-H) have unique characteristics compared withmicrosatellite stability (MSS) cancers, such as proximal anatomic location and severeinflammatory cell infiltration. Several studies have also indicated that patients withMSI-H achieve better prognosis compared with MSS CRCs. In our previous study，tumor samples obtained at baseline from101patients, who were selected from arandom phase II clinical trial of sequential administration of OXA+5-Fu and dendriticcell (DC) vaccine in treatment of metastatic colorectal cancer, were suitable for MSIstatus analysis. Of them60patients received sequential administration ofOXA+5-Fu/LV and DC vaccine (Group A),41received OXA+5-Fu/LV (Group B)only. There were18MSI-H patients, of them,10MSI-H in group A and8MSI-H ingroup B. The results indicated that patients with MSI-H in Group A achieved80%(8/10) objective clinical response, while2patients achieved25%(2/8) objectiveclinical response in Group B.
However, the underlying mechanisms remain to be unidentified. Our researchwas intended to identify the biological features of CRCs with MSI-H, so as to elucidate the underlying mechanisms for the improved prognosis in CRCs withMSI-H. Our major findings are summarized in four parts as followed:
PartⅠ: Establishment of the colorectal cancer cell models with MSI-H and MSSin vitro.
Five colorectal cancer cells with MSI-H（LS174t, LS180, HCT15, HCT116,SW48) were obtained for isolating their corresponding MSS cells according to tumorcell heterogeneity via cloning. The multiple cell clones of each colorectal cancer cellline were obtained by serial dilutions and plating in96-microwell plates. The numberof cell clones of LS174t, LS180, HCT15, HCT16, SW48was71,81,25,65,37,respectively. Genomic DNA was extracted from all separate cell clones. MSI at agiven microsatellite locus was detected by comparison of the allele patterns ofmultiple separate cell clones of each cell line. The microsatellite markers included theBAT25, BAT26, D2S123, D17S250, D5S346. The numbers of different allele patternsfor each microsatellite marker in a given cell line was scored. If different alleles of agiven microsatellite marker were detected in the separate cell clones of a given cellline, the locus was then scored as MSI-positive, and if no different alleles weredetected, then it was MSS. When two or more of the five microsatellite markersshowed MSI, based on the NCI criteria, MSI-high was scored. We isolated multiplecolorectal cancer cells with MSS status, the number of MSS cells in LS174t, LS180,HCT15, HCT16, SW48was21,11,12,10,10and named them LS174t-MSS,LS180-MSS, HCT15-MSS, HCT16-MSS, SW48-MSS, respectively. The rest of celllines were named LS174t-MSI-H, LS180-MSI-H, HCT15-MSI-H, HCT16-MSI-H,SW48-MSI-H, respectively. Finally we established the colorectal cancer cell modelswith MSI-H and MSS in vitro. We detected the different morphological features inMSI-H/S cell models, especially in LS180cell model. We also detected the differentsensitivity of the established colorectal cancer cell models to5-Fu and CPT, andfound that MSI-H cells were more resistant to5-Fu and more sensitive to CPT, inconsistence with previous reports.
PartⅡ: Identification of differentially expressed molecules in MSI-H/S colorectalcancer cell models.
We profiled gene expression in3pairs of MSI-H/S colorectal cancer cell models,consisting of LS180-MSI-H, LS174t-MSI-H, HCT116-MSI-H, and LS180-MSS,LS174t-MSS, HCT116-MSS, and explored the molecular variation in MSI-H andMSS cells through Whole-Genome gene array (Affymetrix). We identified2216,3446,989genes in LS180, LS174t, HCT116cell model, respectively, the expressionof which were significantly different between MSI-H and MSS groups. Mostly relatedpathway about the1456up-regulated genes in LS180-MSI-H is cytokine-cytokinereceptor interaction, in LS174t-MSI-H is polyadenylation of mRNA pathway, and inHCT116MSI-H is cell cycle pathway. Cluster analysis of the up-regulated genes inthree MSI-H cells showed that as few as7genes was up-regulated in MSI-H,including Fas, Versican, Ezrin, translocated promoter region (TPR), chromosome14open reading frame139(C14orf139), similar to ankyrin repeat domain20family,member A1(LOC727770) and chromosome5open reading frame24(C5orf24).
We investigated the protein variation in MSI-H and MSS cells via HumanCytokine Antibody Array (RayBiotech). We found many up-regulated cytokines inMSI-H cells compared with MSS, such as elevated IL-8, TIMP-1, TIMP-2, BMP-4,BMP-5, AR, GH, GDF-15, IGFBP-4, CCL28, CCL20, CXCL10, CXCL5and so on inLS180-MSI-H cells. The mRNA levels of IL-8, TIMP-2, GDF-15, CXCL5andCCL20were consistently upregualated in LS180-MSI-H cells with gene array; withIGFBP-2and IGFBP-3upregulated in LS174t-MSI-H cells.
FACS analysis of Fas expression in three MSI-H/S models indicated48.3%Faspositive cells in MSI-H as compared with29.9%in MSS in LS180cell model;80.4%in MSI-H as compared with70.2%MSS in LS174t cell model, and68.7%MSI-H ascompared with48.6%MSS in HCT116cell model.
We also found up-regulated tumor associated antigen genes in MSI-H cellswithin three MSI-H/S models, including cancer/testis antigen (CTAG), melanomaantigen family A (MAGE-A) and P antigen family. CTAG-1(NY-ESO-1)，CTAG2(NY-ESO-2) and MAGE-A4were increased in LS180-MSI-H cells by qPCR and FACS analysis. QPCR analysis also revealed up-regulated MAGE-3and MAGE-12in LS174t-MSI-H cells, as well as up-regulated Versican in all three MSI-H/S cellmodels.
FACS analysis of the expression of NY-ESO-1，NY-ESO-2，MAGE-A4in10colorectal cancer cell lines (7MSI-H,3MSS), in which MSI status have been perviousascertained, showed that the average expression of NY-ESO-1， NY-ESO-2，MAGE-A4is higher in MSI-H than MSS cells.
Part Ⅲ: Potential mechannism analysis for better prognosis of MSI-H CRC
We measured Fas-agonist induced apoptosis in MSI-H cells by FACS analysis,and found that the percentage of Annexin and PI double positive cells is higher inMSI-H than MSS cells. The cell viability is lower in MSI-H than MSS cells via cellcounting CCK8-assay. Thus, the elevated expression of Fas in MSI-H may enhancethe sensitivity to CTLs.
To explore whether DCs pulsed with antigen specific peptides were capable ofinducing peptide-specific CTLs in vitro. PBMCs from HLA-A2positive volunteerwere separated by density gradient centrifugation using Ficoll-Hypaque. IsolatedPBMCs were plated (1×107cells/ml per well) into a6-well plate in RPMI1640medium supplemented10%fetal calf serum (FCS). After2hours of incubation,non-adherent cells as T-cell-enriched fractions were removed and placed in a new6-well plate. Adherent cells were cultured in RPMI1640medium supplemented with10%FCS,500U/ml GM-CSF and40ng/ml IL-4. On day6, DCs were collected,stimulated with10μg/ml synthesized peptides, NY-ESO-1(HLA-A2restricted),NY-ESO-2(HLA-A2restricted) and MAGE-A4(HLA-A2restricted) for48hoursand then washed twice.2×105peptide-pulsed DCs were co-cultured with2×106autologous T-cell-enriched non-adherent cells in1ml RPMI1640mediumsupplemented with10%FCS. The cells were further re-stimulated with freshpeptide-pulsed DCs every7days. On day3after second stimulation,20U/ml rhIL-2was supplemented. Media were changed every3days with half fresh media inpresence of rhIL-2and expanded as necessary. On day7after the last stimulation,cytotoxicity assays were performed using a standard CFSE-PI assay. The results showed that CTLs induced by NY-ESO-1, NY-ESO-2and MAGE-A4pulsed DCsexhibited a stronger peptide-specific response against LS180-MSI-H as comparedwith LS180-MSS and T2cells. On the contrary, no specific lysis was observed in Tcells co-cultured with OVA peptide pulsed DCs or untreated DC. In ELISPOT assay,LS180-MSI-H cells，LS180-MSS cells were treated with mitomycin and used asstimulators，with T lymphocytes as responders. The results showed that T cellsinduced by DC pulsed with NY-ESO-1, NY-ESO-2and MAGE-A4elicited moreIFN-γ positive blots upon in vitro restimulation of LS180-MSI-H than LS180-MSScells or OVA.
To investigate whether DCs pulsed with antigen specific peptides were capableof inducing peptide-specific CTLs in vivo. HLA-A2/Kbtransgenic mice,6–8weeksold, were subcutaneously immunized three times at a week’s interval with1×106BMDCs pulsed with10μg/ml NY-ESO-1, NY-ESO-2and MAGE-A4or untreatedBMDCs. Splenocytes from immunized mice were re-stimulated withMitomycin-treated LS180-MSI-H, Mitomycin-treated LS180-MSS or OVA peptide.Elispot results showed that more IFN-γ secreting cells were induced in splenocytesgenerated from mice immunized with NY-ESO-1, NY-ESO-2and MAGE-A4pulsedBMDCs restimulated with LS180-MSI-H, than LS180-MSS cells or OVA. Weinvestigated whether peptide-specific CTL could be induced by the immunization ofBMDCs pulsed with NY-ESO-1, NY-ESO-2and MAGE-A4. More potentpeptide-specific lysis of LS180-MSI-H were observed in splenocytes from miceimmunized with DCs pulsed with NY-ESO-1, NY-ESO-2and MAGEA4peptide thanlysis of LS180-MSS or T2cells, while effector cells from mice immunized with OVApulsed BMDCs or untreated BMDCs showed almost no killing of target cells. Overall,these results indicated that immunization of NY-ESO-1, NY-ESO-2and MAGE-A4pulsed BMDCs could induce peptide specific CTLs in vivo.
In order to analyze whether the splenocytes from immunized mice indeed havethe ability of tumor rejection, we created an in vivo model of adoptive transfer inC57BL/6nu/numice bearing human tumors. The human colorectal cancer cell linesLS180-MSI-H (HLA-A2positive) were tested for progressive tumor growth in nude mice. C57BL/6nu/numice were challenged subcutaneously with2×106LS180-MSI-Htumor cells in the flank area. The mice were injected intravenously with1×108splenocytes per animal on day5after tumor bearing. This adoptive transfer wasperformed one time, splenocytes induced by NY-ESO-1or NY-ESO-2peptide pulsedBMDCs in HLA-A2/Kbtransgenic mice were able to suppress LS180-MSI-H growthin nude mice. All the control mice developed palpable tumor11days after tumorchallenge. All mice in control groups died between day37and day50after tumorchallenge. In this two groups,1,2of the eight animals were tumor free ever since,respectively, while no significant protection or improvement in survival was observedin control groups. The experiments demonstrated that immunization of mice with theNY-ESO-1or NY-ESO-2pulsed DCs induces potent protective immune responseagainst LS180-MSI-H tumor cells.Part Ⅳ: Identification of cancer associated antigen expression on CRC
Tumor samples obtained from186patients with colorectal cancer regardless oftreatments, who were selected from four hospitals, from2005to2009.63MSI-H and123MSS were identified. In this study, the primary tumor lesion was resected bysurgery and IHC was performed using NY-ESO-1or NY-ESO-2antibodies.4-μmsections of paraffin-embedded tissue were used for immunostaining NY-ESO-1(1:250,Bioss), NY-ESO-2(1:700, Bioss) and slides were then deparaffinized and rehydrated.Endogenous peroxidase was blocked with3%hydrogen peroxide in methanol for20min. Antigen retrieval was achieved with heating in a pressure cooker using10mmol/L of sodium-citrate buffer (pH6.0). Sections were incubated with20%goatblood serum at room temperature for blocking nonspecific binding. After washingwith PBS, sections were incubated with primary antibody at37℃for two hours, andthen incubated with secondary antibody (HRP-R/M, EnVision, Dako) at37℃for30mins. All antibodies were visualized using3,3-diaminobenzidine as chromagen(DAB). Negative controls omitted the primary antibody.
NY-ESO-1+expression were detected as positive in36from63MSI-H patients(57.14%), as compared with53negative from123MSS patients (43.09%).NY-ESO-2+expression were detected as positive in39from63MSI-H patients (61.90%), significantly higher than MSS patients who achieved38.21%(47/123)(P=0.0022).
We analyzed the3-year survival rate of patients with NY-ESO-1and NY-ESO-2protein expression, according to MSI status. The results showed that3-year survivalrate of MSI-H patients with NY-ESO-1positive was74.18%, compared with55.71%survival in patients with NY-ESO-1negative (P=0.2133).
The results also indicated that3-year survival rate of MSI-H patients withNY-ESO-2positive was80.96%, significantly higher than in patients with NY-ESO-2negative46.05%(95%CI,0.08-0.62; P=0.0109). Kaplan-Meier Analysis also showedthat3-year survival rate of MSI-H patients with NY-ESO-2positive was significantlyhigher than the patients with NY-ESO-2negative (P=0.0044).
These results showed that the expression of NY-ESO-2in MSI-H patients wassignificantly higher than MSS, and3-year survival rate of MSI-H patients withNY-ESO-2positive was also significantly higher than that of patients with NY-ESO-2negative. The results suggested that MSI-H CRCs had a better prognosis, mayresulting from the expression of NY-ESO-2protein, which promotes theimmunogenicity of MSI-H colorectal cancer cells.
In conclusion, we successfully established the colorectal cancer cell models withMSI-H and MSS in vitro, and identified the differences in drug sensitivity and MSIstatus between MSI-H/S cell types. We also identified and characterized thedifferentially expressed molecules in MSI-H/S colorectal cancer cell models throughWhole-Genome gene array, showing that Fas as well as some tumor antigen associategenes are up-regulated in MSI-H cells within the MSI-H/S cell models.
MSI-H cells with tumor antigen expression (NY-ESO-1, NY-ESO-2, MAGE-A4)could elite more effective immune response than MSS. Fas protein up-regulated inMSI-H tumor cells may enhance the sensitivity to effector cells. The expression ofNY-ESO-2in MSI-H colorectal cancer was significantly higher than in MSS, and3-year survival rate of MSI-H patients with NY-ESO-2positive was also significantlyhigher than NY-ESO-2negative patients. The results indicated patients with MSI-H colorectal cancer could promote its immunogenicity through the expression ofNY-ESO-2protein, enhance the sensitivity to effector cells by upregulation of Fasprotein, all these effects would contribute to its improved prognosis.
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