两个新胃癌细胞表面分子标志物的发现及鉴定
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摘要
胃癌（gastric cancer，GC）是一种常见的消化系统恶性肿瘤，在世界范围内是肿瘤相关死亡的重要原因。由于早期诊断技术以及治疗手段的改进，近年来胃癌的总体预后有所改善，但是晚期胃癌的临床结局并不令人满意，5年生存率较低。在后基因组时代，寻找胃癌新的分子标志物，构建早期敏感、特异、即时检测（point of care testing, POCT）的诊断方法以及研发单克隆抗体靶向治疗药物将是胃癌基础以及临床研究领域的热点。
目前筛选胃癌分子标志物的方法以传统蛋白质组学策略为主，虽然已鉴定出一些新的胃癌分子标志物，但是由于这些分子特异性较差而停滞于实验室研究阶段，难以临床应用。筛选鉴定特异性强且定位于肿瘤细胞表面的靶点分子标志物将为胃癌的抗体靶向治疗开辟新的思路。
本研究拟采用以制备的抗胃癌单克隆抗体反向筛选鉴定靶抗原的方法，旨在实现“一步法”同时制备抗胃癌细胞表面天然构象抗原的特异性单抗以及鉴定特异性胃癌分子标志物。由于传统的抗体筛选制备方法很难获得识别活细胞表面天然构象抗原的抗体，所以本研究采用“鸟枪法”活细胞免疫小鼠，杂交瘤技术结合流式细胞术高通量筛选的创新方法，在活细胞水平直接筛选抗肿瘤细胞表面天然构象抗原的特异性抗体，在进行抗体的特异性鉴定及分析特异性抗体识别的胃癌细胞表面天然抗原与胃癌临床病理学参数的相关性和临床意义之后，采用人蛋白质组芯片对制备的特异性抗体结合的靶抗原蛋白分子进行快捷鉴定，从而确定新的胃癌细胞表面分子标志物。本研究分为以下4个部分：
一．抗胃癌细胞表面天然抗原特异性抗体的高通量筛选、制备
研究方法：胃癌细胞株（SGC7901、BGC823、MKN28、MKN45）混合后免疫6-8周龄BALB/c小鼠(1×107个细胞)，取脾脏B细胞与SP2/0细胞融合，杂交瘤细胞大批量铺板，选择性培养；利用BD FACS Calibur的高通量检测系统（fluorescence activating cell sorter-highthroughput screening system，FACS-HTS），在活细胞水平通过FACS高通量检测预处理的杂交瘤细胞培养上清与胃癌细胞（免疫用4株胃癌细胞）以及正常对照细胞（健康志愿者外周血单个核细胞，PBMC）的结合反应情况；根据ELISA-HTS(enzyme linked immunosorbentassay-HTS)的基本筛选原则，挑取杂交瘤细胞阳性克隆，再进行FACS交叉鉴定后建立能稳定分泌抗胃癌细胞表面天然构象抗原的单克隆抗体杂交瘤细胞株。制备的单克隆抗体进行亚型鉴定及ProteinA/G亲和层析纯化。
研究结果：杂交瘤技术与活细胞水平FACS-HTS相结合的筛选方法制备获得两株抗胃癌细胞表面天然构象抗原的单克隆抗体，分别命名为MS38-2.1及MS40-3.1（根据高通量筛选鉴定过程中对杂交瘤细胞的特定编号）。两株抗体均为IgG1/κ型。
研究结论：建立了“鸟枪法”胃癌活细胞免疫，杂交瘤技术结合活细胞水平FACS-HTS的创新方法，制备了两株抗胃癌细胞表面天然构象抗原的特异性抗体。本研究建立的创新方法也可用于其它实体肿瘤细胞表面天然构象抗原特异性抗体的制备、筛选。
二．抗胃癌细胞表面天然抗原单抗的特异性鉴定
研究方法：培养38种细胞系，包括15种胃癌细胞，以及结直肠癌、肝细胞肝癌、乳腺癌、胆囊癌以及急性单核细胞白血病细胞，人胎肝细胞和GES-1、EA.hy926细胞，采用间接免疫荧光染色、FACS检测MS38-2.1及MS40-3.1的标记情况。收集12例手术切除的胃癌新鲜组织及同一病例癌旁组织，部分组织分别进行酶消化后制备单细胞悬液，间接免疫荧光染色后FACS检测MS38-2.1及MS40-3.1与肿瘤细胞及癌旁组织细胞的免疫反应情况；剩余新鲜组织常规10%福尔马林固定，石蜡包埋，免疫组化EnVision法检测胃癌和癌旁组织两株抗体的标记情况。
研究结果：FACS结果显示，MS38-2.1与胃癌细胞、GES-1细胞、结肠癌、肝细胞肝癌、L-02细胞、急性单核细胞白血病、乳腺癌以及胆囊癌细胞均有不同强度的结合反应，与EA.hy926细胞也有结合反应；MS40-3.1与胃癌细胞、GES-1细胞、结直肠癌、肝细胞肝癌及L-02细胞均有结合反应；两株抗体与对照细胞（包括健康志愿者PBMC以及正常胃黏膜上皮细胞）均无结合反应。免疫组化结果显示MS38-2.1及MS40-3.1可特异性标记胃癌细胞，并且MS38-2.1可特异性标记中小血管。FACS分析显示MS38-2.1及MS40-3.1与新鲜胃癌以及癌旁组织细胞结合反应的差异具有统计学意义（P <0.05）。
研究结论：MS38-2.1识别的靶抗原蛋白分子在胃癌细胞、GES-1细胞、结肠癌、肝细胞肝癌、L-02细胞、急性单核细胞白血病、乳腺癌、胆囊癌细胞表面均有表达，并且在中小血管内皮细胞也有特异性表达；MS40-3.1识别的靶抗原蛋白分子在胃癌细胞、GES-1细胞、结直肠癌、肝细胞肝癌及L-02细胞表面均有表达。提示MS38-2.1和MS40-3.1均能特异性识别胃癌细胞，与正常胃黏膜上皮细胞无交叉反应。MS38-2.1还可特异性识别中小血管内皮细胞。
三．特异性抗体识别的胃癌细胞表面天然抗原蛋白分子与胃癌临床病理学参数的相关性及临床意义
研究方法：选取90例石蜡包埋的胃癌及对应癌旁组织标本，免疫组化EnVision法检测MS38-2.1及MS40-3.1的标记情况，并分析单抗识别的胃癌细胞表面天然抗原蛋白分子与胃癌临床病理学参数的相关性及临床意义。
研究结果：90例胃癌及对应的癌旁组织中，28例胃癌组织MS38-2.1阳性（31.11%），8例癌旁组织阳性（8.89%），差异具有统计学意义（P＜0.05）；42例胃癌组织MS40-3.1阳性（46.67%），3例癌旁组织阳性（3.33%），差异具有统计学意义（P＜0.05）。胃癌组织MS38-2.1及MS40-3.1标记阳性即MS38-2.1及MS40-3.1特异性识别的靶抗原蛋白分子均与肿瘤浸润深度、淋巴结转移、远处转移及TNM分期有关（P＜0.05），而与年龄、性别、肿瘤大小、肿瘤部位以及肿瘤分化程度无明显相关性。生存分析显示，胃癌组织中MS38-2.1及MS40-3.1高阳性率即MS38-2.1及MS40-3.1特异性识别的靶抗原蛋白分子的高表达均与患者生存时间短有明显相关性（P＜0.0001）。
研究结论：MS38-2.1及MS40-3.1特异性识别的靶抗原蛋白分子在胃癌组织中均特异性高表达，且两个靶抗原蛋白分子的高表达均与胃癌的浸润、转移、临床分期和预后有关，可作为胃癌诊断及预后评估的潜在分子标志物。
四．抗胃癌细胞表面天然抗原特异性抗体结合靶抗原蛋白的鉴定
研究方法：培养胃癌细胞（BGC823、MKN28、SGC7901、MKN45），胃黏膜上皮细胞（GES-1），结肠癌细胞（SW1116），乳腺癌细胞（MCF-7）以及肝癌细胞（Hep3B），Western blot方法检测上述细胞中MS38-2.1及MS40-3.1特异性结合靶抗原蛋白的分子量及表达情况，以正常胃黏膜组织裂解物作为对照。采用包含16,368种不同人全长重组蛋白的蛋白质组芯片，鉴定MS38-2.1及MS40-3.1的特异性以及MS38-2.1及MS40-3.1特异性结合的靶抗原蛋白分子，ELISA及Western blot验证MS38-2.1及MS40-3.1与靶抗原蛋白分子的结合反应。
研究结果：Western blot结果显示，抗胃癌单抗MS38-2.1识别的靶抗原蛋白分子在胃癌细胞BGC823、SGC7901、MKN45以及胃黏膜上皮细胞GES-1细胞表面高表达，在胃癌细胞MKN28细胞表面低表达；在结肠癌细胞SW1116，乳腺癌细胞MCF-7以及肝癌细胞Hep3B细胞表面均有表达；正常胃黏膜组织裂解物中不含这一蛋白分子。上述细胞表达的MS38-2.1特异性结合靶抗原蛋白的分子量约为128kDa。
MS40-3.1识别的靶抗原蛋白分子在胃癌细胞BGC823、SGC7901、MKN28以及MKN45细胞表面高表达；在胃黏膜上皮细胞GES-1细胞表面高表达；在结肠癌细胞SW1116及肝癌细胞Hep3B细胞表面均表达；正常胃黏膜组织裂解物中不含这一蛋白分子。上述细胞表达的MS40-3.1特异性结合靶抗原蛋白的分子量约为60kDa。
人蛋白质组芯片（human proteome microarray）鉴定结果显示，MS38-2.1特异性识别的蛋白分子为富含脯氨酸的小分子蛋白3（small proline-rich protein3，SPRR3）， MS40-3.1特异性识别的蛋白分子为鸟氨酸脱羧酶1（ornithinedecarboxylase1，ODC-1），两株抗体与人蛋白质组芯片中其它细胞蛋白均无交叉反应。ELISA及Western blot显示MS38-2.1与人SPRR3重组蛋白，MS40-3.1与人ODC-1重组蛋白均有良好的特异性结合反应。
研究结论：制备的MS38-2.1及MS40-3.1均为单一特异性单克隆抗体，MS38-2.1、MS40-3.1识别的靶抗原蛋白分子是与SPRR3、ODC-1类似或至少具有相同抗原表位的分子，为两个新的胃癌细胞表面候选分子标志物。
综上所述，本研究建立了“鸟枪法”胃癌活细胞免疫，杂交瘤技术结合活细胞水平FACS-HTS，筛选制备抗胃癌细胞表面天然构象抗原特异性抗体的创新方法；发现了两个新的可作为胃癌诊断及预后评估的胃癌细胞表面分子标志物。本文建立的创新研究方法和结果对于其它恶性肿瘤分子标志物的研究具有重要的借鉴作用和参考价值。
Gastric cancer (GC) is a common neoplasm in digestive system, which is leadingthe cause of cancer-related deaths worldwide. With the progression of earlydiagnostic techniques and therapeutic strategies, the overall survival rate of GCpatients has been improved in recent years. The outcome survival rate of theadvanced GC still remains poor while the5-year overall survival rate is diminishing.In post-genomic era, the discovery and identification of novel GC biomarkers isrequired for more sensitive, specific and functional targets, which could be easilyused for diagnosis and therapeutic purposes. The generaton and the development ofmonoclonal antibody would be played a key role in the basic and clinical researchesof GC.
The development of proteomics has been set the feasible approches to identifythe GC biomarkers and the associated molecules although many known biomarkers ofGC are non-specific and limited. Therefore, the HTS (high throughput screening) andidentification of specific molecules, especially the membrane bound proteins fromdifferent cancer cells, would provide the new hopes besed on monoclonal antibodytechnology to diagnosis and treat the GC patients.
In this study, we have used an antibody-based approach to identify antigen withthe counter screening strategy. The aim of our study was to generate monoclonalantibodies (mAbs) against the native antigens or receptors as the cancer biomarkerson the surface of gastric cancer cells through our unique “one step” colony pickingand HTS screening methods. It is difficult to prepare mAbs against the nativeantigens on cell surface if still using the current screening approaches. Therefore, we have developed a streamlined strategy to make mAbs binding to native antigens oncell surface, which we used live cell immunization(so-called “shot gun” manner) andlive cell screening FACS-HTS (fluorescence activating cell sorter-highthrough putscreening) strategy with the unique hybridoma technology. The identifed and selectedmAbs were implemented in the Immunohistochemstry, ELISA, FACS analysis and ahuman proteome microarray etc. These biomarkers were verified at least twice by thetwo mAbs.
The four sections of our study as follows:
Ⅰ. The specific mAbs against native antigens on cell-surface of gastric cancercells were generated and screened by FACS-HTS
METHODS: Gastric cancer cell lines (SGC7901, BGC823, MKN28, MKN45)were cultured and mixed together to be served as an immunogen in about1×107cellsper mouse were sc.(subcutaneous) and ip.(intraperitoneal) injections into6-8weeksold BALB/C mice. The splenocytes from three times immunized mouse were fusedwith SP2/0myeloma cells and inoculated in96-wells cell culture plates with a largescale manner. The immunoreactivity hybridoma from the culture supernatants of HATconditional media were used to screen with live gastric cancer cells, as well as thenormal control cells (peripheral blood mononuclear cells from healthy individuals) inFACS-HTS system. We selected many positive hybrids according to ELISA-HTSprinciple and double confirm the signals, positive and stable hybridoma clones werevalidated and picked and then the immunoglobulin isotyping were performed. Thepurification of mAbs by Protein A/G-Sepharose chromatography were conducted.
RESULTS: Two mAbs against native antigens on cell-surface of gastric cancercells, termed as MS38-2.1and MS40-3.1, were generated via hybridoma technologyin combination with viable cell-based FACS-HTS. Both of the two mAbs were IgG1/ κ.
CONCLUSIONS: The series data indicated that our strategy for the generationand validation of anti-GC mAbs is working well. The “shot gun” mannerimmunization and the combination screening methods that the supernatants ofhybridoma colonies were for both ELISA-HTS and the live cells based FACS-HTSare the pivotal high throughput approaches in a robust platform for the mAbgenerations that would be specifically for the native antigens on cell surface of gastriccancer and other solid tumors.
Ⅱ. The validation of mAbs specificity against native antigen on the surface ofgastric cancer cells
METHODS: Having38cell lines including15gastric cancer cell lines,colorectal cancer, liver cancer, breast cancer, gallbladder cancer, acute monocyticleukemia and GES-1, EA.hy926cells were used to analyse the binding profiles ofMS38-2.1and MS40-3.1through indirect immunofluorescence (IF) staining andFACS assay. The12fresh tumor tissues and the adjacent normal mucosa tissues werefreshly obtained from the gastric cancer patients who were undertaken the surgeryresection. A proportion of12fresh gastric tissues were separated into single-cellsuspension by a cocktail enzymes digestion that would be immediately for FACSanalysis with indirect IF staining by MS38-2.1and MS40-3.1mAb. Anotherproportion of12fresh gastric tissues were used for immunohistochemical (IHC)analysis with formalin-fixed and paraffin-embedded (FFPE) to identify theimmunoreactivity of two mAbs in tissues.
RESULTS: The MS38-2.1and MS40-3.1mAbs demonstrated bothimmunofluorescence and immunohistochemistry grades from the assays. The twopurified mAbs specifically recognized the native antigens on cell surface of gastric cancer and other malignancies, and the MS38-2.1could interestingly haveimmunoreactivity with EA.hy926cells (human umbilical vein endothelial cells). Thetwo mAbs have no cross reactions to the normal control cells including humanperipheral blood mononuclear cells (PBMC) and normal primary gastric cells. Thesignificant IHC staining on the gastric cancer cells were observed when detected bythe two mAbs and strong staining of small and medium blood vessels were observedwhen detected with MS38-2.1. FACS analysis demonstrated that there weresignificant differences between fresh prepared gastric cancer cells and correspondingadjacent normal gastric cells when detected by MS38-2.1and MS40-3.1mAbsrespectively (P <0.05).
CONCLUSIONS: The target antigen recognized by MS38-2.1was expressed ingastric cancer, colorectal cancer, liver cancer, breast cancer, gallbladder cancer, acutemonocytic leukemia, GES-1and L-02cells, and also in vascular endothelial cells ofsmall and medium blood vessels.
Ⅲ. The correlation and clinical significance between the expression of gastriccancer cell-surfaced native antigens defined by specific mAbs andclinicopathological characteristics of gastric cancer
METHODS:90FFPE gastric cancer tissues and corresponding adjacent normalmucosa tissues were selected to the applications of the immunoreactivity ofMS38-2.1and MS40-3.1mAbs in IHC. The relationship among the staining intensityof two mAbs, clinicopathologic characteristics and prognosis were evaluated.
RESULTS:28in90cases were positive (31.11%) on both gastric cancer tissuesand their small blood vessels, and8in90cases were positive (8.89%) on tumoradjacent tissues and their small blood vessels by MS38-2.1mAb (P<0.05).42in90cases were positive (46.67%) on gastric cancer tissues and only3out of90cases arepositive (3.33%) on tumor adjacent tissues by MS40-3.1mAb (P<0.05). The IHC staining of both MS38-2.1and MS40-3.1mAbs showed the specificifity on thetargets that are related to degrees of tumor invasion, lymph nodes metastasis andTNM stage, but not correlated to age, gender, the size and location of tumor andtumor differentiation. The survival analysis demonstrated that there was a significantcorrelation between short survival and high expression of target antigens thatMS38-2.1and MS40-3.1specifically binding to (P＜0.0001).
CONCLUSIONS: The biomarkers that were recognized by MS38-2.1andMS40-3.1mAbs were both specifically expressed in gastric cancer tissues and relatedto tumor invasion, metastasis, clinical staging and prognosis, which could bepotentially applied to the prognosis assessment and diagnosis of gastric cancer.
Ⅳ. The identification of the native antigens on cell surface of gastric cancerspecifically recognized by mAbs
METHODS: Gastric cancer cell lines (BGC823, MKN28, SGC7901, MKN45),human gastric epithelial cells (GES-1), colon adenocarcinoma cells (SW1116),breast cancer cells (MCF-7) and hepatocellular adenocarcinoma cells (Hep3B) wereall cultured and treated with mammalian cell membrane protein lysis buffer. TheWestern blot method was utilized to determine the expressed proteins and theirmolecular weights of these cell lines, which MS38-2.1and MS40-3.1were used forspecific binding. The lysates of normal gastric tissues were served as the control. The16,368unique full-length human proteins in proteome microarray were used toanalyze the specificity of the two mAbs and to identify the target candidates or theprotein ID of these two mAbs. The immunoreactivity of the potential target antigenswith the two mAbs was validated with ELISA and Western blot.
RESULTS: The results of Western blot suggested the biomarker candidates thatMS38-2.1specifically binding to was highly expressed on cell surface, includinggastric cancer cell lines (BGC823, SGC7901, MKN45) and human gastric epithelial cell line GES-1, but was lowly expressed on gastric cancer cell line MKN28. Inaddition, the biomarker candidate was expressed on colon adenocarcinoma cell lineSW1116, breast cancer cell line MCF-7and hepatocellular adenocarcinoma cell lineHep3B. However, the lysates of normal gastric tissues not comprised the biomarkercandidates which were detected by MS38-2.1. The approximate molecular weight ofthe biomarker candidate was128kDa.
As for MS40-3.1, the Western blot demonstrated that the biomarker candidatethat the mAb could specifically bind to was highly expressed on the cancer cellsurface, including gastric cancer cell lines BGC823, SGC7901, MKN28, MKN45andhuman gastric epithelial cells GES-1. The biomarker candidate was also expressed oncolon adenocarcinoma cells SW1116and hepatocellular adenocarcinoma cells Hep3B.However, the lysates of normal gastric tissues not comprised the biomarkercandidates which was detected with MS40-3.1. The approximate molecular weight ofthe biomarker candidate was60kDa.
The results of human proteome microarray showed that the both generated mAbsare monospecific antibodies. The biomarker candidate that MS38-2.1specificallybinding to is a small proline-rich protein3(SPRR3). The biomarker candidate thatMS40-3.1specifically binding to is an ornithine decarboxylase1(ODC-1). TheELISA and Western blot assay showed that the favourable immunoreactivitiesbetween human recombinant proteins SPRR3, ODC-1and MS38-2.1, MS40-3.1respectively.
CONCLUSIONS: Both MS38-2.1and MS40-3.1are monospecific antibodies.The biomarker candidates that MS38-2.1, MS40-3.1specifically binding to are somemolecules similar to SPRR3, ODC-1respectively, or at least molecules share thecommon epitopes of SPRR3and ODC-1. According to current results, The twobiomarker candidates might be novel biomarkers expressed on gastric cancer cell surface.
In general, our study has been developed a streamlined strategy for the screeningof the monospecific mAbs against gastric cancer cell surface native conformationalantigens with the combination of viable cell immunization, hybridoma techniques andthe live cell based FACS-HTS assays. The two potential biomarkers on gastric cancercell surface were preliminarily identified and might be applied for diagnosis andprognosis assessment of gastric cancer. The novel approaches developed in our studyand the current results will be of great importance to the research work of molecularbiomarkers of various human malignancies.
目前筛选胃癌分子标志物的方法以传统蛋白质组学策略为主，虽然已鉴定出一些新的胃癌分子标志物，但是由于这些分子特异性较差而停滞于实验室研究阶段，难以临床应用。筛选鉴定特异性强且定位于肿瘤细胞表面的靶点分子标志物将为胃癌的抗体靶向治疗开辟新的思路。
本研究拟采用以制备的抗胃癌单克隆抗体反向筛选鉴定靶抗原的方法，旨在实现“一步法”同时制备抗胃癌细胞表面天然构象抗原的特异性单抗以及鉴定特异性胃癌分子标志物。由于传统的抗体筛选制备方法很难获得识别活细胞表面天然构象抗原的抗体，所以本研究采用“鸟枪法”活细胞免疫小鼠，杂交瘤技术结合流式细胞术高通量筛选的创新方法，在活细胞水平直接筛选抗肿瘤细胞表面天然构象抗原的特异性抗体，在进行抗体的特异性鉴定及分析特异性抗体识别的胃癌细胞表面天然抗原与胃癌临床病理学参数的相关性和临床意义之后，采用人蛋白质组芯片对制备的特异性抗体结合的靶抗原蛋白分子进行快捷鉴定，从而确定新的胃癌细胞表面分子标志物。本研究分为以下4个部分：
一．抗胃癌细胞表面天然抗原特异性抗体的高通量筛选、制备
研究方法：胃癌细胞株（SGC7901、BGC823、MKN28、MKN45）混合后免疫6-8周龄BALB/c小鼠(1×107个细胞)，取脾脏B细胞与SP2/0细胞融合，杂交瘤细胞大批量铺板，选择性培养；利用BD FACS Calibur的高通量检测系统（fluorescence activating cell sorter-highthroughput screening system，FACS-HTS），在活细胞水平通过FACS高通量检测预处理的杂交瘤细胞培养上清与胃癌细胞（免疫用4株胃癌细胞）以及正常对照细胞（健康志愿者外周血单个核细胞，PBMC）的结合反应情况；根据ELISA-HTS(enzyme linked immunosorbentassay-HTS)的基本筛选原则，挑取杂交瘤细胞阳性克隆，再进行FACS交叉鉴定后建立能稳定分泌抗胃癌细胞表面天然构象抗原的单克隆抗体杂交瘤细胞株。制备的单克隆抗体进行亚型鉴定及ProteinA/G亲和层析纯化。
研究结果：杂交瘤技术与活细胞水平FACS-HTS相结合的筛选方法制备获得两株抗胃癌细胞表面天然构象抗原的单克隆抗体，分别命名为MS38-2.1及MS40-3.1（根据高通量筛选鉴定过程中对杂交瘤细胞的特定编号）。两株抗体均为IgG1/κ型。
研究结论：建立了“鸟枪法”胃癌活细胞免疫，杂交瘤技术结合活细胞水平FACS-HTS的创新方法，制备了两株抗胃癌细胞表面天然构象抗原的特异性抗体。本研究建立的创新方法也可用于其它实体肿瘤细胞表面天然构象抗原特异性抗体的制备、筛选。
二．抗胃癌细胞表面天然抗原单抗的特异性鉴定
研究方法：培养38种细胞系，包括15种胃癌细胞，以及结直肠癌、肝细胞肝癌、乳腺癌、胆囊癌以及急性单核细胞白血病细胞，人胎肝细胞和GES-1、EA.hy926细胞，采用间接免疫荧光染色、FACS检测MS38-2.1及MS40-3.1的标记情况。收集12例手术切除的胃癌新鲜组织及同一病例癌旁组织，部分组织分别进行酶消化后制备单细胞悬液，间接免疫荧光染色后FACS检测MS38-2.1及MS40-3.1与肿瘤细胞及癌旁组织细胞的免疫反应情况；剩余新鲜组织常规10%福尔马林固定，石蜡包埋，免疫组化EnVision法检测胃癌和癌旁组织两株抗体的标记情况。
研究结果：FACS结果显示，MS38-2.1与胃癌细胞、GES-1细胞、结肠癌、肝细胞肝癌、L-02细胞、急性单核细胞白血病、乳腺癌以及胆囊癌细胞均有不同强度的结合反应，与EA.hy926细胞也有结合反应；MS40-3.1与胃癌细胞、GES-1细胞、结直肠癌、肝细胞肝癌及L-02细胞均有结合反应；两株抗体与对照细胞（包括健康志愿者PBMC以及正常胃黏膜上皮细胞）均无结合反应。免疫组化结果显示MS38-2.1及MS40-3.1可特异性标记胃癌细胞，并且MS38-2.1可特异性标记中小血管。FACS分析显示MS38-2.1及MS40-3.1与新鲜胃癌以及癌旁组织细胞结合反应的差异具有统计学意义（P <0.05）。
研究结论：MS38-2.1识别的靶抗原蛋白分子在胃癌细胞、GES-1细胞、结肠癌、肝细胞肝癌、L-02细胞、急性单核细胞白血病、乳腺癌、胆囊癌细胞表面均有表达，并且在中小血管内皮细胞也有特异性表达；MS40-3.1识别的靶抗原蛋白分子在胃癌细胞、GES-1细胞、结直肠癌、肝细胞肝癌及L-02细胞表面均有表达。提示MS38-2.1和MS40-3.1均能特异性识别胃癌细胞，与正常胃黏膜上皮细胞无交叉反应。MS38-2.1还可特异性识别中小血管内皮细胞。
三．特异性抗体识别的胃癌细胞表面天然抗原蛋白分子与胃癌临床病理学参数的相关性及临床意义
研究方法：选取90例石蜡包埋的胃癌及对应癌旁组织标本，免疫组化EnVision法检测MS38-2.1及MS40-3.1的标记情况，并分析单抗识别的胃癌细胞表面天然抗原蛋白分子与胃癌临床病理学参数的相关性及临床意义。
研究结果：90例胃癌及对应的癌旁组织中，28例胃癌组织MS38-2.1阳性（31.11%），8例癌旁组织阳性（8.89%），差异具有统计学意义（P＜0.05）；42例胃癌组织MS40-3.1阳性（46.67%），3例癌旁组织阳性（3.33%），差异具有统计学意义（P＜0.05）。胃癌组织MS38-2.1及MS40-3.1标记阳性即MS38-2.1及MS40-3.1特异性识别的靶抗原蛋白分子均与肿瘤浸润深度、淋巴结转移、远处转移及TNM分期有关（P＜0.05），而与年龄、性别、肿瘤大小、肿瘤部位以及肿瘤分化程度无明显相关性。生存分析显示，胃癌组织中MS38-2.1及MS40-3.1高阳性率即MS38-2.1及MS40-3.1特异性识别的靶抗原蛋白分子的高表达均与患者生存时间短有明显相关性（P＜0.0001）。
研究结论：MS38-2.1及MS40-3.1特异性识别的靶抗原蛋白分子在胃癌组织中均特异性高表达，且两个靶抗原蛋白分子的高表达均与胃癌的浸润、转移、临床分期和预后有关，可作为胃癌诊断及预后评估的潜在分子标志物。
四．抗胃癌细胞表面天然抗原特异性抗体结合靶抗原蛋白的鉴定
研究方法：培养胃癌细胞（BGC823、MKN28、SGC7901、MKN45），胃黏膜上皮细胞（GES-1），结肠癌细胞（SW1116），乳腺癌细胞（MCF-7）以及肝癌细胞（Hep3B），Western blot方法检测上述细胞中MS38-2.1及MS40-3.1特异性结合靶抗原蛋白的分子量及表达情况，以正常胃黏膜组织裂解物作为对照。采用包含16,368种不同人全长重组蛋白的蛋白质组芯片，鉴定MS38-2.1及MS40-3.1的特异性以及MS38-2.1及MS40-3.1特异性结合的靶抗原蛋白分子，ELISA及Western blot验证MS38-2.1及MS40-3.1与靶抗原蛋白分子的结合反应。
研究结果：Western blot结果显示，抗胃癌单抗MS38-2.1识别的靶抗原蛋白分子在胃癌细胞BGC823、SGC7901、MKN45以及胃黏膜上皮细胞GES-1细胞表面高表达，在胃癌细胞MKN28细胞表面低表达；在结肠癌细胞SW1116，乳腺癌细胞MCF-7以及肝癌细胞Hep3B细胞表面均有表达；正常胃黏膜组织裂解物中不含这一蛋白分子。上述细胞表达的MS38-2.1特异性结合靶抗原蛋白的分子量约为128kDa。
MS40-3.1识别的靶抗原蛋白分子在胃癌细胞BGC823、SGC7901、MKN28以及MKN45细胞表面高表达；在胃黏膜上皮细胞GES-1细胞表面高表达；在结肠癌细胞SW1116及肝癌细胞Hep3B细胞表面均表达；正常胃黏膜组织裂解物中不含这一蛋白分子。上述细胞表达的MS40-3.1特异性结合靶抗原蛋白的分子量约为60kDa。
人蛋白质组芯片（human proteome microarray）鉴定结果显示，MS38-2.1特异性识别的蛋白分子为富含脯氨酸的小分子蛋白3（small proline-rich protein3，SPRR3）， MS40-3.1特异性识别的蛋白分子为鸟氨酸脱羧酶1（ornithinedecarboxylase1，ODC-1），两株抗体与人蛋白质组芯片中其它细胞蛋白均无交叉反应。ELISA及Western blot显示MS38-2.1与人SPRR3重组蛋白，MS40-3.1与人ODC-1重组蛋白均有良好的特异性结合反应。
研究结论：制备的MS38-2.1及MS40-3.1均为单一特异性单克隆抗体，MS38-2.1、MS40-3.1识别的靶抗原蛋白分子是与SPRR3、ODC-1类似或至少具有相同抗原表位的分子，为两个新的胃癌细胞表面候选分子标志物。
综上所述，本研究建立了“鸟枪法”胃癌活细胞免疫，杂交瘤技术结合活细胞水平FACS-HTS，筛选制备抗胃癌细胞表面天然构象抗原特异性抗体的创新方法；发现了两个新的可作为胃癌诊断及预后评估的胃癌细胞表面分子标志物。本文建立的创新研究方法和结果对于其它恶性肿瘤分子标志物的研究具有重要的借鉴作用和参考价值。
Gastric cancer (GC) is a common neoplasm in digestive system, which is leadingthe cause of cancer-related deaths worldwide. With the progression of earlydiagnostic techniques and therapeutic strategies, the overall survival rate of GCpatients has been improved in recent years. The outcome survival rate of theadvanced GC still remains poor while the5-year overall survival rate is diminishing.In post-genomic era, the discovery and identification of novel GC biomarkers isrequired for more sensitive, specific and functional targets, which could be easilyused for diagnosis and therapeutic purposes. The generaton and the development ofmonoclonal antibody would be played a key role in the basic and clinical researchesof GC.
The development of proteomics has been set the feasible approches to identifythe GC biomarkers and the associated molecules although many known biomarkers ofGC are non-specific and limited. Therefore, the HTS (high throughput screening) andidentification of specific molecules, especially the membrane bound proteins fromdifferent cancer cells, would provide the new hopes besed on monoclonal antibodytechnology to diagnosis and treat the GC patients.
In this study, we have used an antibody-based approach to identify antigen withthe counter screening strategy. The aim of our study was to generate monoclonalantibodies (mAbs) against the native antigens or receptors as the cancer biomarkerson the surface of gastric cancer cells through our unique “one step” colony pickingand HTS screening methods. It is difficult to prepare mAbs against the nativeantigens on cell surface if still using the current screening approaches. Therefore, we have developed a streamlined strategy to make mAbs binding to native antigens oncell surface, which we used live cell immunization(so-called “shot gun” manner) andlive cell screening FACS-HTS (fluorescence activating cell sorter-highthrough putscreening) strategy with the unique hybridoma technology. The identifed and selectedmAbs were implemented in the Immunohistochemstry, ELISA, FACS analysis and ahuman proteome microarray etc. These biomarkers were verified at least twice by thetwo mAbs.
The four sections of our study as follows:
Ⅰ. The specific mAbs against native antigens on cell-surface of gastric cancercells were generated and screened by FACS-HTS
METHODS: Gastric cancer cell lines (SGC7901, BGC823, MKN28, MKN45)were cultured and mixed together to be served as an immunogen in about1×107cellsper mouse were sc.(subcutaneous) and ip.(intraperitoneal) injections into6-8weeksold BALB/C mice. The splenocytes from three times immunized mouse were fusedwith SP2/0myeloma cells and inoculated in96-wells cell culture plates with a largescale manner. The immunoreactivity hybridoma from the culture supernatants of HATconditional media were used to screen with live gastric cancer cells, as well as thenormal control cells (peripheral blood mononuclear cells from healthy individuals) inFACS-HTS system. We selected many positive hybrids according to ELISA-HTSprinciple and double confirm the signals, positive and stable hybridoma clones werevalidated and picked and then the immunoglobulin isotyping were performed. Thepurification of mAbs by Protein A/G-Sepharose chromatography were conducted.
RESULTS: Two mAbs against native antigens on cell-surface of gastric cancercells, termed as MS38-2.1and MS40-3.1, were generated via hybridoma technologyin combination with viable cell-based FACS-HTS. Both of the two mAbs were IgG1/ κ.
CONCLUSIONS: The series data indicated that our strategy for the generationand validation of anti-GC mAbs is working well. The “shot gun” mannerimmunization and the combination screening methods that the supernatants ofhybridoma colonies were for both ELISA-HTS and the live cells based FACS-HTSare the pivotal high throughput approaches in a robust platform for the mAbgenerations that would be specifically for the native antigens on cell surface of gastriccancer and other solid tumors.
Ⅱ. The validation of mAbs specificity against native antigen on the surface ofgastric cancer cells
METHODS: Having38cell lines including15gastric cancer cell lines,colorectal cancer, liver cancer, breast cancer, gallbladder cancer, acute monocyticleukemia and GES-1, EA.hy926cells were used to analyse the binding profiles ofMS38-2.1and MS40-3.1through indirect immunofluorescence (IF) staining andFACS assay. The12fresh tumor tissues and the adjacent normal mucosa tissues werefreshly obtained from the gastric cancer patients who were undertaken the surgeryresection. A proportion of12fresh gastric tissues were separated into single-cellsuspension by a cocktail enzymes digestion that would be immediately for FACSanalysis with indirect IF staining by MS38-2.1and MS40-3.1mAb. Anotherproportion of12fresh gastric tissues were used for immunohistochemical (IHC)analysis with formalin-fixed and paraffin-embedded (FFPE) to identify theimmunoreactivity of two mAbs in tissues.
RESULTS: The MS38-2.1and MS40-3.1mAbs demonstrated bothimmunofluorescence and immunohistochemistry grades from the assays. The twopurified mAbs specifically recognized the native antigens on cell surface of gastric cancer and other malignancies, and the MS38-2.1could interestingly haveimmunoreactivity with EA.hy926cells (human umbilical vein endothelial cells). Thetwo mAbs have no cross reactions to the normal control cells including humanperipheral blood mononuclear cells (PBMC) and normal primary gastric cells. Thesignificant IHC staining on the gastric cancer cells were observed when detected bythe two mAbs and strong staining of small and medium blood vessels were observedwhen detected with MS38-2.1. FACS analysis demonstrated that there weresignificant differences between fresh prepared gastric cancer cells and correspondingadjacent normal gastric cells when detected by MS38-2.1and MS40-3.1mAbsrespectively (P <0.05).
CONCLUSIONS: The target antigen recognized by MS38-2.1was expressed ingastric cancer, colorectal cancer, liver cancer, breast cancer, gallbladder cancer, acutemonocytic leukemia, GES-1and L-02cells, and also in vascular endothelial cells ofsmall and medium blood vessels.
Ⅲ. The correlation and clinical significance between the expression of gastriccancer cell-surfaced native antigens defined by specific mAbs andclinicopathological characteristics of gastric cancer
METHODS:90FFPE gastric cancer tissues and corresponding adjacent normalmucosa tissues were selected to the applications of the immunoreactivity ofMS38-2.1and MS40-3.1mAbs in IHC. The relationship among the staining intensityof two mAbs, clinicopathologic characteristics and prognosis were evaluated.
RESULTS:28in90cases were positive (31.11%) on both gastric cancer tissuesand their small blood vessels, and8in90cases were positive (8.89%) on tumoradjacent tissues and their small blood vessels by MS38-2.1mAb (P<0.05).42in90cases were positive (46.67%) on gastric cancer tissues and only3out of90cases arepositive (3.33%) on tumor adjacent tissues by MS40-3.1mAb (P<0.05). The IHC staining of both MS38-2.1and MS40-3.1mAbs showed the specificifity on thetargets that are related to degrees of tumor invasion, lymph nodes metastasis andTNM stage, but not correlated to age, gender, the size and location of tumor andtumor differentiation. The survival analysis demonstrated that there was a significantcorrelation between short survival and high expression of target antigens thatMS38-2.1and MS40-3.1specifically binding to (P＜0.0001).
CONCLUSIONS: The biomarkers that were recognized by MS38-2.1andMS40-3.1mAbs were both specifically expressed in gastric cancer tissues and relatedto tumor invasion, metastasis, clinical staging and prognosis, which could bepotentially applied to the prognosis assessment and diagnosis of gastric cancer.
Ⅳ. The identification of the native antigens on cell surface of gastric cancerspecifically recognized by mAbs
METHODS: Gastric cancer cell lines (BGC823, MKN28, SGC7901, MKN45),human gastric epithelial cells (GES-1), colon adenocarcinoma cells (SW1116),breast cancer cells (MCF-7) and hepatocellular adenocarcinoma cells (Hep3B) wereall cultured and treated with mammalian cell membrane protein lysis buffer. TheWestern blot method was utilized to determine the expressed proteins and theirmolecular weights of these cell lines, which MS38-2.1and MS40-3.1were used forspecific binding. The lysates of normal gastric tissues were served as the control. The16,368unique full-length human proteins in proteome microarray were used toanalyze the specificity of the two mAbs and to identify the target candidates or theprotein ID of these two mAbs. The immunoreactivity of the potential target antigenswith the two mAbs was validated with ELISA and Western blot.
RESULTS: The results of Western blot suggested the biomarker candidates thatMS38-2.1specifically binding to was highly expressed on cell surface, includinggastric cancer cell lines (BGC823, SGC7901, MKN45) and human gastric epithelial cell line GES-1, but was lowly expressed on gastric cancer cell line MKN28. Inaddition, the biomarker candidate was expressed on colon adenocarcinoma cell lineSW1116, breast cancer cell line MCF-7and hepatocellular adenocarcinoma cell lineHep3B. However, the lysates of normal gastric tissues not comprised the biomarkercandidates which were detected by MS38-2.1. The approximate molecular weight ofthe biomarker candidate was128kDa.
As for MS40-3.1, the Western blot demonstrated that the biomarker candidatethat the mAb could specifically bind to was highly expressed on the cancer cellsurface, including gastric cancer cell lines BGC823, SGC7901, MKN28, MKN45andhuman gastric epithelial cells GES-1. The biomarker candidate was also expressed oncolon adenocarcinoma cells SW1116and hepatocellular adenocarcinoma cells Hep3B.However, the lysates of normal gastric tissues not comprised the biomarkercandidates which was detected with MS40-3.1. The approximate molecular weight ofthe biomarker candidate was60kDa.
The results of human proteome microarray showed that the both generated mAbsare monospecific antibodies. The biomarker candidate that MS38-2.1specificallybinding to is a small proline-rich protein3(SPRR3). The biomarker candidate thatMS40-3.1specifically binding to is an ornithine decarboxylase1(ODC-1). TheELISA and Western blot assay showed that the favourable immunoreactivitiesbetween human recombinant proteins SPRR3, ODC-1and MS38-2.1, MS40-3.1respectively.
CONCLUSIONS: Both MS38-2.1and MS40-3.1are monospecific antibodies.The biomarker candidates that MS38-2.1, MS40-3.1specifically binding to are somemolecules similar to SPRR3, ODC-1respectively, or at least molecules share thecommon epitopes of SPRR3and ODC-1. According to current results, The twobiomarker candidates might be novel biomarkers expressed on gastric cancer cell surface.
In general, our study has been developed a streamlined strategy for the screeningof the monospecific mAbs against gastric cancer cell surface native conformationalantigens with the combination of viable cell immunization, hybridoma techniques andthe live cell based FACS-HTS assays. The two potential biomarkers on gastric cancercell surface were preliminarily identified and might be applied for diagnosis andprognosis assessment of gastric cancer. The novel approaches developed in our studyand the current results will be of great importance to the research work of molecularbiomarkers of various human malignancies.
引文
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