舌鳞癌组织内CEACAM1过表达调控中性粒细胞浸润、功能影响其恶性表型及预后的研究
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摘要
前言
口腔鳞状细胞癌(Oral squamous cell carcinoma, OSCC)属全球十大常见恶性肿瘤之一,也是头颈部最常见的恶性肿瘤。舌鳞状细胞癌(Tongue squamosus cell carcinoma, TSCC)是最常见的口腔鳞癌类型,且由于其手术范围相对局限,肿瘤易发生侵袭、转移,导致舌鳞癌易复发预后不良。因此,深入阐述、揭示舌鳞癌恶性生物学表型产生的机制,寻找有效的阻断靶点是提高舌鳞癌临床治疗疗效的重要途径。
越来越多的资料表明,炎症参与了恶性肿瘤的演进和异质化,并且是被称为肿瘤发展史上的第七大标志。炎细胞的种类繁多,其中中性粒细胞是外周血白细胞中占据最大比例的细胞成分。由于其抗病原微生物特性,中性粒细胞传统上被认为是抗肿瘤的。然而,目前已有很多资料表明,在一系列恶性肿瘤内(如肾癌,肝癌,胃癌等)存在着丰富的中性粒细胞浸润,并且其浸润与较差的临床结局和较短的生存期有关。另外,相关研究还发现中性粒细胞在肿瘤恶性生物学行为中发挥着重要的作用,它可以促进肿瘤的侵袭、转移和血管生成等,因此许多学者称其为肿瘤相关中性粒细胞(Tumor associated neutrophils, TANs)。而中性粒细胞在舌鳞癌组织中的浸润情况及意义却鲜见报道。并且大量中性粒细胞浸润的原因以及中性粒细胞促进肿瘤演进的机制研究甚少。
癌胚抗原相关细胞粘附分子1(CEACAM1)是属于超级球蛋白家族的一名成员,并且广泛表达于多种细胞,包括上皮细胞,内皮细胞,血细胞等。CEACAM1蛋白的功能呈多样化特点,比如调节细胞生长,血管生成,免疫反应,肿瘤侵袭及病原微生物感染等。最近的一个研究表明：细胞因子诱导的角质细胞上高表达的CEACAM1可以促进中性粒细胞的生存；而另外一个研究显示：黑色素瘤细胞表达的CEACAM1可以抑制NK细胞的细胞毒性功能。以上结果提示：肿瘤组织来源的CEACAM1对于调控肿瘤内中性粒细胞的浸润及其功能可能有重要的作用,并从而能影响肿瘤的生物学行为。然而肿瘤细胞来源的CEACAM1蛋白对于中性粒细胞作用的相关研究报道甚少。因此在本课题中,我们利用免疫组化技术研究了舌鳞癌组织中中性粒细胞的浸润及CEACAM1的表达情况,分析了二者相关性,以及二者与病人生存预后的关系。体外实验中,利用慢病毒技术,研究了舌鳞癌细胞系Cal-27过表达CEACAM1-4L和CEACAM1-4S两亚型后对癌细胞自身增殖、侵袭和迁移能力的影响,以及对中性粒细胞的浸润和其功能改变的影响。以期为舌鳞癌恶性生物学行为的遏制找到关键的靶分子。
第一部分
中性粒细胞在舌鳞癌中的浸润及其与CEACAM1表达的相关性研究
目的
1.研究中性粒细胞在舌鳞癌组织中的浸润情况及意义,
2.CEACAM1在舌鳞癌中的表达及意义；
3.探讨CEACAM1的表达与中性粒细胞浸润的相关性。
方法
1.标本收集：收集2005-2010年间,在青岛大学医学院附属医院进行舌鳞癌首次根治性手术切除的肿瘤标本共74例,并在取得书面知情同意书后,对病人进行了随访。所有病人均进行了淋巴结清扫术。所有的病理诊断参照《头颈部肿瘤病理学和遗传学》(世界卫生组织分类及诊断标准)进行。患者术前均未行放疗、化疗以及其它干预治疗。
2.组织芯片和免疫组化：选取74例舌鳞癌组织及17例癌旁组织内典型的区域各两块(避开出血和坏死组织),制作成组织芯片。利用免疫组织化学方法检测了舌鳞癌及癌旁组织中CD15+中性粒细胞的浸润密度和CEACAM1的表达情况,分析了二者与临床病理参数及病人生存预后的关系,以及两者的相关性。
结果1.CEACAM1在舌鳞癌中的表达及与临床病理参数及病人生存的关系。CEACAM1主要表达于癌细胞的胞浆内,少数呈胞膜表达。CEACAM1在舌鳞癌组织中的表达较癌旁组织明显升高(P=0.003)。在淋巴结转移组内CEACAM1的表达高于非淋巴结转移组(P=0.000),并且较高临床分期组的CEACAM1表达亦高于低临床分期组(P=0.001)。而CEACAM1的表达与病人性别、年龄、病理学分级、肿瘤大小及有无复发均无关。单因素及多因素生存分析结果显示：CEACAM1的过表达与较短的累计癌症相关生存期有关(P=0.032),但它不是一个独立的生存预后因子。
2.CD15阳性中性粒细胞在舌鳞癌中的浸润情况及其意义
在舌鳞癌组织中,存在着大量的中性粒细胞浸润,其浸润丰度显著高于癌旁组织(P=0.038)。大量的中性粒细胞浸润与舌鳞癌的高临床分期(P=0.037)、淋巴结转移(P=0.01)和肿瘤复发(P=0.024)有关系,而与病人的性别、年龄、病理学分级机肿瘤大小无关。单因素和多因素生存分析显示：大量中性粒细胞浸润与缩短的累计生存期有关(P=0.020),并且是舌鳞癌病人独立的生存预后因子(P=0.019)。
3.舌鳞癌组织中,CEACAM1的表达与中性粒细胞浸润的关系
利用Spearman等级相关分析发现：CEACAM1在癌细胞上的表达强弱与CD15阳性中性粒细胞浸润的丰度呈正相关(P=0.002)。
结论
在舌鳞癌组织内存在着CEACAM1过表达和CD15阳性中性粒细胞的大量浸润。二者均与较差的临床结果有关系,并且与较短的生存期相关；大量中性粒细胞浸润是舌鳞癌独立的生存预后因子。舌鳞癌内,CEACAM1的高表达与中性粒细胞的浸润丰度呈正相关。
第二部分
过表达CEACAM1对舌鳞癌Cal-27细胞生物学行为的影响
目的
1.研究CEACAM1过表达对Cal-27细胞增殖能力的影响；
2.研究CEACAM1过表达对Cal-27细胞迁移及侵袭能力的影响。
方法
1.细胞培养：舌鳞癌细胞系Cal-27在37℃,5%CO2饱和湿度条件下,常规培养于含10%胎牛血清的DMEM培养液中。
2.CEACAM1-4L及CEACAM1-4S过表达慢病毒载体的构建：其中CEACAM1-4L及-4S的全长cDNA序列由一美国学者赠予,慢病毒的构建及包装工作由上海吉凯公司完成。两种慢病毒载体及空载体分别命名为CC1-4L-LV,CC1-4S-LV和V-LV；
3.将三种慢病毒载体分别转染Ca1-27细胞系,并设立空白对照组(Blank).3-4天后,用荧光显微镜观察慢病毒转染率；并同时提取四组细胞的RNA和蛋白,利用半定量PCR及Western-blot方法验证慢病毒的转染效率；
4.MTT方法检测CEACAM1过表达对Cal-27细胞增殖能力的影响：取指数生长期的CC1-4L-LV,CC1-4S-LV,V-LV和Blank四组细胞,消化计数后按接种于96孔细胞培养板中,培养7天,于第1、3、5、7天,用酶联免疫检测仪检测各组细胞在490nm处的吸光度值。
5.划痕实验观察CEACAM1过表达对Ca1-27细胞迁移能力的影响：取四组Cal-27细胞以相同的细胞密度分别接种入六孔板内,待第二天细胞汇合度达到90%以上时,用枪头进行划痕,PBS冲洗后换成无血清培养基；在24h后,进行拍照分析。
6.Transwell侵袭实验观察CEACAMI过表达对Cal-27细胞侵袭能力的影响：取四组Ca1-27无血清细胞悬液100u1,加入包被有Matrigel胶的上室内,下室加入600μl含10%胎牛血清的DMEM培养液,20h后光学显微镜下计数穿膜细胞数目,评估各组细胞的侵袭能力。
结果
1.CEACAM1-4L和CEACAM1-4S过表达慢病毒转染可以显著升高Cal-27细胞内CEACAM1-4L和-4S的mRNA及蛋白表达的水平；
2.应用MTT法检钡0CEACAM1-4L和CEACM1-4S基因过表达后Cal-27细胞的增殖活性,结果发现：第一天四组细胞的增殖活性未见明显差异,但是从第三天开始,CC1-4L-LV和CC1-4S-LV组的细胞增殖活性较V-LV和Blank组开始下降(P<0.05),随着时间的延长差距增大；
3.划痕实验结果显示：Cal-27细胞过表达CEACAM1-4L后其划痕距离跟V-LV和Blank组相比明显缩短(P<0.05)；而CEACAM1-4S组的划痕距离较V-LV和Blank组相比无明显差别。
4.Transwell侵袭实验结果：与V-LV和Blank组相比,CEACAM1-4L过表达组穿膜的Ca1-27细胞数均明显增多(P<0.05),CEACAM1-4S组与V-LV与Blank组相比穿膜数无明显差异；
结论
1. CEACAM1-4L和CEACAM1-4S过表达慢病毒转染可以明显提高两亚型在Cal-27细胞内的表达水平；
2. CEACAM1-4L和CEACAM1-4S基因在Cal-27细胞内过表达后,其增殖活性减弱；
3. CEACAM1-4L基因在Cal-27细胞内过表达可以增强其迁移及侵袭能力。
第三部分
过表达CEACAM1舌鳞癌Ca1-27细胞对中性粒细胞的影响及机制
目的
1.研究癌细胞内高表达的CEACAM1对中性粒细胞趋化和浸润的影响；
2.研究癌细胞内高表达的CEACAM1对中性粒细胞的功能及类型转化的影响；
方法
1.细胞培养：舌鳞癌细胞系Cal-27在37℃,5%C02饱和湿度条件下,常规培养于含10%胎牛血清的DMEM培养液中。人前髓白血病HL-60细胞在37℃,5%CO2饱和湿度条件下,悬浮培养于IMDM培养基中；
2.利用qRT-PCR方法,检测四组细胞内IL-8,CXCL-6,MCP-1的mRNA表达情况；
3.HL-60细胞系在全反式视黄酸的诱导下,三天后,经流式细胞术检测总CEACAM的表达,发现可以向中性粒细胞系分化,将其命名为iHL-60；
4.将iHL-60细胞与四组Cal-27细胞直接和间接共培养,以探讨CEACAM1在Cal-27过表达后对中性粒细胞功能的影响。
(1)将转染后的四组Cal-27细胞以2×106细胞/ml的密度常规培养于DMEM中,24h后,收集各组细胞的培养上清,经高速离心去除细胞碎片后,与iHL-60细胞共培养。24h后,将各组iHL-60及未共培养的iHL-60,共5组,一块提取总RNA。利用qRT-PCR方法检测五组iHL-60内IL-8,MMP-9,VEGF-A及TNF-α的表达变化。
(2)将四组Cal-27细胞与iHL-60细胞按1：5的比例分别共培养,24h后,利用MTT方法,检测各共培养组内Cal-27细胞在490nm处的吸光度值,以分析iHL-60对各组Cal-27细胞杀伤能力的改变。
5. qRT-PCR方法,检测四组Cal-27细胞内TGF-B及IFN-β的mRNA表达；
6. Elisa法检测慢四组Ca1-27细胞24小时培养上清内TGF-β蛋白的含量；
7.免疫组化方法检测舌鳞癌组织内TGF-β的表达,并分析TGF-β与CEACAM1表达相关性；利用荧光双标观察舌鳞癌内组织内CEACAM1和TGF-β的共定位情况；
8.将上述间接和直接共培养体系内加入TGF-β的中和抗体,再检测五组iHL-60内各种因子的表达情况及其对四组Ca1-27细胞杀伤能力的改变。
结果
1. qRT-PCR结果显示：Ca1-27细胞过表达CEACAM14L后可以上调IL-8和CXCL-6的mRNA表达(P<0.05),而对MCP-1的表达则无显著影响；
2.流式细胞术结果显示：HL-60细胞经全反式视黄酸诱导三天后,其细胞表面总CEACAM的表达显著上调,说明其向中性粒细胞方向分化；
3.Ca1-27细胞与iHL-60细胞共培养结果：
(1)qRT-PCR结果显示：两组细胞间接共培养后,可是iHL-60细胞内IL-8,MMP-9及VEGF-A的mRNA呈上调趋势,而TNF-α的表达则下调。这种上调及下调趋势在CC1-4L-LV和CC1-4S-LV组尤为显著,与V-LV和Blank组相比,差异有统计学意义(P<0.05)。而当各组中加入TGF-β的中和抗体后,则各共培养组之间的差异,以及与未共培养组之间的差异均明显缩小,差别无统计学意义。
(2)MTT结果显示：直接共培养后,CC1-4L-LV和CC1-4S-LV组癌细胞的吸光度值显著高于V-LV和Blank组,即iHL-60对CC1-4L-LV和CC1-4S-LV组癌细胞的杀伤力明显减弱(P<0.05)。而当各组加入TGF-β中和抗体后,iHL-60细胞对各组癌细胞的杀伤能力均明显提高,各组间差别无统计学意义。
4.舌鳞癌组织内CEACAM1表达与TGF-β表达的相关性。
(1) qRT-PCR及Elisa结果显示：Cal-27细胞内CEACAM1-4L和CEACAM1-4S两亚型过表达均能上调TGF-β的mRNA表达及蛋白的分泌。
(2)免疫组化及荧光双标结果显示：TGF-β在舌癌组织的表达高于癌旁组织,并且与CEACAM1的表达成正相关(P=0.014),舌癌组织内存在着CEACAM1和TGF-β表达的共定位现象；
结论
1.CEACAM1-4L在Ca1-27内过表达,可以上调趋化因子IL-8和CXCL-6的mRNA表达。
2.在舌鳞癌组织内CEACAM1的表达与TGF-β的表达呈正相关,存在着共定位现象；CEACAM1-4L和CEACAM1-4S两亚型在Cal-27细胞过表达后,均可以上调TGF-β的mRN/及蛋白水平的表达；
3.CEACAM1在Cal-27细胞内过表达,可以使共培养的iHL-60细胞的肿瘤杀伤能力减弱,并且更容易使其由抗肿瘤类型(N1)向促肿瘤类型(N2)转化。
4.Cal-27细胞内高表达的CEACAM1对中性粒细胞类型转化的促进作用主要是通过上调TGF-β的表达来实现的。
Bankground
Oral squamous cell carcinoma (OSCC) is one of the ten most frequently diagnosed cancers in the world, and is the most common malignant tumor in head and neck. The tongue squamous cells carcinoma (TSCC) is the most common type in OSCC. The prognosis of TSCC is not well due to the relatively limited scope of surgery and the invasive, metastatic and recurrent biological characters of tumor. So it is particularly important to explore the mechanisms of malignant phenotype, and to find the effective blocking target for clinical therapy.
Accumulating studies have suggested that inflammation was involved in cancer progression and was the seventh hallmark of cancer. Inflammatory cells include a great variety of cells. Among them, neutrophils represent the maximum fraction of total circulating leukocytes. Neutrophils are traditionally considered antitumoral in the context of their anti-bacterial functions. However, in a series of tumors (renal cell carcinoma, hepatocellular carcinoma, gastric adenocarcinoma etc), there were abundant neutrophils infiltration and the presence of intratumoral neutrophils was associated with poorer clinical outcomes and cancer-related survival. Some researches demonstrated that neutrophils play important roles in cancer biology. Neutrophils have been reported to be closely associated with tumor progression and tumor vasculature. So they were called tumor-associated neutrophils (TANs) by some researchers. Whereas, the research of neutrophils infiltration in TSCC tissues has rarely been reported. Further more, the mechanism research of neutroophils infiltration and its protumoral effects were relatively rare.
Carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1) belongs to the immunoglobulin superfamily and is expressed on a variety of epithelial, endothelial and hematopoietic cells. CEACAM1protein has multiple functions, such as regulating cell proliferation, angiogenesis, immunoreaction, tumor invasion and infection of microorganisms etc. A recent study showed that cytokine-induced CEACAM1expression on keratinocytes contributes to a prolonged lifespan of neutrophils. Markel G et al. has also demonstrated that CEACAM1from melanoma cells can inhibit cytotoxicity of NK cells in a class I MHC-independent way. The above studies implied that CEACAM1protein in cancerous tissues may have pivotal roles in regulating neutrophils' infiltration and functions, and so as to influence the cancer biology. However, the research of the effect of CEACAM1from tumor cells on neutrophils are limited. In this study, we investigated neutrophils infiltration and CEACAM1expression in TSCC tissues using IHC, analyzed the relationship of them with clinical pathological features and cancer-related survival of patients, and explored the association between them.
Section I
Neutrophils infiltration in tongue squamous cell carcinoma and its relationship with CEACAM1expression on tumor cells
Objectives
1. To explore neutrophils infiltration in TSCC tissues and its clinical significance.
2. To explore CEACAM1expression on TSCC tissues and its clinical significance.
3. To probe the association between neutrophils infiltration and CEACAM1expression on tumor cells.
Materials and methods
1. Patients and specimens. Between2005and2010, a total of74patients underwent primary and curative resection for TSCC at the Affiliated Hospital of Qingdao University were selected for the study population and reviewed retrospectively, after obtaining written informed consent from74patients. All the74patients underwent neck dissection. All the diagnoses were made following the Pathology and Genetics of Head and Neck Tumors of World Health Organization Classification of Tumors. The patients had no treatment of radiotherapy, chemical therapy or other intervention before operation.
2. Tissue Microarray and Immunohistochemistry Tow representative areas (away from necrotic and hemorrhagic materials) in each specimen were premarked and selected for tissue microarray. Immunohistochemistry was used to detect CD15+ neutrophils infiltration and CEACAM1expression on tumor cells. The association of neutrophils infiltration and CEACAM1expression with clinical pathological parameters and cancer-related survival was analysed. The correlation between neutrophils infiltration and CEACAM1expression was also analysed.
Results
1. CEACAM1expression on TSCC and its correlation with clinicopathologic parameters and patients' survival.
IHC results showed that CEACAM1protein expressed mainly on cytoplasm of tumor cells. Some expressed on the cell membrane. The expression of CEACAM1was obviously stronger in TSCC tissues than in peritumoral tissues (P<0.05). In lymph node metastasis group, CEACAM1expression was higher than in without LN metastasis group (P<0.05). Likewise, its expressiom was higher in stage Ⅲ/Ⅳ groups than in stage Ⅰ/Ⅱ groups (P<0.05). While there were no correlation between CEACAM1expression with patients'gender, age, grade, tumor size and recurrence. Univariant and multivariant survival analysis revealed that overexpression of CEACAM1was associated with shorter cancer-related survival, but not an independent prognostic factor.
2. CD15+neutrophils infiltration in TSCC and its clinical significance. There were abundant neutrophils infiltration in TSCC. The density of neutrophils was higher in TSCC than in peritumoral tissues. Abundant neutrophils infiltration was associated with higher clinical stage, LN metastasis and tumor recurrence. While there was no correlation between neutrophils infiltration with patients'gender, age, grade and tumor size. Univariant and multivariant survival analysis revealed that abuandant neutrophils infiltration was associated with shorter cancer-related survival, and was an independent prognostic factor.
3. Correlation of neutrophils infiltration with CEACAM1expression on tumor cells. Using Spearman's rho coefficient test, we found that there was a positive correlation between CEACAM1expression and neutrophils infiltration.
Conclusions
There were overexpression of CEACAM1protein and abuandant neutrophils infiltration in TSCC tissues. Both of them was associated with poor clinical outcomes and shorter cancer-related survival. What's more, abundant neutrophils infiltration was an independent prognostic factor. There was a positive correlation between CEACAM1expression and neutrophils infiltration in TSCC tissues.
Section Ⅱ
The effect of CEACAMl overexpression on the biological behavior of the tongue squamous cell carcinoma Cal-27cells
Objective
1. To detect the effect of CEACAM1overexpression on proliferation of Cal-27cells;
2. To detect the effect of CEACAM1overexpression on invasion and migration of Cal-27cells;
Materials and methods
1.Cell culture:human tongue squamous cell carcinoma cell lines (Cal-27cells) were routinely cultured in DMEM containing10%fetal bovine serum, at37℃in a humidified air atmosphere containing5%carbon dioxide.
2. Construction of CEACAM1-4L and CEACAM1-4S overexpression Lenti-virus vectors. The cDNA sequence of CEACAM1-4L and CEACAM1-4S was a kind gift from an American researcher. The vector construction and virus packaging was finished by Shanghai Genechem Company. The three kinds of Lenti-virus vector were named as CC1-4L-LV,CC1-4S-LV and V-LV respectively.
3. Transfection of the three kinds of Lenti-virus into Cal-27cells. Use untrasfected cells cells as the blank control. After3or4day, using semiquantitative RT-PCR and Western blot to testify the transfection efficiency.
4. Cell proliferation assay MTT was used to detect cell proliferation in CC1-4L-LV,CC1-4S-LV, V-LV and Blank groups. After digestion and counting, the cells in four groups were inoculated into a96-well plate. Then the growth curve was draw after1,3,5,7days to assess Tca8113cell proliferation activity.
5. Wound healing assay The Cal-27cells in4groups were inoculated into a6-well plate. When the cells got90%confluence, made a linear scratch in each well with a tip. After24h, took photos in each well and analysed the healing length.
6. Transwell invasion assay The Cal-27cells in4groups were added into the transwell inserts and were incubated for20hours. After incubation, the invaded cells were counted in five fields for each filter under a light microscope at400x magnification.
Results
1. Transfection of CEACAM1-4L and CEACAM1-4S overexpression Lenti-virus vector could enhance the expression level of CEACAM1-4L and CEACAM1-4S both in mRNA and protein level, using RT-PCR and Western blot analysis.
2. MTT result showed that proliferative activity of the four Cal-27cells had no obvious difference in the firstday. However, CC1-4L-LV and CC1-4S-LV cells grew more slowly than V-LV and Blank group after3,5,7days (P<0.05). There was no significant difference between the V-LV and Blank group.
3. Wound healing assay result showed that the scratch distance was obviously shorter in CC1-4L-LV group than V-LV and Blank groups (P<0.05) after24h. There was no significant difference between CC1-4S-LV and V-LV, Blank group.
4. Transwell invasion assay results showed that the count of the invaded cells was obviously higher in CC1-4L-LV group than in V-LV and Blank group (P<0.05). There was no significant difference between CC1-4S-LV and V-LV, Blank group.
Conclusion
1. Transfection of CEACAM1-4L and CEACAM1-4S overexpression Lenti-virus vector could obviously enhance the expression level of CEACAM1-4L and CEACAM1-4S both in mRNA and protein level.
2. Overexpression of CEACAM1-4L and CEACAM1-4S in Cal-27cells could inhibit cell proliferation.
3. Overexpression of CEACAM1-4L in Cal-27cells could enhance the ability of cell migration and invasion.
Section Ⅲ
The effect of CEACAM1overexpession in Cal-27on neutrophilsand its mechanisms
Objective
1. To explore the effect of CEACAM1overexpession in Cal-27on neutrophils infiltration;
2. to probe the possible effect of CEACAM1overexpession in Cal-27on the function and type change of neutrophils.
Materials and methods
1.Cell culture:
1) Human tongue squamous cell carcinoma cell line (Cal-27cells) were routinely cultured in DMEM containing10%fetal bovine serum, at37℃in a humidified air atmosphere containing5%carbon dioxide.
2) Human pro-myeloid cell line (HL-60) were routinely suspended cultured in IMEM containing10%fetal bovine serum, at37℃in a humidified air atmosphere containing5%carbon dioxide.
2. Quantitative real-time RT-PCR(qRT-PCR) was used to detect the mRNA expression of IL-8, CXCL-6and MCP-1.
3. HL-60cells was induced by all trans retinoid acid for3days. Flow cytometry (FC) was used to detect the total CEACAM expression on HL-60cell surface. HL-60cells could be induced toward neutrophils differentiation, and was named as iHL-60.
4. Direct and indirect coculture of4groups of Cal-27and iHL-60cells.
(1) Supernatant of the4group cells was collected. After centrifugation, the supernatant was use to coculturwith iHL-60. After24h, the total RNA of iHL-60from4groups and untreated iHL-60was extracted. qRT-PCR was used to detect the mRNA expression of IL-8, MMP-9, VEGF and TNF-αin5groups of iHL-60.
(2) Let iHL-60cells directed co-cultured with4groups of Cal-27cells in a ratio of1:5. After24h, MTT was used to detect the490nm absorbance values of Cal-27cells in different groups.
5. qRT-PCR was used to detect the mRNA expression of TGF-β and IFN-β.
6. Eliasa was used to detect the secretory TGF-βprotein in4group of Cal-27.
7. IHC was used to exlore the TGF-β protein expression on TSCC. The correlation of TGF-p and CEACAM1expression was evaluated. Immunofluorescent double-labeling was used to observe the co-localization of TGF-p and CEACAM1.
8. Add TGF-P neutrolizing antibodies to the above direct and indirect coculture systems, and redetect the mRNA expression of various factors in iHL-60and its tumor-killing ability in different groups.
Results
1. qRT-PCR result showed that overexpression of CEACAM1-4L could upregualte mRNA expression of IL-8and CXCL-6, while has no influence to MCP-1.
2. FC result showed that after induction of all trans retinoid acid for3days, the CEACAM expression on HL-60cell surface was obviously upregualted, which was a marker of neutrophil differentiation.
3. Results of direct and indirect coculture of Cal-27and iHL-60.
(1) qRT-PCR result showed that co-culture with tumor cells could upregulate the mRAN expression of IL-8, MMP-9and VEGF and downregulate TNF-ain iHL-60cells.This trend was particularly obvious in CC1-4L-LV and CC1-4S-LV group,and the difference had statistical significane compared with V-LV and Blank group (P<0.05). After adding of TGF-β neutrolizing antibodies, the differences in different groups was obviously reduced, and had no statistical significance.
(2) MTT result showed that co-culture with tumor cells from CC1-4L-LV and CC1-4S-LV group could weaken the tumor-killing ability of iHL-60compared with V-LV and Blank group (P<0.05). After adding of TGF-β neutrolizing antibodies, the tumor-killing ability of iHL-60was obviously enhanced in all groups. And the differences in various groups was obviously reduced, and had no statistical significance.
4. Correlation of CEACAM1exprssion and TGF-β expression.
(1) qRT-PCR and Elisa results showed that overexpression of both CEACAM1-4L and-4S could upregulate mRNA and protein expression of TGF-β.
(2) IHC result showed that TGF-βexpression was higher in TSCC than in peritumoral tissues, and was positively related to CEACAM1expression. Immunofluorescent double-labeling result showed that there were co-localization of TGF-β and CEACAM1.
Conclusion
1. Overexpression of CEACAM1-4L could upregulate mRNA expression of IL-8and CXCL-6.
2. In TSCC tissues, there was a positive relationship between CEACAM1and TGF-β expression, and had co-localization of them two. Overexpression of both CEACAM1-4L and CEACAM1-4S could upregulate TGF-β expression.
3. Overexpression of CEACAM1in Cal-27cells could weaken the tumor-killing ability of iHL-60that Co-cultured with tumor cells, and can more easily to transform iHL-60cells from antitumoral type (N1) to protumoral type (N2).
4. The effect of CE AC AMI overexpression in Cal-27on the type conversion of iHL-60was mainly through upregulating TGF-β.
口腔鳞状细胞癌(Oral squamous cell carcinoma, OSCC)属全球十大常见恶性肿瘤之一,也是头颈部最常见的恶性肿瘤。舌鳞状细胞癌(Tongue squamosus cell carcinoma, TSCC)是最常见的口腔鳞癌类型,且由于其手术范围相对局限,肿瘤易发生侵袭、转移,导致舌鳞癌易复发预后不良。因此,深入阐述、揭示舌鳞癌恶性生物学表型产生的机制,寻找有效的阻断靶点是提高舌鳞癌临床治疗疗效的重要途径。
越来越多的资料表明,炎症参与了恶性肿瘤的演进和异质化,并且是被称为肿瘤发展史上的第七大标志。炎细胞的种类繁多,其中中性粒细胞是外周血白细胞中占据最大比例的细胞成分。由于其抗病原微生物特性,中性粒细胞传统上被认为是抗肿瘤的。然而,目前已有很多资料表明,在一系列恶性肿瘤内(如肾癌,肝癌,胃癌等)存在着丰富的中性粒细胞浸润,并且其浸润与较差的临床结局和较短的生存期有关。另外,相关研究还发现中性粒细胞在肿瘤恶性生物学行为中发挥着重要的作用,它可以促进肿瘤的侵袭、转移和血管生成等,因此许多学者称其为肿瘤相关中性粒细胞(Tumor associated neutrophils, TANs)。而中性粒细胞在舌鳞癌组织中的浸润情况及意义却鲜见报道。并且大量中性粒细胞浸润的原因以及中性粒细胞促进肿瘤演进的机制研究甚少。
癌胚抗原相关细胞粘附分子1(CEACAM1)是属于超级球蛋白家族的一名成员,并且广泛表达于多种细胞,包括上皮细胞,内皮细胞,血细胞等。CEACAM1蛋白的功能呈多样化特点,比如调节细胞生长,血管生成,免疫反应,肿瘤侵袭及病原微生物感染等。最近的一个研究表明：细胞因子诱导的角质细胞上高表达的CEACAM1可以促进中性粒细胞的生存；而另外一个研究显示：黑色素瘤细胞表达的CEACAM1可以抑制NK细胞的细胞毒性功能。以上结果提示：肿瘤组织来源的CEACAM1对于调控肿瘤内中性粒细胞的浸润及其功能可能有重要的作用,并从而能影响肿瘤的生物学行为。然而肿瘤细胞来源的CEACAM1蛋白对于中性粒细胞作用的相关研究报道甚少。因此在本课题中,我们利用免疫组化技术研究了舌鳞癌组织中中性粒细胞的浸润及CEACAM1的表达情况,分析了二者相关性,以及二者与病人生存预后的关系。体外实验中,利用慢病毒技术,研究了舌鳞癌细胞系Cal-27过表达CEACAM1-4L和CEACAM1-4S两亚型后对癌细胞自身增殖、侵袭和迁移能力的影响,以及对中性粒细胞的浸润和其功能改变的影响。以期为舌鳞癌恶性生物学行为的遏制找到关键的靶分子。
第一部分
中性粒细胞在舌鳞癌中的浸润及其与CEACAM1表达的相关性研究
目的
1.研究中性粒细胞在舌鳞癌组织中的浸润情况及意义,
2.CEACAM1在舌鳞癌中的表达及意义；
3.探讨CEACAM1的表达与中性粒细胞浸润的相关性。
方法
1.标本收集：收集2005-2010年间,在青岛大学医学院附属医院进行舌鳞癌首次根治性手术切除的肿瘤标本共74例,并在取得书面知情同意书后,对病人进行了随访。所有病人均进行了淋巴结清扫术。所有的病理诊断参照《头颈部肿瘤病理学和遗传学》(世界卫生组织分类及诊断标准)进行。患者术前均未行放疗、化疗以及其它干预治疗。
2.组织芯片和免疫组化：选取74例舌鳞癌组织及17例癌旁组织内典型的区域各两块(避开出血和坏死组织),制作成组织芯片。利用免疫组织化学方法检测了舌鳞癌及癌旁组织中CD15+中性粒细胞的浸润密度和CEACAM1的表达情况,分析了二者与临床病理参数及病人生存预后的关系,以及两者的相关性。
结果1.CEACAM1在舌鳞癌中的表达及与临床病理参数及病人生存的关系。CEACAM1主要表达于癌细胞的胞浆内,少数呈胞膜表达。CEACAM1在舌鳞癌组织中的表达较癌旁组织明显升高(P=0.003)。在淋巴结转移组内CEACAM1的表达高于非淋巴结转移组(P=0.000),并且较高临床分期组的CEACAM1表达亦高于低临床分期组(P=0.001)。而CEACAM1的表达与病人性别、年龄、病理学分级、肿瘤大小及有无复发均无关。单因素及多因素生存分析结果显示：CEACAM1的过表达与较短的累计癌症相关生存期有关(P=0.032),但它不是一个独立的生存预后因子。
2.CD15阳性中性粒细胞在舌鳞癌中的浸润情况及其意义
在舌鳞癌组织中,存在着大量的中性粒细胞浸润,其浸润丰度显著高于癌旁组织(P=0.038)。大量的中性粒细胞浸润与舌鳞癌的高临床分期(P=0.037)、淋巴结转移(P=0.01)和肿瘤复发(P=0.024)有关系,而与病人的性别、年龄、病理学分级机肿瘤大小无关。单因素和多因素生存分析显示：大量中性粒细胞浸润与缩短的累计生存期有关(P=0.020),并且是舌鳞癌病人独立的生存预后因子(P=0.019)。
3.舌鳞癌组织中,CEACAM1的表达与中性粒细胞浸润的关系
利用Spearman等级相关分析发现：CEACAM1在癌细胞上的表达强弱与CD15阳性中性粒细胞浸润的丰度呈正相关(P=0.002)。
结论
在舌鳞癌组织内存在着CEACAM1过表达和CD15阳性中性粒细胞的大量浸润。二者均与较差的临床结果有关系,并且与较短的生存期相关；大量中性粒细胞浸润是舌鳞癌独立的生存预后因子。舌鳞癌内,CEACAM1的高表达与中性粒细胞的浸润丰度呈正相关。
第二部分
过表达CEACAM1对舌鳞癌Cal-27细胞生物学行为的影响
目的
1.研究CEACAM1过表达对Cal-27细胞增殖能力的影响；
2.研究CEACAM1过表达对Cal-27细胞迁移及侵袭能力的影响。
方法
1.细胞培养：舌鳞癌细胞系Cal-27在37℃,5%CO2饱和湿度条件下,常规培养于含10%胎牛血清的DMEM培养液中。
2.CEACAM1-4L及CEACAM1-4S过表达慢病毒载体的构建：其中CEACAM1-4L及-4S的全长cDNA序列由一美国学者赠予,慢病毒的构建及包装工作由上海吉凯公司完成。两种慢病毒载体及空载体分别命名为CC1-4L-LV,CC1-4S-LV和V-LV；
3.将三种慢病毒载体分别转染Ca1-27细胞系,并设立空白对照组(Blank).3-4天后,用荧光显微镜观察慢病毒转染率；并同时提取四组细胞的RNA和蛋白,利用半定量PCR及Western-blot方法验证慢病毒的转染效率；
4.MTT方法检测CEACAM1过表达对Cal-27细胞增殖能力的影响：取指数生长期的CC1-4L-LV,CC1-4S-LV,V-LV和Blank四组细胞,消化计数后按接种于96孔细胞培养板中,培养7天,于第1、3、5、7天,用酶联免疫检测仪检测各组细胞在490nm处的吸光度值。
5.划痕实验观察CEACAM1过表达对Ca1-27细胞迁移能力的影响：取四组Cal-27细胞以相同的细胞密度分别接种入六孔板内,待第二天细胞汇合度达到90%以上时,用枪头进行划痕,PBS冲洗后换成无血清培养基；在24h后,进行拍照分析。
6.Transwell侵袭实验观察CEACAMI过表达对Cal-27细胞侵袭能力的影响：取四组Ca1-27无血清细胞悬液100u1,加入包被有Matrigel胶的上室内,下室加入600μl含10%胎牛血清的DMEM培养液,20h后光学显微镜下计数穿膜细胞数目,评估各组细胞的侵袭能力。
结果
1.CEACAM1-4L和CEACAM1-4S过表达慢病毒转染可以显著升高Cal-27细胞内CEACAM1-4L和-4S的mRNA及蛋白表达的水平；
2.应用MTT法检钡0CEACAM1-4L和CEACM1-4S基因过表达后Cal-27细胞的增殖活性,结果发现：第一天四组细胞的增殖活性未见明显差异,但是从第三天开始,CC1-4L-LV和CC1-4S-LV组的细胞增殖活性较V-LV和Blank组开始下降(P<0.05),随着时间的延长差距增大；
3.划痕实验结果显示：Cal-27细胞过表达CEACAM1-4L后其划痕距离跟V-LV和Blank组相比明显缩短(P<0.05)；而CEACAM1-4S组的划痕距离较V-LV和Blank组相比无明显差别。
4.Transwell侵袭实验结果：与V-LV和Blank组相比,CEACAM1-4L过表达组穿膜的Ca1-27细胞数均明显增多(P<0.05),CEACAM1-4S组与V-LV与Blank组相比穿膜数无明显差异；
结论
1. CEACAM1-4L和CEACAM1-4S过表达慢病毒转染可以明显提高两亚型在Cal-27细胞内的表达水平；
2. CEACAM1-4L和CEACAM1-4S基因在Cal-27细胞内过表达后,其增殖活性减弱；
3. CEACAM1-4L基因在Cal-27细胞内过表达可以增强其迁移及侵袭能力。
第三部分
过表达CEACAM1舌鳞癌Ca1-27细胞对中性粒细胞的影响及机制
目的
1.研究癌细胞内高表达的CEACAM1对中性粒细胞趋化和浸润的影响；
2.研究癌细胞内高表达的CEACAM1对中性粒细胞的功能及类型转化的影响；
方法
1.细胞培养：舌鳞癌细胞系Cal-27在37℃,5%C02饱和湿度条件下,常规培养于含10%胎牛血清的DMEM培养液中。人前髓白血病HL-60细胞在37℃,5%CO2饱和湿度条件下,悬浮培养于IMDM培养基中；
2.利用qRT-PCR方法,检测四组细胞内IL-8,CXCL-6,MCP-1的mRNA表达情况；
3.HL-60细胞系在全反式视黄酸的诱导下,三天后,经流式细胞术检测总CEACAM的表达,发现可以向中性粒细胞系分化,将其命名为iHL-60；
4.将iHL-60细胞与四组Cal-27细胞直接和间接共培养,以探讨CEACAM1在Cal-27过表达后对中性粒细胞功能的影响。
(1)将转染后的四组Cal-27细胞以2×106细胞/ml的密度常规培养于DMEM中,24h后,收集各组细胞的培养上清,经高速离心去除细胞碎片后,与iHL-60细胞共培养。24h后,将各组iHL-60及未共培养的iHL-60,共5组,一块提取总RNA。利用qRT-PCR方法检测五组iHL-60内IL-8,MMP-9,VEGF-A及TNF-α的表达变化。
(2)将四组Cal-27细胞与iHL-60细胞按1：5的比例分别共培养,24h后,利用MTT方法,检测各共培养组内Cal-27细胞在490nm处的吸光度值,以分析iHL-60对各组Cal-27细胞杀伤能力的改变。
5. qRT-PCR方法,检测四组Cal-27细胞内TGF-B及IFN-β的mRNA表达；
6. Elisa法检测慢四组Ca1-27细胞24小时培养上清内TGF-β蛋白的含量；
7.免疫组化方法检测舌鳞癌组织内TGF-β的表达,并分析TGF-β与CEACAM1表达相关性；利用荧光双标观察舌鳞癌内组织内CEACAM1和TGF-β的共定位情况；
8.将上述间接和直接共培养体系内加入TGF-β的中和抗体,再检测五组iHL-60内各种因子的表达情况及其对四组Ca1-27细胞杀伤能力的改变。
结果
1. qRT-PCR结果显示：Ca1-27细胞过表达CEACAM14L后可以上调IL-8和CXCL-6的mRNA表达(P<0.05),而对MCP-1的表达则无显著影响；
2.流式细胞术结果显示：HL-60细胞经全反式视黄酸诱导三天后,其细胞表面总CEACAM的表达显著上调,说明其向中性粒细胞方向分化；
3.Ca1-27细胞与iHL-60细胞共培养结果：
(1)qRT-PCR结果显示：两组细胞间接共培养后,可是iHL-60细胞内IL-8,MMP-9及VEGF-A的mRNA呈上调趋势,而TNF-α的表达则下调。这种上调及下调趋势在CC1-4L-LV和CC1-4S-LV组尤为显著,与V-LV和Blank组相比,差异有统计学意义(P<0.05)。而当各组中加入TGF-β的中和抗体后,则各共培养组之间的差异,以及与未共培养组之间的差异均明显缩小,差别无统计学意义。
(2)MTT结果显示：直接共培养后,CC1-4L-LV和CC1-4S-LV组癌细胞的吸光度值显著高于V-LV和Blank组,即iHL-60对CC1-4L-LV和CC1-4S-LV组癌细胞的杀伤力明显减弱(P<0.05)。而当各组加入TGF-β中和抗体后,iHL-60细胞对各组癌细胞的杀伤能力均明显提高,各组间差别无统计学意义。
4.舌鳞癌组织内CEACAM1表达与TGF-β表达的相关性。
(1) qRT-PCR及Elisa结果显示：Cal-27细胞内CEACAM1-4L和CEACAM1-4S两亚型过表达均能上调TGF-β的mRNA表达及蛋白的分泌。
(2)免疫组化及荧光双标结果显示：TGF-β在舌癌组织的表达高于癌旁组织,并且与CEACAM1的表达成正相关(P=0.014),舌癌组织内存在着CEACAM1和TGF-β表达的共定位现象；
结论
1.CEACAM1-4L在Ca1-27内过表达,可以上调趋化因子IL-8和CXCL-6的mRNA表达。
2.在舌鳞癌组织内CEACAM1的表达与TGF-β的表达呈正相关,存在着共定位现象；CEACAM1-4L和CEACAM1-4S两亚型在Cal-27细胞过表达后,均可以上调TGF-β的mRN/及蛋白水平的表达；
3.CEACAM1在Cal-27细胞内过表达,可以使共培养的iHL-60细胞的肿瘤杀伤能力减弱,并且更容易使其由抗肿瘤类型(N1)向促肿瘤类型(N2)转化。
4.Cal-27细胞内高表达的CEACAM1对中性粒细胞类型转化的促进作用主要是通过上调TGF-β的表达来实现的。
Bankground
Oral squamous cell carcinoma (OSCC) is one of the ten most frequently diagnosed cancers in the world, and is the most common malignant tumor in head and neck. The tongue squamous cells carcinoma (TSCC) is the most common type in OSCC. The prognosis of TSCC is not well due to the relatively limited scope of surgery and the invasive, metastatic and recurrent biological characters of tumor. So it is particularly important to explore the mechanisms of malignant phenotype, and to find the effective blocking target for clinical therapy.
Accumulating studies have suggested that inflammation was involved in cancer progression and was the seventh hallmark of cancer. Inflammatory cells include a great variety of cells. Among them, neutrophils represent the maximum fraction of total circulating leukocytes. Neutrophils are traditionally considered antitumoral in the context of their anti-bacterial functions. However, in a series of tumors (renal cell carcinoma, hepatocellular carcinoma, gastric adenocarcinoma etc), there were abundant neutrophils infiltration and the presence of intratumoral neutrophils was associated with poorer clinical outcomes and cancer-related survival. Some researches demonstrated that neutrophils play important roles in cancer biology. Neutrophils have been reported to be closely associated with tumor progression and tumor vasculature. So they were called tumor-associated neutrophils (TANs) by some researchers. Whereas, the research of neutrophils infiltration in TSCC tissues has rarely been reported. Further more, the mechanism research of neutroophils infiltration and its protumoral effects were relatively rare.
Carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1) belongs to the immunoglobulin superfamily and is expressed on a variety of epithelial, endothelial and hematopoietic cells. CEACAM1protein has multiple functions, such as regulating cell proliferation, angiogenesis, immunoreaction, tumor invasion and infection of microorganisms etc. A recent study showed that cytokine-induced CEACAM1expression on keratinocytes contributes to a prolonged lifespan of neutrophils. Markel G et al. has also demonstrated that CEACAM1from melanoma cells can inhibit cytotoxicity of NK cells in a class I MHC-independent way. The above studies implied that CEACAM1protein in cancerous tissues may have pivotal roles in regulating neutrophils' infiltration and functions, and so as to influence the cancer biology. However, the research of the effect of CEACAM1from tumor cells on neutrophils are limited. In this study, we investigated neutrophils infiltration and CEACAM1expression in TSCC tissues using IHC, analyzed the relationship of them with clinical pathological features and cancer-related survival of patients, and explored the association between them.
Section I
Neutrophils infiltration in tongue squamous cell carcinoma and its relationship with CEACAM1expression on tumor cells
Objectives
1. To explore neutrophils infiltration in TSCC tissues and its clinical significance.
2. To explore CEACAM1expression on TSCC tissues and its clinical significance.
3. To probe the association between neutrophils infiltration and CEACAM1expression on tumor cells.
Materials and methods
1. Patients and specimens. Between2005and2010, a total of74patients underwent primary and curative resection for TSCC at the Affiliated Hospital of Qingdao University were selected for the study population and reviewed retrospectively, after obtaining written informed consent from74patients. All the74patients underwent neck dissection. All the diagnoses were made following the Pathology and Genetics of Head and Neck Tumors of World Health Organization Classification of Tumors. The patients had no treatment of radiotherapy, chemical therapy or other intervention before operation.
2. Tissue Microarray and Immunohistochemistry Tow representative areas (away from necrotic and hemorrhagic materials) in each specimen were premarked and selected for tissue microarray. Immunohistochemistry was used to detect CD15+ neutrophils infiltration and CEACAM1expression on tumor cells. The association of neutrophils infiltration and CEACAM1expression with clinical pathological parameters and cancer-related survival was analysed. The correlation between neutrophils infiltration and CEACAM1expression was also analysed.
Results
1. CEACAM1expression on TSCC and its correlation with clinicopathologic parameters and patients' survival.
IHC results showed that CEACAM1protein expressed mainly on cytoplasm of tumor cells. Some expressed on the cell membrane. The expression of CEACAM1was obviously stronger in TSCC tissues than in peritumoral tissues (P<0.05). In lymph node metastasis group, CEACAM1expression was higher than in without LN metastasis group (P<0.05). Likewise, its expressiom was higher in stage Ⅲ/Ⅳ groups than in stage Ⅰ/Ⅱ groups (P<0.05). While there were no correlation between CEACAM1expression with patients'gender, age, grade, tumor size and recurrence. Univariant and multivariant survival analysis revealed that overexpression of CEACAM1was associated with shorter cancer-related survival, but not an independent prognostic factor.
2. CD15+neutrophils infiltration in TSCC and its clinical significance. There were abundant neutrophils infiltration in TSCC. The density of neutrophils was higher in TSCC than in peritumoral tissues. Abundant neutrophils infiltration was associated with higher clinical stage, LN metastasis and tumor recurrence. While there was no correlation between neutrophils infiltration with patients'gender, age, grade and tumor size. Univariant and multivariant survival analysis revealed that abuandant neutrophils infiltration was associated with shorter cancer-related survival, and was an independent prognostic factor.
3. Correlation of neutrophils infiltration with CEACAM1expression on tumor cells. Using Spearman's rho coefficient test, we found that there was a positive correlation between CEACAM1expression and neutrophils infiltration.
Conclusions
There were overexpression of CEACAM1protein and abuandant neutrophils infiltration in TSCC tissues. Both of them was associated with poor clinical outcomes and shorter cancer-related survival. What's more, abundant neutrophils infiltration was an independent prognostic factor. There was a positive correlation between CEACAM1expression and neutrophils infiltration in TSCC tissues.
Section Ⅱ
The effect of CEACAMl overexpression on the biological behavior of the tongue squamous cell carcinoma Cal-27cells
Objective
1. To detect the effect of CEACAM1overexpression on proliferation of Cal-27cells;
2. To detect the effect of CEACAM1overexpression on invasion and migration of Cal-27cells;
Materials and methods
1.Cell culture:human tongue squamous cell carcinoma cell lines (Cal-27cells) were routinely cultured in DMEM containing10%fetal bovine serum, at37℃in a humidified air atmosphere containing5%carbon dioxide.
2. Construction of CEACAM1-4L and CEACAM1-4S overexpression Lenti-virus vectors. The cDNA sequence of CEACAM1-4L and CEACAM1-4S was a kind gift from an American researcher. The vector construction and virus packaging was finished by Shanghai Genechem Company. The three kinds of Lenti-virus vector were named as CC1-4L-LV,CC1-4S-LV and V-LV respectively.
3. Transfection of the three kinds of Lenti-virus into Cal-27cells. Use untrasfected cells cells as the blank control. After3or4day, using semiquantitative RT-PCR and Western blot to testify the transfection efficiency.
4. Cell proliferation assay MTT was used to detect cell proliferation in CC1-4L-LV,CC1-4S-LV, V-LV and Blank groups. After digestion and counting, the cells in four groups were inoculated into a96-well plate. Then the growth curve was draw after1,3,5,7days to assess Tca8113cell proliferation activity.
5. Wound healing assay The Cal-27cells in4groups were inoculated into a6-well plate. When the cells got90%confluence, made a linear scratch in each well with a tip. After24h, took photos in each well and analysed the healing length.
6. Transwell invasion assay The Cal-27cells in4groups were added into the transwell inserts and were incubated for20hours. After incubation, the invaded cells were counted in five fields for each filter under a light microscope at400x magnification.
Results
1. Transfection of CEACAM1-4L and CEACAM1-4S overexpression Lenti-virus vector could enhance the expression level of CEACAM1-4L and CEACAM1-4S both in mRNA and protein level, using RT-PCR and Western blot analysis.
2. MTT result showed that proliferative activity of the four Cal-27cells had no obvious difference in the firstday. However, CC1-4L-LV and CC1-4S-LV cells grew more slowly than V-LV and Blank group after3,5,7days (P<0.05). There was no significant difference between the V-LV and Blank group.
3. Wound healing assay result showed that the scratch distance was obviously shorter in CC1-4L-LV group than V-LV and Blank groups (P<0.05) after24h. There was no significant difference between CC1-4S-LV and V-LV, Blank group.
4. Transwell invasion assay results showed that the count of the invaded cells was obviously higher in CC1-4L-LV group than in V-LV and Blank group (P<0.05). There was no significant difference between CC1-4S-LV and V-LV, Blank group.
Conclusion
1. Transfection of CEACAM1-4L and CEACAM1-4S overexpression Lenti-virus vector could obviously enhance the expression level of CEACAM1-4L and CEACAM1-4S both in mRNA and protein level.
2. Overexpression of CEACAM1-4L and CEACAM1-4S in Cal-27cells could inhibit cell proliferation.
3. Overexpression of CEACAM1-4L in Cal-27cells could enhance the ability of cell migration and invasion.
Section Ⅲ
The effect of CEACAM1overexpession in Cal-27on neutrophilsand its mechanisms
Objective
1. To explore the effect of CEACAM1overexpession in Cal-27on neutrophils infiltration;
2. to probe the possible effect of CEACAM1overexpession in Cal-27on the function and type change of neutrophils.
Materials and methods
1.Cell culture:
1) Human tongue squamous cell carcinoma cell line (Cal-27cells) were routinely cultured in DMEM containing10%fetal bovine serum, at37℃in a humidified air atmosphere containing5%carbon dioxide.
2) Human pro-myeloid cell line (HL-60) were routinely suspended cultured in IMEM containing10%fetal bovine serum, at37℃in a humidified air atmosphere containing5%carbon dioxide.
2. Quantitative real-time RT-PCR(qRT-PCR) was used to detect the mRNA expression of IL-8, CXCL-6and MCP-1.
3. HL-60cells was induced by all trans retinoid acid for3days. Flow cytometry (FC) was used to detect the total CEACAM expression on HL-60cell surface. HL-60cells could be induced toward neutrophils differentiation, and was named as iHL-60.
4. Direct and indirect coculture of4groups of Cal-27and iHL-60cells.
(1) Supernatant of the4group cells was collected. After centrifugation, the supernatant was use to coculturwith iHL-60. After24h, the total RNA of iHL-60from4groups and untreated iHL-60was extracted. qRT-PCR was used to detect the mRNA expression of IL-8, MMP-9, VEGF and TNF-αin5groups of iHL-60.
(2) Let iHL-60cells directed co-cultured with4groups of Cal-27cells in a ratio of1:5. After24h, MTT was used to detect the490nm absorbance values of Cal-27cells in different groups.
5. qRT-PCR was used to detect the mRNA expression of TGF-β and IFN-β.
6. Eliasa was used to detect the secretory TGF-βprotein in4group of Cal-27.
7. IHC was used to exlore the TGF-β protein expression on TSCC. The correlation of TGF-p and CEACAM1expression was evaluated. Immunofluorescent double-labeling was used to observe the co-localization of TGF-p and CEACAM1.
8. Add TGF-P neutrolizing antibodies to the above direct and indirect coculture systems, and redetect the mRNA expression of various factors in iHL-60and its tumor-killing ability in different groups.
Results
1. qRT-PCR result showed that overexpression of CEACAM1-4L could upregualte mRNA expression of IL-8and CXCL-6, while has no influence to MCP-1.
2. FC result showed that after induction of all trans retinoid acid for3days, the CEACAM expression on HL-60cell surface was obviously upregualted, which was a marker of neutrophil differentiation.
3. Results of direct and indirect coculture of Cal-27and iHL-60.
(1) qRT-PCR result showed that co-culture with tumor cells could upregulate the mRAN expression of IL-8, MMP-9and VEGF and downregulate TNF-ain iHL-60cells.This trend was particularly obvious in CC1-4L-LV and CC1-4S-LV group,and the difference had statistical significane compared with V-LV and Blank group (P<0.05). After adding of TGF-β neutrolizing antibodies, the differences in different groups was obviously reduced, and had no statistical significance.
(2) MTT result showed that co-culture with tumor cells from CC1-4L-LV and CC1-4S-LV group could weaken the tumor-killing ability of iHL-60compared with V-LV and Blank group (P<0.05). After adding of TGF-β neutrolizing antibodies, the tumor-killing ability of iHL-60was obviously enhanced in all groups. And the differences in various groups was obviously reduced, and had no statistical significance.
4. Correlation of CEACAM1exprssion and TGF-β expression.
(1) qRT-PCR and Elisa results showed that overexpression of both CEACAM1-4L and-4S could upregulate mRNA and protein expression of TGF-β.
(2) IHC result showed that TGF-βexpression was higher in TSCC than in peritumoral tissues, and was positively related to CEACAM1expression. Immunofluorescent double-labeling result showed that there were co-localization of TGF-β and CEACAM1.
Conclusion
1. Overexpression of CEACAM1-4L could upregulate mRNA expression of IL-8and CXCL-6.
2. In TSCC tissues, there was a positive relationship between CEACAM1and TGF-β expression, and had co-localization of them two. Overexpression of both CEACAM1-4L and CEACAM1-4S could upregulate TGF-β expression.
3. Overexpression of CEACAM1in Cal-27cells could weaken the tumor-killing ability of iHL-60that Co-cultured with tumor cells, and can more easily to transform iHL-60cells from antitumoral type (N1) to protumoral type (N2).
4. The effect of CE AC AMI overexpression in Cal-27on the type conversion of iHL-60was mainly through upregulating TGF-β.
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