VEGF-A，C，D作为恶性浆膜腔积液诊断、预后及疗效预测标志物的研究
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摘要
肺癌、乳腺癌和恶性淋巴瘤是导致恶性胸腔积液的前３位病因，一旦出现了恶性胸腔积液，患者平均生存期往往不到6个月。恶性腹腔积液最常见于消化道肿瘤和妇科肿瘤，一旦肿瘤患者出现了恶性腹腔积液，即预示中位生存期不足20周，尤其以消化道肿瘤引起的恶性腹腔积液预后最差，生存期仅12-20周。心包积液一般为患者临终前表现，预后更差。恶性浆膜腔积液的治疗常常是临床上的一个难题和挑战。目前恶性浆膜腔积液的常规治疗主要是利尿、限盐、浆膜腔穿刺抽液术、腔内化疗，生物反应调节剂，中药，热疗等，但疗效均不理想，治疗后腔内积液往往减少不明显、复发较快，同时几乎所有治疗都有不同程度的毒副反应。研究表明：肿瘤浸润或转移至胸腹膜后，血管内皮生长因子VEGF（Vascularendothelial growth factor)水平的升高、肿瘤新生血管的生成以及血管通透性增加是浆膜腔内积液形成的重要机制。VEGF家族包括6种类型:VEGF-A、B、C、D、E和胎盘生长因子PIGF（placental growth factor），主要由肿瘤细胞和巨噬细胞分泌，VEGF-A是肿瘤血管生成中最重要的调控因子，VEGF-C和VEGF-D是肿瘤淋巴管生成中最重要的调控因子。应用血管生成抑制剂，特别是靶向VEGF的药物，已经成为基础和临床研究的热点。目前对浆膜腔积液中VEGF的研究主要集中于代表血管生成的VEGF-A研究，代表淋巴管生成的VEGF-C，VEGF-D研究甚少。国产一类创新药物重组人血管内皮抑素(rh-endostatin，商品名endostar，恩度)是一种人源化广谱的抗血管生成药物，可以下调多种促血管生成因子对血管内皮的保护作用，直接作用靶点为血管内皮细胞，发挥直接和间接抗血管作用。近年来，有多位学者探索应用恩度单药或者联合化疗药物治疗恶性浆膜腔积液，结果发现，这种方法高效低毒，控制积液作用强，耐受性高，并能明显地改善患者的生活质量。但是恩度等血管生成抑制剂的疗效评价体系、探索针对性的分子标志物来预测恩度的疗效，还有大量的工作需要人们去做。如何完善并利用分子标记物来指导恶性浆膜腔积液的临床诊断、预后、预测其疗效、优化其治疗是未来工作的方向。本研究通过检测原发于肺癌及胃癌的恶性浆膜腔积液上清及细胞水平的VEGF-A,C,D的蛋白含量及表达，探究三因子在恶性浆膜腔积液诊断，预后及恩度疗效预测的临床价值。并建立了稳定整合荧光素酶（luciferase）的胃癌未分化NUGC-4-luc细胞系，并以该细胞系为基础，建立胃癌裸鼠腹水瘤模型，结合生物发光活体成像（in vivo bioluminescent imaging system）动态监测腹水癌细胞的变化，并检测腹膜结节微血管密度MVD（microvesseldensity）、淋巴管密度LVD (1ymphatic vessel density)的数量，综合评价分子标志物VEGF-A,C,D在恶性浆膜腔积液靶向治疗中的价值。
第一部分VEGF-A,C,D在恶性浆膜腔积液中诊断及预后的临床意义
目的：评价VEGF-A,C,D在恶性浆膜腔积液中诊断，预后的临床价值，寻找合适的检测水平，为局部抗血管抗淋巴管治疗提供依据。
方法：应用酶联免疫吸附实验（ELISAenzyme-linked immunosorbentassay）方法检测浆膜腔积液患者外周血清79例（肺癌30例，胃癌21例，良性积液28例），积液上清96例（肺癌38例，胃癌30例，良性积液28例） VEGF-A,C,D的蛋白含量；免疫细胞化学方法（ICCimmunocytochemistry）检测积液细胞水平71例（肺癌34例，胃癌17例，良性积液20例）VEGF-A,C,D的蛋白表达，通过SPSS17.0分析其诊断，预后，临床病理因素及其相关性的临床意义。
结果：
1VEGF-A
1.1VEGF-A含量及表达：血清sVEGF-A含量在癌组及良性组无统计学差别(p>0.05)。积液上清pVEGF-A含量在肺癌及胃癌组均高于良性积液组(p<0.05)，且pVEGF-A的升高在肺癌组和胃癌组间无差别(p>0.05)。pVEGF-A与sVEGF-A含量在良性积液组相近，但在恶性积液中显示pVEGF-A明显高于sVEGF-A水平(p<0.05)。20例良性积液未见癌细胞，染色部位主要位于间皮细胞胞浆，淋巴细胞及粒细胞均未着色。细胞水平（cVEGF-A）在51例恶性积液患者中表达率为52.94%，均主要表达于癌细胞的胞浆。
1.2VEGF-A与临床病理因素联系：pVEGF-A与年龄呈负相关，与恶性积液，血性积液及单发胸腹膜转移成正相关(p<0.05)。cVEGF-A与年龄呈负相关，回归系数B为0.340(p<0.05)。
1.3血清sVEGF-A，积液上清pVEGF-A与生存关系：以恶性积液患者血清及上清三指标含量的中位值为临界值，分为高含量及低含量组，对比其1年生存率情况。 pVEGF-A水平>406.19pg/ml较pVEGF-A≤406.19pg/ml患者1年生存期短，统计学差异处于边缘值(p=0.066)。sVEGF-A水平未显示1年生存差别(p>0.05)。
2VEGF-C, VEGF-D
2.1VEGF-C，VEGF-D的含量及表达：血清sVEGF-C，sVEGF-D含量在癌组及良性组无统计学差别(p>0.05)。积液上清pVEGF-C，pVEGF-D含量在癌组及良性组无统计学差别(p>0.05)。细胞水平cVEGF-C，cVEGF-D在51例恶性患者中表达率分别为70.58%和82.35%，均主要表达与癌细胞的胞浆。cVEGF-D在肺癌及胃癌恶性浆膜腔积液中的阳性表达，尤其是强阳性表达明显高于cVEGF-A，cVEGF-C (p<0.05)。cVEGF-A,C,D在恶性浆膜腔积液中肺癌组与胃癌组无病种差别(p>0.05)。
2.2VEGF-C, VEGF-D与临床病理因素联系：pVEGF-C与年龄呈负相关(p<0.05)。pVEGF-D与年龄正相关，与恶性积液及单发浆膜转移成负相关(p<0.05)。sVEGF-C，sVEGF-D蛋白水平与临床病理因素无相关。cVEGF-C，cVEGF-D蛋白表达与临床病理因素无相关。
2.3血清sVEGF-C，sVEGF-D，积液上清pVEGF-C，pVEGF-D与生存关系：上清pVEGF-C，pVEGF-D,及血清sVEGF-C，sVEGF-D水平均未显示1年生存差别(p>0.05)。
结论：
1积液上清VEGF-A在良恶性积液有差异，提示可能成为良恶性积液的肿瘤标志物。上清VEGF-A在1年生存预后中达到统计学边缘值，需引起重视，需扩大样本量及延长随访时间进一步探讨其预后价值。
2积液上清及细胞水平VEGF-A与年龄呈负相关，提示随年龄增长，VEGF-A水平下降；积液上清VEGF-A在恶性、血性积液及单发浆膜转移中，含量高于良性、非血性及非单发浆膜转移患者。
3积液上清VEGF-C，VEGF-D在良恶性诊断方面无统计学差异，在癌细胞水平却高表达。临床需扩大良性积液样本，进行病种分层后进一步研究其积液上清的诊断价值。
4血清VEGF-A,C,D水平在浆膜腔积液诊断及预后中均无明显临床意义，亦与临床病理因素无明显关联。提示血清学标志物在浆膜腔积液的诊断预后价值可能不及局部标志物。
第二部分VEGF-A,C,D在恩度治疗恶性浆膜腔积液中疗效预测的临床意义
目的：评价VEGF-A,C,D在恩度治疗恶性浆膜腔积液中的疗效预测价值。
方法：应用酶联免疫吸附实验（ELISA）方法检测，20对恶性浆膜腔积液患者恩度腔内治疗前后，积液上清VEGF-A,C,D蛋白含量，通过SPSS17.0分析以上三因子在恩度疗效预测中的价值。
结果：
1局部浆膜腔单药恩度组，局部浆膜腔恩度联合顺铂组及局部恩度联合全身抗肿瘤治疗组，在客观有效率RR(CR+PR)，临床获益率DCR(CR+PR+SD)及局部进展PD方面均未见明显差异，（p>0.05）。
2积液上清VEGF-A,C,D蛋白含量与疗效的关系：以疗效评价指标RR，SD，PD进行分组：三因子治疗前后变化差值（d0-d7）及治疗前后变化差值比（d0-d7/d0），在RR，SD，PD组间均未达到统计学差别，p>0.05，其中上清VEGF-A差值比（d0-d7/d0）组间p=0.085。
3积液上清VEGF-A,C,D蛋白含量在治疗组间的差别：积液上清VEGF-A,C,D的治疗前后变化差值比（d0-d7/d0）在三治疗组间无明显统计学差异（p>0.05）。
结论：
1积液上清VEGF-A,C,D的治疗前后变化差值（d0-d7）及治疗前后变化差值比（d0-d7/d0）在RR, NC, PD组间均无统计学差别，提示以上三因子尚不能预测局部恩度疗效。
2积液上清VEGF-A,C,D治疗前后变化差值比（d0-d7/d0）在恩度不同治疗组间无统计学差别，提示以上三因子尚不能指导临床选择不同的药物治疗方式。
第三部分VEGF-A,C,D在恩度不同给药方式治疗胃癌腹水瘤模型中的研究
目的：结合癌细胞监测及血管、淋巴管内皮标记，综合评价分子标志物VEGF-A,C,D在胃癌腹水瘤模型中的价值；探讨恩度顺铂不同给药方式的疗效。
方法：培养NUGC-4-luc细胞（RPMI1640培养基+10%FBS+1%P/S），取5x106数量级细胞在0.5ml PBS中，腹腔接种28只雌性裸鼠，接种后每周行动物生物发光活体成像。接种一周后随机分组：Group1:顺铂(1mg/kg)d1-3+恩度(8mg/kg)d4-7；Group2:恩度(8mg/kg) d1-4+顺铂(1mg/kg)d5-7；Group3:恩度(8mg/kg)+顺铂(1mg/kg)d1,4,7；Group4即对照组：生理盐水50ul d1-7，均腹腔给药，连用2周（每周成像一次），停药观察一周成像后处死。处死后取腹水上清，应用ELISA方法，检测积液上清VEGF-A,C,D蛋白含量；腹膜结节包被蜡块后，HE确认癌细胞，应用IHC方法，检测VEGF-A,C,D及MVD，LVD。动物发光数据先经IVIS系统处理，后通过SPSS17.0，分析其活体成像，VEGF-A,C,D蛋白表达及含量，LVD，MVD数量在四个治疗组间的差别。
结果：
1成瘤情况
5×106数量级NUGC-4-luc细胞接种于裸鼠腹腔。7天后，据测量的癌细胞光子数进行分组，四组无明显统计学差异（p>0.05）。Group2中1只动物在用药后第18天意外死亡。裸鼠用药后隔日测量四组小鼠的体重并记录，绘制小鼠体重变化曲线，4组无明显区别（p>0.05）。裸鼠处死后显示血性腹水明显，腹膜细小结节多而广泛。
2用药疗效
2.1四组腹水量(ml)：Group4（4.87±0.45）>Group1（3.1±0.53）>Group2（2.0±0.08）>Group3（1.8±0.16）。
2.2四组腹膜结节数量：Group4（33.75±2.5）> Group2（21.66±5.77）> Group3（18.75±2.5）> Group1（8.75±4.78）。
2.3癌细胞光子数
据每次测量的癌细胞光子数绘制肿瘤的生长曲线，结果经统计分析后显示，在第1天及第8天四治疗组无明显区别，p>0.05。第15天及21天时显示，Group1, Group2, Group3均优于Group4，p<0.01。其中第15天时（用药后2周），恩度联合顺铂组（Group3）及顺铂序贯恩度组（Group1）均优于恩度序贯顺铂组（Group2），p<0.05；但Group3与Group1无明显区别，p>0.05。第21天时（用药后2周，观察1周），Group3仍优于Group2，p<0.05。
2.4腹膜结节MVD,LVD数量
D2-40和CD34以染成棕黑色管腔的判定为淋巴管及血管。MVD在四组间有差异（p<0.01），经过两两比较，MVD数量Group4>Group1> Group2（p<0.01），Group2与Group3无差别（p>0.05）。而LVD数量在四组之间无统计学差别（p>0.05）。
3VEGF-A,C,D的蛋白含量及表达
3.1腹水上清VEGF-A,C,D的蛋白含量：上清VEGF-A在四组有差别（p<0.05）；经过两两比较，四组均有差别（p<0.05），VEGF-A蛋白含量在Group4> Group1> Group2> Group3。而腹水上清VEGF-C, VEGF-D的蛋白含量在四组间无明显差别（p>0.05）。
3.2腹膜结节VEGF-A,C,D蛋白表达：VEGF-A,C,D均在癌细胞胞浆着色，统计后发现VEGF-A在四组表达有差别（p<0.05），两两比较，Group4>Group2>Group1（p<0.05），Group1与Group3无区别（p>0.05）。而腹膜结节VEGF-C, VEGF-D的蛋白表达在四组间无明显差别（p>0.05）。
4VEGF-A蛋白含量与腹水量高度相关，相关系数r=0.994（p<0.01）。VEGF-A蛋白表达与腹膜结节数量高度相关，相关系数r=0.972（p<0.01）；与MVD及癌细胞光子数均无相关关系（p>0.05）。
结论：
1本研究首次建立了稳定整合luciferase的胃癌未分化NUGC-4-luc细胞系，并建立了以该细胞系为基础的胃癌裸鼠腹水瘤模型，为利用生物发光活体成像技术，研究胃癌腹膜转移瘤的生长转移机制及抗癌药物的研发，提供了稳定可靠、直观、方便、灵敏的动物模型。
2本研究中恩度联合顺铂及序贯组均耐受性好，治疗应至少2周后差别显著。首次结合活体成像及MVD，提示恩度联合顺铂优于序贯，联合组对癌细胞的杀伤及对血管内皮的抑制作用均最大；顺铂序贯恩度杀伤癌细胞优于恩度序贯顺铂；恩度序贯顺铂对腹膜血管内皮的抑制优于顺铂序贯恩度，故提示可以通过分子标志物预测靶点变化及功能成像监测癌细胞数量，共同指导临床合理选择靶向及细胞毒药物的用药时机。
3VEGF-A与腹水量及腹膜结节数量正相关，提示VEGF-A不仅可以预测腹水量还可以预测腹膜结节的变化，突破了临床只依据腹水量判断疗效的局限性。VEGF-A与癌细胞光子数及MVD无相关关系，提示VEGF-A优化治疗作用局限。
4未发现腹水及腹膜结节VEGF-C，VEGF-D，LVD在四治疗组间差别，尚不能提示其疗效预测作用。
The first three causes of malignant pleural effusion are lung cancer,breast cancer and malignant lymphoma. Once malignant pleural effusion hasdeveloped, the average survival time of the patient is often less than6months.Malignant ascites is most common in gastrointestinal and gynecologicalcancers, which indicates that the median survival is less than20weeks,particularly in the patients with malignant ascites caused by gastrointestinalcancers, who have the worst prognosis and whose survival times are only12-20weeks. Pericardial effusion is generally observed in dying patients, andits prognosis is worse. The treatment of malignant effusions is often a clinicalchallenge. Currently, the conventional treatments of malignant effusions aremainly composed of diuresis, salt restriction, serous cavity paracentesis,intracavitary chemotherapy, biological response modifiers, traditional chinesemedicine, thermotherapy, etc. However, the effects of the above-mentionedtreatments are all unsatisfactory. After therapy with these methods, there is nosignificant decrease of effusions while it often relapses fast, and almost all ofthe treatments have toxic side effects of various degrees.Studies have shownthat elevated levels of VEGF (vascular endothelial growth factor), tumorangiogenesis, and increased vascular permeability after tumor invading ormetastasizing to the pleuroperitoneum are important formation mechanisms ofserous cavity effusions. The VEGF family includes six types: VEGF-A, B, C,D, E, and PIGF (placental growth factor). These factors are mainly secreted bymacrophages and tumor cells. VEGF-A is the most important regulatory factorin the tumor angiogenesis, and VEGF-C and VEGF-D are the most importantregulatory factors in the tumor lymphangiogenesis. The use of angiogenesisinhibitors, particularly VEGF targeted drugs has become a hot topic of thebasic and clinical researches.Currently, the study on the VEGF in serous cavity effusions is mainly focused on VEGF-A representing the angiogenesis,and there are few studies on the lymphangiogenesis representative factorsVEGF-C and VEGF-D.The domestic first class new drug recombinant humanendostatin (rh-endostatin, endostar) is a broad spectrum humanizedanti-angiogenic drug which targets vascular endothelial cells but not tumorcells and can down-regulate the protecting effect of a variety of proangiogenicfactors on the vascular endothelium, thus playing direct and indirectantiangiogenic effects. In recent years, there are a number of scholars hasexplored the treatment effect of endostar used alone or combined withchemotherapy on malignant serous cavity effusions. They found it efficientand low toxic, not only being able to produce a strong effusions controllingeffect with a high tolerance, but also being able to significantly improve thequality of life of patients. However, there are a lot of work to do in thefollowing areas: the efficacy evaluation system of angiogenesis inhibitors suchas endostar, and exploring new molecular markers to predict and evaluate theefficacy of endostar. How to improve and use molecular markers to guide theclinical diagnosis, evaluate the prognosis, predict the efficacy and optimize thetreatment of malignant serous cavity effusions is the direction of futureworks.In this study, we detected the contents and expression levels ofVEGF-A,C,D in the supernatants and cells from the malignant serous cavityeffusions caused by lung cancer and gastric cancer, to explore the clinicalsignificance of the three factors in the diagnosis and prognosis of malignantserous cavity effusions and in the prediction of efficacy of endostar on theeffusions. We established a stably expressing luciferase gastric NUGC-4-luccell line, and on the basis of this cell line we established gastric cancer ascitestumor model in nude mice for the first time. By combining dynamicmonitoring changes of gastric cancer ascites tumor cells by in vivobioluminescence imaging with detecting the peritoneal nodules MVD(microvessel density), LVD (1ymphatic vessel density), we comprehensivelyevaluated the value of the molecular markers VEGF-A, VEGF-C and VEGF-Din the targeted therapy of malignant serous cavity effusions.
Part1Diagnostic and prognostic values of vascular endothelial growthfactor (VEGF)-A,-C and-D in malignant effusion of lung andgastric cancer patients
Objective: To evaluate VEGF-A,C,D the diagnosis, clinical prognosticvalue in malignant effusion
Methods: Enzyme-linked immunosorbent assay(ELISA) was used todetect VEGF-A,C,Dprotein content from patients with effusion of the serum79cases (30lung cancer,21gastric cancer,28benign effusions), thesupernatant fluid96cases (38lung cancer,30gastric cancer,28benigneffusion); Immunocytochemistry (ICC) detected VEGF-A,C,D proteinexpression from71cases of effusion cell level (34lung cancer,17gastriccancer,20benign effusions), by SPSS17.0to analyze the diagnosis, prognosis,clinicopathological factors and correlation of clinical significance.
Results:
1VEGF-A
1.1The level of sVEGF-A proteins was not statistically different betweencancer and benign patients (p>0.05). Cancer patients had higher pVEGF-Alevel than those with benign effusion (p <0.05). The upregulated level ofpVEGF-A was not associated with tumor histological types (p>0.05).pVEGF-A level was similar to the corresponding sVEGF-A level in patientswith benign effusions. Nevertheless, pVEGF-A level in malignant effusionwas significantly higher than its corresponding sVEGF-A level (p<0.05).
We detected VEGF-A,-C, and-D expressions in20cytological smearsfrom benign effusions and the data showed that there was no positive stainingof anti-VEGF-A,-C, and-D antibodies in these20benign cases. VEGF-Aexpression rate was52.94%and mainly expressed in the cytoplasm ofpositively expressed tumor cells.
1.2The data showed that pVEGF-A and cVEGF-A were negativelyassociated with age and pVEGF-A was positively associated with malignantand bloody effusion, pleural or peritoneal metastasis (p<0.05).
1.3With the median levels as the cut-off values, patients were divided into high and low pleural effusion parameter groups. One-year survival timeanalysis showed a relatively shorter survival time for patients with pVEGF-Alevels>406.19pg/ml as compared with those presenting with pVEGF-a levels≤406.19pg/ml, although this effect was only of marginal statisticalsignificance (p=0.066). sVEGF-A with the median levels as the cut-off, didnot exhibited statistical significance in predicting survival for patients withmalignant effusions (p>0.05).
2VEGF-C, VEGF-D
2.1Levels of sVEGF-C， sVEGF-D proteins were not statisticallydifferent between cancer and benign patients (p>0.05). Levels of pVEGF-C，pVEGF-D proteins were not statistically different between cancer and benignpatients (p>0.05).cVEGF-C，cVEGF-D expression rates were70.58%and82.35, respectively, in51patients with lung or gastric cancer. These proteinswere mainly expressed in the cytoplasm of positively expressed tumor cells.Regarding their expression levels of in tumor patients, the expression rate ofcVEGF-D was significantly higher than that of cVEGF-A and cVEGF-C(p<0.05). However, there was no statistically significant difference incVEGF-A,-C and-D expression between lung and gastric cancers (p>0.05).
2.2The data showed that pVEGF-C was negatively associated with age.pVEGF-D expression was positively associated with age and negativelyassociated with malignant effusion and pleural metastasis (p<0.05). However,there was no association of sVEGF-C， sVEGF-D proteins with theclinicopathological factors (p>0.05).
2.3sVEGF-C,sVEGF-D and pVEGF-C,pVEGF-D with the median levelsas the cut-off, did not exhibited statistical significance in predicting survivalfor patients with malignant effusions (p>0.05).
Conclusions:
1The supernatant fluid VEGF-A was significantly different betweenbenign and malignant effusion, suggesting that VEGF-A may be used as atumor marker in the clinical setting to discriminate benign from malignanteffusions.VEGF-A levels reached marginal statistical significance as a potential predictor of poor clinical outcome. Given the fact that VEGF-Aexpression might have a survival impact, attention should be paid to thepatients with high VEGF-A expression.
2The supernatant fluid and cellular level of VEGF-A were negativelycorrelated with age, probably reflecting with age growing, malignant effusionsand level of VEGF-A are reduced. The supernatant fluid levels of VEGF-A inthe patients with bloody pleural effusion and only pleural metastasis are higherthan that in patients with non-bloody pleural effusion and not only pleuralmetastasis.
3The supernatant fluid VEGF-C and VEGF-D had no significantdifference in the clinical diagnosis of benign and malignant, but highlyexpressed at the cellular level. It needs expand samples of benign effusions inclinical practice to further study its diagnostic value after disease stratification.
4Serum VEGF-A,C,D levels had no obvious clinical significance in thediagnosis and prognosis of serous cavity effusions, and they were also notsignificantly associated with the clinicalpathological factors. It is suggestedthe serological markers’ diagnosis and prognostic values for serous cavityeffusions may be not as good as that of the local effusion markers.
Part2The predictive value of VEGF-A, C, D for the clinical efficacy ofendostar on malignant effusions
Objective: To evaluate the predictive value of VEGF-A,C,D for theclinical efficacy of endostar on malignant effusions.
Methods: ELISA was used to detect VEGF-A,C,D protein contents of20pairs of patients with malignant effusions reciving intracavitary endostarbefore and after treatments. The values of the three factors in pridicting theefficacy of endostar were analyzed by SPSS17.0.
Results:
1Between the endostar monotherapy group, endostar plus cisplatin groupand endostar monotherapy combined with systemic anti-tumor therapy group,there were no significant differences in the objective response rate (RR: CR+PR), clinical benefit rate (DCR:CR+PR+NC) or local progression (PD)(p> 0.05).
2The relationships between supernatant fluid VEGF-A,C,D proteincontents with efficacy: Neither the differences between before and aftertreatment （d0-d7） nor the difference ratios before and after treatment（d0-d7/d0）of the three factors reached statistical difference in RR, NC, PDgroups (p>0.05), with the difference ratio of the supernatant VEGF-A(d1-d7/d1) between groups result showed p=0.085.
3The relationships between supernatant fluid VEGF-A,C,D proteincontents with treatment: The difference ratios before and after treatment（d0-d7/d0）of supernatant fluid VEGF-A,C,D had no significant differencebetween the three endostar groups (p>0.05).
Conclusions:
1Neither the differences between before and after treatment（d0-d7） northe difference ratios before and after treatment （d0-d7/d0）of supernatant fluidVEGF-A,C,D had no statistical difference in RR, NC, PD groups, suggestingthe three factors can’t pridict the efficacy of endostar.
2The difference ratios before and after treatment (d0-d7/d0) ofVEGF-A,C,D had no statistical difference in different endostar groups,suggesting the three factors can’t guide the clinical treatment.
Part3The research of VEGF-A, C, D in gastric cancer ascites tumor modeltreated with endostar given by different ways
Objective: To comprehensively evaluate the value of the molecularmarkers VEGF-A,C,D in the gastric cancer ascites model by combiningmonitoring the cancer cells with detecting the vascular and lymphaticendothelial markers.
Methods: Cultured NUGC-4-luc cells (RPMI1640medium+10%FBS+1%P/S), suspended a total of5×106NUGC-4-luc cells in50ml PBS. Thesuspension was pipetted with micropipettor and injected into abdominalcavities of28female nude mice. These intraperitoneally inoculated miceunderwent in vivo bioluminescence imaging weekly. One week afterNUGC-4-luc cells injection, mice were randomly divided into four groups of7 mice each: Group1: cisplatin (1mg/kg) d1-3+endostar (8mg/kg) d4-7;Group2: endostar (8mg/kg) d1-4+cisplatin (1mg/kg) d5-7; Group3: endostar(8mg/kg)+cisplatin (1mg/kg) d1,4,7; Group4(control group):50μl ofnormal saline d1-7. All of the treatments were injected intraperitoneally. Everygroup was treated two consecutive weeks, underwent imaging weekly. Oneweek after the final administration, mice were anesthetized and sacrificed.Then the supernatant fluid of ascites was taken and the contents ofVEGF-A,C,D proteins in it were detected by ELISA. VEGF-A,C,D as well asMVD and LVD in the peritoneal nodules were detected by IHC methods afterthe nodules had been embeded into wax blocks. All bioluminescent data werefirst collected and processed using IVIS and then analyzed by SPSS17.0toexplore the differences of imagings, protein expressions and contents ofVEGF-A,C,D, and LVD and MVD between the four treatment groups.
Results:
1Ascites tumor model
5×106NUGC-4-luc cells were injected into abdominal cavities. One weekafter NUGC-4-luc cells injection, mice were randomly divided into fourgroups by the number of cancer cell photons, with no statistical differences inthe four groups (p>0.05). After initiating treatment an accidental deathsoccured in the first18days in Group2.On every other day there were nosignificant differences in the nude mice weights between the four treatmentgroups (p>0.05).After mice were sacrificed,we found bloody ascites wereobvious and peritoneal nodules were small,numerous and widely.
2Drug efficacy
2.1Volume of ascites in four groups(ml):Group4（4.87±0.45）>Group1（3.1±0.53）>Group2（2.0±0.08）>Group3（1.8±0.16）.
2.2The number of peritoneal nodules: Group4（33.75±2.5）> Group2（21.66±5.77）> Group3（18.75±2.5）> Group1（8.75±4.78）.
2.3The counts of cancer cell photons
By using in vivo imaging processing software, tumor growth curves wereplotted according to the number of photons per measurement. On day1and day8, there were no significant differences between the four groups (p>0.05).On day15and day21, group1,2and3were all better than the control group(p<0.01). On day15(after treatment of two weeks), endostar combinationwith cisplatin group (Group3) and cisplatin sequential endostar group (group1)were better than endostar sequential cisplatin group (group2)(p<0.05),however there was no significant difference between the former two groups(p>0.05). On day21(after treatment of two weeks, one week after withdrawn),endostar combination with cisplatin group (Group3) was still better thanendostar sequential cisplatin group (group2)(p<0.05).
2.4MVD,LVD
The lumens of blood vessels and lymphatics were determined by CD34and D2-40stained brown to black. There were differences in MVD betweenthe four groups (p<0.01), pairwise comparisons showing group4> group1>group2(p<0.01), and there was no difference between group2and group3(p>0.05). However, there were no statistical differences in LVD numberbetween the four groups (p>0.05).
3VEGF-A,C,D
3.1There were differences in supernatant VEGF-A between the fourgroups (p<0.05), pairwise comparisons showing there was difference betweeneach two groups of the four groups with the result ofgroup4>group1>group2>group3(p<0.05). The ascites supernatant levels ofVEGF-C and VEGF-D proteins had no significant difference between the fourgroups (p>0.05).
3.2VEGF-A,C,D proteins were all cytoplasmic staining. There werestatistical differences in VEGF-A between the four groups (p<0.05), pairwisecomparisons showing Group4>Group2>Group1（p<0.05）， and there was nodifference between group1and group3(p>0.05). However, there were nostatistical differences in VEGF-C and VEGF-D expressions between the fourgroups (p>0.05).
4VEGF-A protein content was highly correlated with the volume ofascites (r=0.994, p<0.01). VEGF-A protein expression was highly correlated with the number of peritoneal nodules (r=0.994, p<0.01), but was notcorrelated with the MVD and the counts of cancer cell photons (p>0.05).
Conclusions:
1On the basis of a stably expressing luciferase gastric NUGC-4-luc celllines, we established gastric cancer ascites tumor model in nude mice for thefirst rime. We provided a stable, reliable, intuitive, convenient, and sensitiveanimal model for exploring the growth and metastasis mechanisms of thegastric cancer peritoneal metastatic tumor and the research and developmentof anticancer drugs by using in vivo imaging techniques.
2In this study, the groups of endostar combined and sequentially usedwith cisplatin were all well tolerated, and treatment should be at least twoweeks before observing differences. The present study combined in vivoimaging with the MVD for the first time, and the results suggested:combination group was superior to sequential groups, the former’s killingcapability on cancer cells and inhibiting effect on vascular endothelium wereall the greatest; cisplatin sequential endostar was superior to endostarsequential cisplatin in killing cancer cells, while endostar sequential cisplatinwas superior to cisplatin sequential endostar in inhibiting peritoneal vascularendothelium. It was suggested that we can use molecular markers to forecasttarget changes and use functional imaging to monitor the number of cancercells, to guide the rational choice of the targeted and cytotoxic drugs and theadministration timing.
3VEGF levels were highly correlated with the volume of ascites and thenumber of peritoneal nodules, suggesting that the predictive value of VEGFfor the efficacy of malignant effusion may be not only the volume of ascitesbut the size of peritoneal nodules. VEGF-A expressions were not correlatedwith the MVD and the counts of cancer cell photons. These suggested that itcan’t be used to monitor the activity of cancer cells and lymphatic vascularstatus, and it can’t be used to guide the choice of drugs intraperitoneallyinjected as well.
4No differences in ascites and peritoneal nodules VEGF-C and VEGF-D proteins and LVD between the four treatment groups were found, so they stillcan’t serve as predictors of efficacy.
第一部分VEGF-A,C,D在恶性浆膜腔积液中诊断及预后的临床意义
目的：评价VEGF-A,C,D在恶性浆膜腔积液中诊断，预后的临床价值，寻找合适的检测水平，为局部抗血管抗淋巴管治疗提供依据。
方法：应用酶联免疫吸附实验（ELISAenzyme-linked immunosorbentassay）方法检测浆膜腔积液患者外周血清79例（肺癌30例，胃癌21例，良性积液28例），积液上清96例（肺癌38例，胃癌30例，良性积液28例） VEGF-A,C,D的蛋白含量；免疫细胞化学方法（ICCimmunocytochemistry）检测积液细胞水平71例（肺癌34例，胃癌17例，良性积液20例）VEGF-A,C,D的蛋白表达，通过SPSS17.0分析其诊断，预后，临床病理因素及其相关性的临床意义。
结果：
1VEGF-A
1.1VEGF-A含量及表达：血清sVEGF-A含量在癌组及良性组无统计学差别(p>0.05)。积液上清pVEGF-A含量在肺癌及胃癌组均高于良性积液组(p<0.05)，且pVEGF-A的升高在肺癌组和胃癌组间无差别(p>0.05)。pVEGF-A与sVEGF-A含量在良性积液组相近，但在恶性积液中显示pVEGF-A明显高于sVEGF-A水平(p<0.05)。20例良性积液未见癌细胞，染色部位主要位于间皮细胞胞浆，淋巴细胞及粒细胞均未着色。细胞水平（cVEGF-A）在51例恶性积液患者中表达率为52.94%，均主要表达于癌细胞的胞浆。
1.2VEGF-A与临床病理因素联系：pVEGF-A与年龄呈负相关，与恶性积液，血性积液及单发胸腹膜转移成正相关(p<0.05)。cVEGF-A与年龄呈负相关，回归系数B为0.340(p<0.05)。
1.3血清sVEGF-A，积液上清pVEGF-A与生存关系：以恶性积液患者血清及上清三指标含量的中位值为临界值，分为高含量及低含量组，对比其1年生存率情况。 pVEGF-A水平>406.19pg/ml较pVEGF-A≤406.19pg/ml患者1年生存期短，统计学差异处于边缘值(p=0.066)。sVEGF-A水平未显示1年生存差别(p>0.05)。
2VEGF-C, VEGF-D
2.1VEGF-C，VEGF-D的含量及表达：血清sVEGF-C，sVEGF-D含量在癌组及良性组无统计学差别(p>0.05)。积液上清pVEGF-C，pVEGF-D含量在癌组及良性组无统计学差别(p>0.05)。细胞水平cVEGF-C，cVEGF-D在51例恶性患者中表达率分别为70.58%和82.35%，均主要表达与癌细胞的胞浆。cVEGF-D在肺癌及胃癌恶性浆膜腔积液中的阳性表达，尤其是强阳性表达明显高于cVEGF-A，cVEGF-C (p<0.05)。cVEGF-A,C,D在恶性浆膜腔积液中肺癌组与胃癌组无病种差别(p>0.05)。
2.2VEGF-C, VEGF-D与临床病理因素联系：pVEGF-C与年龄呈负相关(p<0.05)。pVEGF-D与年龄正相关，与恶性积液及单发浆膜转移成负相关(p<0.05)。sVEGF-C，sVEGF-D蛋白水平与临床病理因素无相关。cVEGF-C，cVEGF-D蛋白表达与临床病理因素无相关。
2.3血清sVEGF-C，sVEGF-D，积液上清pVEGF-C，pVEGF-D与生存关系：上清pVEGF-C，pVEGF-D,及血清sVEGF-C，sVEGF-D水平均未显示1年生存差别(p>0.05)。
结论：
1积液上清VEGF-A在良恶性积液有差异，提示可能成为良恶性积液的肿瘤标志物。上清VEGF-A在1年生存预后中达到统计学边缘值，需引起重视，需扩大样本量及延长随访时间进一步探讨其预后价值。
2积液上清及细胞水平VEGF-A与年龄呈负相关，提示随年龄增长，VEGF-A水平下降；积液上清VEGF-A在恶性、血性积液及单发浆膜转移中，含量高于良性、非血性及非单发浆膜转移患者。
3积液上清VEGF-C，VEGF-D在良恶性诊断方面无统计学差异，在癌细胞水平却高表达。临床需扩大良性积液样本，进行病种分层后进一步研究其积液上清的诊断价值。
4血清VEGF-A,C,D水平在浆膜腔积液诊断及预后中均无明显临床意义，亦与临床病理因素无明显关联。提示血清学标志物在浆膜腔积液的诊断预后价值可能不及局部标志物。
第二部分VEGF-A,C,D在恩度治疗恶性浆膜腔积液中疗效预测的临床意义
目的：评价VEGF-A,C,D在恩度治疗恶性浆膜腔积液中的疗效预测价值。
方法：应用酶联免疫吸附实验（ELISA）方法检测，20对恶性浆膜腔积液患者恩度腔内治疗前后，积液上清VEGF-A,C,D蛋白含量，通过SPSS17.0分析以上三因子在恩度疗效预测中的价值。
结果：
1局部浆膜腔单药恩度组，局部浆膜腔恩度联合顺铂组及局部恩度联合全身抗肿瘤治疗组，在客观有效率RR(CR+PR)，临床获益率DCR(CR+PR+SD)及局部进展PD方面均未见明显差异，（p>0.05）。
2积液上清VEGF-A,C,D蛋白含量与疗效的关系：以疗效评价指标RR，SD，PD进行分组：三因子治疗前后变化差值（d0-d7）及治疗前后变化差值比（d0-d7/d0），在RR，SD，PD组间均未达到统计学差别，p>0.05，其中上清VEGF-A差值比（d0-d7/d0）组间p=0.085。
3积液上清VEGF-A,C,D蛋白含量在治疗组间的差别：积液上清VEGF-A,C,D的治疗前后变化差值比（d0-d7/d0）在三治疗组间无明显统计学差异（p>0.05）。
结论：
1积液上清VEGF-A,C,D的治疗前后变化差值（d0-d7）及治疗前后变化差值比（d0-d7/d0）在RR, NC, PD组间均无统计学差别，提示以上三因子尚不能预测局部恩度疗效。
2积液上清VEGF-A,C,D治疗前后变化差值比（d0-d7/d0）在恩度不同治疗组间无统计学差别，提示以上三因子尚不能指导临床选择不同的药物治疗方式。
第三部分VEGF-A,C,D在恩度不同给药方式治疗胃癌腹水瘤模型中的研究
目的：结合癌细胞监测及血管、淋巴管内皮标记，综合评价分子标志物VEGF-A,C,D在胃癌腹水瘤模型中的价值；探讨恩度顺铂不同给药方式的疗效。
方法：培养NUGC-4-luc细胞（RPMI1640培养基+10%FBS+1%P/S），取5x106数量级细胞在0.5ml PBS中，腹腔接种28只雌性裸鼠，接种后每周行动物生物发光活体成像。接种一周后随机分组：Group1:顺铂(1mg/kg)d1-3+恩度(8mg/kg)d4-7；Group2:恩度(8mg/kg) d1-4+顺铂(1mg/kg)d5-7；Group3:恩度(8mg/kg)+顺铂(1mg/kg)d1,4,7；Group4即对照组：生理盐水50ul d1-7，均腹腔给药，连用2周（每周成像一次），停药观察一周成像后处死。处死后取腹水上清，应用ELISA方法，检测积液上清VEGF-A,C,D蛋白含量；腹膜结节包被蜡块后，HE确认癌细胞，应用IHC方法，检测VEGF-A,C,D及MVD，LVD。动物发光数据先经IVIS系统处理，后通过SPSS17.0，分析其活体成像，VEGF-A,C,D蛋白表达及含量，LVD，MVD数量在四个治疗组间的差别。
结果：
1成瘤情况
5×106数量级NUGC-4-luc细胞接种于裸鼠腹腔。7天后，据测量的癌细胞光子数进行分组，四组无明显统计学差异（p>0.05）。Group2中1只动物在用药后第18天意外死亡。裸鼠用药后隔日测量四组小鼠的体重并记录，绘制小鼠体重变化曲线，4组无明显区别（p>0.05）。裸鼠处死后显示血性腹水明显，腹膜细小结节多而广泛。
2用药疗效
2.1四组腹水量(ml)：Group4（4.87±0.45）>Group1（3.1±0.53）>Group2（2.0±0.08）>Group3（1.8±0.16）。
2.2四组腹膜结节数量：Group4（33.75±2.5）> Group2（21.66±5.77）> Group3（18.75±2.5）> Group1（8.75±4.78）。
2.3癌细胞光子数
据每次测量的癌细胞光子数绘制肿瘤的生长曲线，结果经统计分析后显示，在第1天及第8天四治疗组无明显区别，p>0.05。第15天及21天时显示，Group1, Group2, Group3均优于Group4，p<0.01。其中第15天时（用药后2周），恩度联合顺铂组（Group3）及顺铂序贯恩度组（Group1）均优于恩度序贯顺铂组（Group2），p<0.05；但Group3与Group1无明显区别，p>0.05。第21天时（用药后2周，观察1周），Group3仍优于Group2，p<0.05。
2.4腹膜结节MVD,LVD数量
D2-40和CD34以染成棕黑色管腔的判定为淋巴管及血管。MVD在四组间有差异（p<0.01），经过两两比较，MVD数量Group4>Group1> Group2（p<0.01），Group2与Group3无差别（p>0.05）。而LVD数量在四组之间无统计学差别（p>0.05）。
3VEGF-A,C,D的蛋白含量及表达
3.1腹水上清VEGF-A,C,D的蛋白含量：上清VEGF-A在四组有差别（p<0.05）；经过两两比较，四组均有差别（p<0.05），VEGF-A蛋白含量在Group4> Group1> Group2> Group3。而腹水上清VEGF-C, VEGF-D的蛋白含量在四组间无明显差别（p>0.05）。
3.2腹膜结节VEGF-A,C,D蛋白表达：VEGF-A,C,D均在癌细胞胞浆着色，统计后发现VEGF-A在四组表达有差别（p<0.05），两两比较，Group4>Group2>Group1（p<0.05），Group1与Group3无区别（p>0.05）。而腹膜结节VEGF-C, VEGF-D的蛋白表达在四组间无明显差别（p>0.05）。
4VEGF-A蛋白含量与腹水量高度相关，相关系数r=0.994（p<0.01）。VEGF-A蛋白表达与腹膜结节数量高度相关，相关系数r=0.972（p<0.01）；与MVD及癌细胞光子数均无相关关系（p>0.05）。
结论：
1本研究首次建立了稳定整合luciferase的胃癌未分化NUGC-4-luc细胞系，并建立了以该细胞系为基础的胃癌裸鼠腹水瘤模型，为利用生物发光活体成像技术，研究胃癌腹膜转移瘤的生长转移机制及抗癌药物的研发，提供了稳定可靠、直观、方便、灵敏的动物模型。
2本研究中恩度联合顺铂及序贯组均耐受性好，治疗应至少2周后差别显著。首次结合活体成像及MVD，提示恩度联合顺铂优于序贯，联合组对癌细胞的杀伤及对血管内皮的抑制作用均最大；顺铂序贯恩度杀伤癌细胞优于恩度序贯顺铂；恩度序贯顺铂对腹膜血管内皮的抑制优于顺铂序贯恩度，故提示可以通过分子标志物预测靶点变化及功能成像监测癌细胞数量，共同指导临床合理选择靶向及细胞毒药物的用药时机。
3VEGF-A与腹水量及腹膜结节数量正相关，提示VEGF-A不仅可以预测腹水量还可以预测腹膜结节的变化，突破了临床只依据腹水量判断疗效的局限性。VEGF-A与癌细胞光子数及MVD无相关关系，提示VEGF-A优化治疗作用局限。
4未发现腹水及腹膜结节VEGF-C，VEGF-D，LVD在四治疗组间差别，尚不能提示其疗效预测作用。
The first three causes of malignant pleural effusion are lung cancer,breast cancer and malignant lymphoma. Once malignant pleural effusion hasdeveloped, the average survival time of the patient is often less than6months.Malignant ascites is most common in gastrointestinal and gynecologicalcancers, which indicates that the median survival is less than20weeks,particularly in the patients with malignant ascites caused by gastrointestinalcancers, who have the worst prognosis and whose survival times are only12-20weeks. Pericardial effusion is generally observed in dying patients, andits prognosis is worse. The treatment of malignant effusions is often a clinicalchallenge. Currently, the conventional treatments of malignant effusions aremainly composed of diuresis, salt restriction, serous cavity paracentesis,intracavitary chemotherapy, biological response modifiers, traditional chinesemedicine, thermotherapy, etc. However, the effects of the above-mentionedtreatments are all unsatisfactory. After therapy with these methods, there is nosignificant decrease of effusions while it often relapses fast, and almost all ofthe treatments have toxic side effects of various degrees.Studies have shownthat elevated levels of VEGF (vascular endothelial growth factor), tumorangiogenesis, and increased vascular permeability after tumor invading ormetastasizing to the pleuroperitoneum are important formation mechanisms ofserous cavity effusions. The VEGF family includes six types: VEGF-A, B, C,D, E, and PIGF (placental growth factor). These factors are mainly secreted bymacrophages and tumor cells. VEGF-A is the most important regulatory factorin the tumor angiogenesis, and VEGF-C and VEGF-D are the most importantregulatory factors in the tumor lymphangiogenesis. The use of angiogenesisinhibitors, particularly VEGF targeted drugs has become a hot topic of thebasic and clinical researches.Currently, the study on the VEGF in serous cavity effusions is mainly focused on VEGF-A representing the angiogenesis,and there are few studies on the lymphangiogenesis representative factorsVEGF-C and VEGF-D.The domestic first class new drug recombinant humanendostatin (rh-endostatin, endostar) is a broad spectrum humanizedanti-angiogenic drug which targets vascular endothelial cells but not tumorcells and can down-regulate the protecting effect of a variety of proangiogenicfactors on the vascular endothelium, thus playing direct and indirectantiangiogenic effects. In recent years, there are a number of scholars hasexplored the treatment effect of endostar used alone or combined withchemotherapy on malignant serous cavity effusions. They found it efficientand low toxic, not only being able to produce a strong effusions controllingeffect with a high tolerance, but also being able to significantly improve thequality of life of patients. However, there are a lot of work to do in thefollowing areas: the efficacy evaluation system of angiogenesis inhibitors suchas endostar, and exploring new molecular markers to predict and evaluate theefficacy of endostar. How to improve and use molecular markers to guide theclinical diagnosis, evaluate the prognosis, predict the efficacy and optimize thetreatment of malignant serous cavity effusions is the direction of futureworks.In this study, we detected the contents and expression levels ofVEGF-A,C,D in the supernatants and cells from the malignant serous cavityeffusions caused by lung cancer and gastric cancer, to explore the clinicalsignificance of the three factors in the diagnosis and prognosis of malignantserous cavity effusions and in the prediction of efficacy of endostar on theeffusions. We established a stably expressing luciferase gastric NUGC-4-luccell line, and on the basis of this cell line we established gastric cancer ascitestumor model in nude mice for the first time. By combining dynamicmonitoring changes of gastric cancer ascites tumor cells by in vivobioluminescence imaging with detecting the peritoneal nodules MVD(microvessel density), LVD (1ymphatic vessel density), we comprehensivelyevaluated the value of the molecular markers VEGF-A, VEGF-C and VEGF-Din the targeted therapy of malignant serous cavity effusions.
Part1Diagnostic and prognostic values of vascular endothelial growthfactor (VEGF)-A,-C and-D in malignant effusion of lung andgastric cancer patients
Objective: To evaluate VEGF-A,C,D the diagnosis, clinical prognosticvalue in malignant effusion
Methods: Enzyme-linked immunosorbent assay(ELISA) was used todetect VEGF-A,C,Dprotein content from patients with effusion of the serum79cases (30lung cancer,21gastric cancer,28benign effusions), thesupernatant fluid96cases (38lung cancer,30gastric cancer,28benigneffusion); Immunocytochemistry (ICC) detected VEGF-A,C,D proteinexpression from71cases of effusion cell level (34lung cancer,17gastriccancer,20benign effusions), by SPSS17.0to analyze the diagnosis, prognosis,clinicopathological factors and correlation of clinical significance.
Results:
1VEGF-A
1.1The level of sVEGF-A proteins was not statistically different betweencancer and benign patients (p>0.05). Cancer patients had higher pVEGF-Alevel than those with benign effusion (p <0.05). The upregulated level ofpVEGF-A was not associated with tumor histological types (p>0.05).pVEGF-A level was similar to the corresponding sVEGF-A level in patientswith benign effusions. Nevertheless, pVEGF-A level in malignant effusionwas significantly higher than its corresponding sVEGF-A level (p<0.05).
We detected VEGF-A,-C, and-D expressions in20cytological smearsfrom benign effusions and the data showed that there was no positive stainingof anti-VEGF-A,-C, and-D antibodies in these20benign cases. VEGF-Aexpression rate was52.94%and mainly expressed in the cytoplasm ofpositively expressed tumor cells.
1.2The data showed that pVEGF-A and cVEGF-A were negativelyassociated with age and pVEGF-A was positively associated with malignantand bloody effusion, pleural or peritoneal metastasis (p<0.05).
1.3With the median levels as the cut-off values, patients were divided into high and low pleural effusion parameter groups. One-year survival timeanalysis showed a relatively shorter survival time for patients with pVEGF-Alevels>406.19pg/ml as compared with those presenting with pVEGF-a levels≤406.19pg/ml, although this effect was only of marginal statisticalsignificance (p=0.066). sVEGF-A with the median levels as the cut-off, didnot exhibited statistical significance in predicting survival for patients withmalignant effusions (p>0.05).
2VEGF-C, VEGF-D
2.1Levels of sVEGF-C， sVEGF-D proteins were not statisticallydifferent between cancer and benign patients (p>0.05). Levels of pVEGF-C，pVEGF-D proteins were not statistically different between cancer and benignpatients (p>0.05).cVEGF-C，cVEGF-D expression rates were70.58%and82.35, respectively, in51patients with lung or gastric cancer. These proteinswere mainly expressed in the cytoplasm of positively expressed tumor cells.Regarding their expression levels of in tumor patients, the expression rate ofcVEGF-D was significantly higher than that of cVEGF-A and cVEGF-C(p<0.05). However, there was no statistically significant difference incVEGF-A,-C and-D expression between lung and gastric cancers (p>0.05).
2.2The data showed that pVEGF-C was negatively associated with age.pVEGF-D expression was positively associated with age and negativelyassociated with malignant effusion and pleural metastasis (p<0.05). However,there was no association of sVEGF-C， sVEGF-D proteins with theclinicopathological factors (p>0.05).
2.3sVEGF-C,sVEGF-D and pVEGF-C,pVEGF-D with the median levelsas the cut-off, did not exhibited statistical significance in predicting survivalfor patients with malignant effusions (p>0.05).
Conclusions:
1The supernatant fluid VEGF-A was significantly different betweenbenign and malignant effusion, suggesting that VEGF-A may be used as atumor marker in the clinical setting to discriminate benign from malignanteffusions.VEGF-A levels reached marginal statistical significance as a potential predictor of poor clinical outcome. Given the fact that VEGF-Aexpression might have a survival impact, attention should be paid to thepatients with high VEGF-A expression.
2The supernatant fluid and cellular level of VEGF-A were negativelycorrelated with age, probably reflecting with age growing, malignant effusionsand level of VEGF-A are reduced. The supernatant fluid levels of VEGF-A inthe patients with bloody pleural effusion and only pleural metastasis are higherthan that in patients with non-bloody pleural effusion and not only pleuralmetastasis.
3The supernatant fluid VEGF-C and VEGF-D had no significantdifference in the clinical diagnosis of benign and malignant, but highlyexpressed at the cellular level. It needs expand samples of benign effusions inclinical practice to further study its diagnostic value after disease stratification.
4Serum VEGF-A,C,D levels had no obvious clinical significance in thediagnosis and prognosis of serous cavity effusions, and they were also notsignificantly associated with the clinicalpathological factors. It is suggestedthe serological markers’ diagnosis and prognostic values for serous cavityeffusions may be not as good as that of the local effusion markers.
Part2The predictive value of VEGF-A, C, D for the clinical efficacy ofendostar on malignant effusions
Objective: To evaluate the predictive value of VEGF-A,C,D for theclinical efficacy of endostar on malignant effusions.
Methods: ELISA was used to detect VEGF-A,C,D protein contents of20pairs of patients with malignant effusions reciving intracavitary endostarbefore and after treatments. The values of the three factors in pridicting theefficacy of endostar were analyzed by SPSS17.0.
Results:
1Between the endostar monotherapy group, endostar plus cisplatin groupand endostar monotherapy combined with systemic anti-tumor therapy group,there were no significant differences in the objective response rate (RR: CR+PR), clinical benefit rate (DCR:CR+PR+NC) or local progression (PD)(p> 0.05).
2The relationships between supernatant fluid VEGF-A,C,D proteincontents with efficacy: Neither the differences between before and aftertreatment （d0-d7） nor the difference ratios before and after treatment（d0-d7/d0）of the three factors reached statistical difference in RR, NC, PDgroups (p>0.05), with the difference ratio of the supernatant VEGF-A(d1-d7/d1) between groups result showed p=0.085.
3The relationships between supernatant fluid VEGF-A,C,D proteincontents with treatment: The difference ratios before and after treatment（d0-d7/d0）of supernatant fluid VEGF-A,C,D had no significant differencebetween the three endostar groups (p>0.05).
Conclusions:
1Neither the differences between before and after treatment（d0-d7） northe difference ratios before and after treatment （d0-d7/d0）of supernatant fluidVEGF-A,C,D had no statistical difference in RR, NC, PD groups, suggestingthe three factors can’t pridict the efficacy of endostar.
2The difference ratios before and after treatment (d0-d7/d0) ofVEGF-A,C,D had no statistical difference in different endostar groups,suggesting the three factors can’t guide the clinical treatment.
Part3The research of VEGF-A, C, D in gastric cancer ascites tumor modeltreated with endostar given by different ways
Objective: To comprehensively evaluate the value of the molecularmarkers VEGF-A,C,D in the gastric cancer ascites model by combiningmonitoring the cancer cells with detecting the vascular and lymphaticendothelial markers.
Methods: Cultured NUGC-4-luc cells (RPMI1640medium+10%FBS+1%P/S), suspended a total of5×106NUGC-4-luc cells in50ml PBS. Thesuspension was pipetted with micropipettor and injected into abdominalcavities of28female nude mice. These intraperitoneally inoculated miceunderwent in vivo bioluminescence imaging weekly. One week afterNUGC-4-luc cells injection, mice were randomly divided into four groups of7 mice each: Group1: cisplatin (1mg/kg) d1-3+endostar (8mg/kg) d4-7;Group2: endostar (8mg/kg) d1-4+cisplatin (1mg/kg) d5-7; Group3: endostar(8mg/kg)+cisplatin (1mg/kg) d1,4,7; Group4(control group):50μl ofnormal saline d1-7. All of the treatments were injected intraperitoneally. Everygroup was treated two consecutive weeks, underwent imaging weekly. Oneweek after the final administration, mice were anesthetized and sacrificed.Then the supernatant fluid of ascites was taken and the contents ofVEGF-A,C,D proteins in it were detected by ELISA. VEGF-A,C,D as well asMVD and LVD in the peritoneal nodules were detected by IHC methods afterthe nodules had been embeded into wax blocks. All bioluminescent data werefirst collected and processed using IVIS and then analyzed by SPSS17.0toexplore the differences of imagings, protein expressions and contents ofVEGF-A,C,D, and LVD and MVD between the four treatment groups.
Results:
1Ascites tumor model
5×106NUGC-4-luc cells were injected into abdominal cavities. One weekafter NUGC-4-luc cells injection, mice were randomly divided into fourgroups by the number of cancer cell photons, with no statistical differences inthe four groups (p>0.05). After initiating treatment an accidental deathsoccured in the first18days in Group2.On every other day there were nosignificant differences in the nude mice weights between the four treatmentgroups (p>0.05).After mice were sacrificed,we found bloody ascites wereobvious and peritoneal nodules were small,numerous and widely.
2Drug efficacy
2.1Volume of ascites in four groups(ml):Group4（4.87±0.45）>Group1（3.1±0.53）>Group2（2.0±0.08）>Group3（1.8±0.16）.
2.2The number of peritoneal nodules: Group4（33.75±2.5）> Group2（21.66±5.77）> Group3（18.75±2.5）> Group1（8.75±4.78）.
2.3The counts of cancer cell photons
By using in vivo imaging processing software, tumor growth curves wereplotted according to the number of photons per measurement. On day1and day8, there were no significant differences between the four groups (p>0.05).On day15and day21, group1,2and3were all better than the control group(p<0.01). On day15(after treatment of two weeks), endostar combinationwith cisplatin group (Group3) and cisplatin sequential endostar group (group1)were better than endostar sequential cisplatin group (group2)(p<0.05),however there was no significant difference between the former two groups(p>0.05). On day21(after treatment of two weeks, one week after withdrawn),endostar combination with cisplatin group (Group3) was still better thanendostar sequential cisplatin group (group2)(p<0.05).
2.4MVD,LVD
The lumens of blood vessels and lymphatics were determined by CD34and D2-40stained brown to black. There were differences in MVD betweenthe four groups (p<0.01), pairwise comparisons showing group4> group1>group2(p<0.01), and there was no difference between group2and group3(p>0.05). However, there were no statistical differences in LVD numberbetween the four groups (p>0.05).
3VEGF-A,C,D
3.1There were differences in supernatant VEGF-A between the fourgroups (p<0.05), pairwise comparisons showing there was difference betweeneach two groups of the four groups with the result ofgroup4>group1>group2>group3(p<0.05). The ascites supernatant levels ofVEGF-C and VEGF-D proteins had no significant difference between the fourgroups (p>0.05).
3.2VEGF-A,C,D proteins were all cytoplasmic staining. There werestatistical differences in VEGF-A between the four groups (p<0.05), pairwisecomparisons showing Group4>Group2>Group1（p<0.05）， and there was nodifference between group1and group3(p>0.05). However, there were nostatistical differences in VEGF-C and VEGF-D expressions between the fourgroups (p>0.05).
4VEGF-A protein content was highly correlated with the volume ofascites (r=0.994, p<0.01). VEGF-A protein expression was highly correlated with the number of peritoneal nodules (r=0.994, p<0.01), but was notcorrelated with the MVD and the counts of cancer cell photons (p>0.05).
Conclusions:
1On the basis of a stably expressing luciferase gastric NUGC-4-luc celllines, we established gastric cancer ascites tumor model in nude mice for thefirst rime. We provided a stable, reliable, intuitive, convenient, and sensitiveanimal model for exploring the growth and metastasis mechanisms of thegastric cancer peritoneal metastatic tumor and the research and developmentof anticancer drugs by using in vivo imaging techniques.
2In this study, the groups of endostar combined and sequentially usedwith cisplatin were all well tolerated, and treatment should be at least twoweeks before observing differences. The present study combined in vivoimaging with the MVD for the first time, and the results suggested:combination group was superior to sequential groups, the former’s killingcapability on cancer cells and inhibiting effect on vascular endothelium wereall the greatest; cisplatin sequential endostar was superior to endostarsequential cisplatin in killing cancer cells, while endostar sequential cisplatinwas superior to cisplatin sequential endostar in inhibiting peritoneal vascularendothelium. It was suggested that we can use molecular markers to forecasttarget changes and use functional imaging to monitor the number of cancercells, to guide the rational choice of the targeted and cytotoxic drugs and theadministration timing.
3VEGF levels were highly correlated with the volume of ascites and thenumber of peritoneal nodules, suggesting that the predictive value of VEGFfor the efficacy of malignant effusion may be not only the volume of ascitesbut the size of peritoneal nodules. VEGF-A expressions were not correlatedwith the MVD and the counts of cancer cell photons. These suggested that itcan’t be used to monitor the activity of cancer cells and lymphatic vascularstatus, and it can’t be used to guide the choice of drugs intraperitoneallyinjected as well.
4No differences in ascites and peritoneal nodules VEGF-C and VEGF-D proteins and LVD between the four treatment groups were found, so they stillcan’t serve as predictors of efficacy.
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