超声微泡破坏抑制肿瘤生长及其作用机制研究
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摘要
研究背景
恶性肿瘤是目前威胁人类健康的主要疾病之一,在某些国家甚至成为首要的死亡原因。现在恶性肿瘤主要的治疗手段有手术、放疗和化疗,其作用的主要机制在于直接切除肿瘤组织,杀灭肿瘤细胞和诱导细胞凋亡。但是手术、放疗未能从根本上解决抑制肿瘤新生血管形成,控制肿瘤的生长,阻止肿瘤细胞侵袭和转移等问题。而目前用于肿瘤治疗的化疗药物大多缺乏肿瘤细胞的靶向性并且具有较大的毒副作用,在杀伤肿瘤细胞的同时,也杀伤了大量的骨髓细胞及其他增殖旺盛的正常细胞,因此以上治疗在临床应用中有很大的局限性。
有研究表明,实体肿瘤的生长依赖于持续和大量的血管生成,血管生成为肿瘤组织提供营养物质和氧气,同时血管又是肿瘤细胞向远处转移的主要途径,因此针对血管形成的某些因子及其关键步骤进行干预,有望切断肿瘤血供及其转移途径,对肿瘤的治疗和防止肿瘤向远处转移有重要意义。近年来,抗血管治疗成为肿瘤治疗领域的研究热点,但是,目前的抗血管新生药物在临床的应用中有很大的局限性,不同肿瘤或者不同患者的同一肿瘤在抗血管生成的治疗中,对药物的反应不同；同时,单一抗肿瘤药物必须和化疗药物联合使用才能起到抗肿瘤的作用,即便如此肿瘤患者的临床获益也非常有限。因此,寻求一种新的安全有效的抗肿瘤血管治疗方法便显得尤为必要。
近些年来,随着超声学的发展,超声及微泡造影剂在临床中应用的更加广泛,他们不仅在诊断方面具有极其重要的科学意义和临床价值,而且随着对超声领域的深入研究,其在治疗方面也显示出比较诱人的前景。
超声微泡在疾病治疗中的主要作用机制是超声空化效应,所谓超声空化效应就是液体中内源性或外源性的微气泡空化核在超声作用下发生振荡、膨胀、收缩及内爆等一系列的动力学过程,它是超声在液态物质中传播所特有的现象。既往研究表明微泡破坏产生的空化效应可以损伤微血管内皮,并对周围的组织细胞产生影响,而这种作用有可能为肿瘤的治疗提供一种新的方法。接下来的一系列研究显示：超声微泡破坏可以损伤肿瘤血管,从而使肿瘤区域的血流灌注得到明显减少,其病理变化主要表现为出血、血管扩张、组织间水肿、血栓形成。但是这样的空化效应使血流灌注明显减少能持续多长时间,是否能使肿瘤的生长得到抑制,以及是否可以明显改善荷瘤动物的生存率,至今仍未见报道。同时,我们预实验显示,超声微泡破坏治疗1分钟后的病理变化中未发现明显的血栓形成,Bunte等在其研究中也显示同样的实验结果。那么,什么样的病理变化使肿瘤在治疗后血流灌注明显减少,目前还不太清楚。
目前有大量文献表明,肿瘤血管在形态结构和功能上与正常血管明显不同。正常的微血管有动脉、静脉和毛细血管之分,而实体瘤的血管没有动静脉的区别,其形态结构主要表现在扩张、分叉、迂曲没有规律,管壁直径不均匀,基底膜缺陷,血管外周细胞不完整等特点。基于以上理论依据,我们假设肿瘤血管对空化效应的机械性损伤可能更为敏感,超声微泡破坏有可能更容易损伤肿瘤血管,在达到抑制肿瘤生长的同时而不损伤正常组织。以上假设可以使我们在保证疗效的同时更确保其安全性,这些都可以给肿瘤的治疗带来重要的临床价值,但类似的设想和研究鲜见报道,更缺乏实验验证和机理解释。
研究目的
(1)通过超声微泡空化效应,观察微泡破坏能使肿瘤血流灌注减少持续多长时间；观察微泡破坏是否抑制肿瘤的生长；观察肿瘤小鼠的生存率是否得到明显提高。(2)探索超声微泡破坏使肿瘤血流灌注减少的原因及其机制(3)观察正常组织血管和肿瘤血管对超声微泡破坏反应的差异及其机制。从而为肿瘤的治疗提供一种安全有效的治疗方法及理论依据。
材料和方法
1.实验材料
主要实验仪器：(1)小型脉冲式聚焦超声空化治疗仪：由第三军医大学新桥医院超声科提供,治疗探头频率：0.94MHZ,超声发射占空比0.1%,声压：1500MPa, t=1min。脉冲重复频率(PRF)10HZ。(2)超声诊断仪(Sequoia512,德国Siemens公司)；15L8w探头,频率7.0～14.0MHz,配有增强脉冲序列(contrast pulse sequencing, CPS)造影成像技术。主要实验试剂：白蛋白微泡造影剂：由南方医科大学药学院自行研制,其成膜材料为白蛋白,核心气体为全氟丙烷,外观呈乳白色凝乳状MBc平均粒径为2.07±1.13um,浓度约为2.00-4.25x109个/ml。
2.实验方法：
(一)超声微泡破坏抑制肿瘤生长疗效的研究
实验分组：将建成的肿瘤模型随机分为两组,即超声治疗组(MB+US组)36只,单纯微泡组29只。超声微泡组采用超声照射联合经尾静脉微泡注射；直接抽取“白蛋白”微泡悬液0.02ml,经尾静脉直接注入,进行对比增强超声(CEU)和二维超声成像,待对比增强超声成像信号完全消失后,再次经尾静脉注入0.1ml的白蛋白微泡,在肿瘤区域行超声破坏治疗,治疗后再次注入0.02ml的白蛋白微泡,再次进行对比增强超声(CEU)和二维超声成像。单纯微泡组(MB)：操作步骤同上,但超声探头不发射超声脉冲波。
肿瘤对比增强超声检查：对所有肿瘤模型组经尾静脉随机注射MB,处理后对肿瘤组织行对比超声检查。动物模型制备后,用自制支架固定超声探头(17L5)于肿瘤上,调整探头位置获得良好肿瘤显像后保持探头位置在整个实验过程中不变,仪器的各项参数在整个实验过程中保持不变。CEU采用经尾静脉微泡弹丸式注射法进行,经过一段时间待血池中循环微泡完全消失后,经尾静脉再次注入0.1ml造影剂,用上述超声治疗条件进行治疗,治疗后即刻、24h和72h再次注入0.02ml,对实验小鼠进行肿瘤CEU检查,获取实验肿瘤的显影图像。CEU检查采用二次谐波成像(second harmonic imaging)技术进行,探头发射频率(transmission frequency)和接收频率分(receiving frequency)别为7.0MHz和14MHz,机械指数(Mechanical Index, MI)为0.18。
肿瘤二维超声检查：每隔5-7天对肿瘤进行B-型超声检查,每次测量肿瘤最大切面的长径和短径,肿瘤体积V=1/2ab2(a：长径,b：短径)探头发射频率15MHz,机械指数(Mechanical Index, MI)为0.57。全部声学图像存于MO盘,以备脱机分析。
对比超声图像的分析：应用MCE软件对超声图像进行分析,在两组中随机选择5只小鼠测量治疗前、治疗后即刻、24h、72h显影的声强度(video intensity,Ⅵ)。
(二)微泡破坏抑制肿瘤生长作用机制的研究
实验分组：20只S180肿瘤模型随即分为治疗前组、治疗后即刻组、治疗后24h组和治疗后72h组。动物模型制备后,经尾静脉注入0.1ml的白蛋白微泡,在肿瘤区域行超声破坏治疗。
免疫组化及组织病理学检测：分别在治疗前、治疗后即刻、24h、72h等四个时间点将肿瘤取出,行免疫组化、HE染色。观察MPO、CD31表达情况。
微血管密度测定方法：采用CD31抗体染色,将与邻近肿瘤细胞和结缔组织分离的被染成棕色的内皮细胞、内皮细胞簇或分支状血管结构视为一个微血管,每个样本先在低倍镜(×100)下观察阳性染色的微血管,择染色最多的区域(即“热点”),然后选择5个不重复高倍镜视野,最后计算单位视野(×200)的平均微血管数,即MVD。
MPO半定量评估：组织免疫组化采用以下得分方法评估,免疫染色的强度分为以下四个等级：0(无)、1(弱)、2(中等)、3(强)；染色范围得分可分为以下四个等级：0(0%)、1(10%-25%)、2(26%-50%)、3(51%-75%)、4(76%-100%),两者评分结果相加即为最终得分。
(三)微泡破坏对正常组织及肿瘤组织的不同影响及其机制
实验分组：实验分为肿瘤治疗组和正常组织治疗组小鼠各5只,分别在小鼠的肿瘤区域和腿部骨骼肌行相同条件超声空化治疗,实验步骤及条件同前。
免疫组化组织病理学检测：分别在治疗前、治疗后即刻、时间点将肿瘤取出,行免疫组化、HE染色、免疫荧光检测。
血管成熟程度评估：用cd31抗体特异性标记血管内皮细胞,用α-sma特异性标记外周细胞/平滑肌细胞。血管如果同时标记两种抗体,表明血管发育较为成熟,如果只标记cd31抗体,表明血管发育较为幼稚。
结果
1.超声微泡破坏抑制肿瘤生长疗效的研究
CEU检查结果：CEU图像显示肿瘤治疗组和对照组均可见显著的超声显影,而肿瘤对照组单纯给予微泡造影剂后,超声显影无明显的变化,而超声治疗组在超声破坏后即刻超声显影明显减弱,治疗后24h和72h时间点造影图虽可见肿瘤内血流灌注信号部分恢复,但与治疗前比较,仍具有显著性差异。
CEU图像分析及统计学分析结果：应用CEU软件对图像进行分析,测量实验组肿瘤组织不同时间点显影的声强度(rideo intensity,Ⅵ),并采用彩色编码技术制作显影的彩色编码图像。治疗前肿瘤区域的Ⅵ值较高,为66.012±4.129,应用超声微泡治疗后即刻、24h、72h与治疗前的VI值相比较明显减少,分别是16.677±3.297、26.524±2.193和33.225±19.952(p<0.001),有显著性差异。
二维超声检查：实验结果显示,单纯超声微泡组肿瘤的生长没有得到抑制,而肿瘤治疗组在超声微泡治疗后,显著抑制了肿瘤生长。在肿瘤种植第七天,两组肿瘤的体积分别为0.315±0.080cm3和0.364±0.135cm3(p=0.098),两者无明显的统计学差异,在单纯微泡组,肿瘤种植第11、17、25天,肿瘤的平均体积分别为1.112±0.340cm3、2.526±0.483cm3和5.718±1.078cm3,而在超声治疗组,肿瘤种植第11、17、25天,肿瘤的平均体积分别为0.662±0.186cm3、1.856±0.349cm3和3.290±0.772cm3(p<0.001),与单纯微泡组相比较,明显抑制了肿瘤的生长。
肿瘤小鼠的生存状况：与单纯的微泡组相比较,超声微泡破坏显著延长了肿瘤小鼠的生存时间。单纯微泡组小鼠的平均生存时间是50.931±8.426天,而肿瘤治疗组小鼠的平均生存时间是72.722±10.870天(p<0.001),与单纯微泡组相比较,有显著性差异。
2.微泡破坏抑制肿瘤生长作用机制的研究
免疫组化结果：免疫组化CD31表达：肿瘤组织在治疗前,血管的形态结构完整,内皮细胞排列比较紧密。而在超声微泡破坏后即刻,血管管腔结构遭到严重破坏,血管内皮细胞部分缺失,呈弥散性分布,超声治疗后24小时、72小时,仍然看到大量的血管没有管腔结构,血管内皮细胞连续性中断,呈弥散性分布,但在肿瘤的周边区域,有少量的新生血管。
测量治疗前后肿瘤微血管密度结果显示：在肿瘤治疗前,肿瘤组织有大量的微血管存在,肿瘤治疗后即刻、24h、72h后微血管密度明显减少,和治疗前相比较,有明显的统计学差异(p<0.001)
免疫组化MPO表达：微泡破坏前,肿瘤组织髓过氧化物酶(MPO)的表达并不明显,中性粒细胞的侵润并不严重,在肿瘤治疗后即刻,髓过氧化物酶的表达并没有明显增加,但治疗24h,髓过氧化物酶的表达明显增加,到72小时后,其表达有所下降,但与治疗前相比,仍然是明显增加。
半定量分析显示：肿瘤治疗前,MPO表达不明显,染色评分为：1.400±0.548,治疗后即刻,评分结果为：1.600±0.548(p=0.572),没有显著性差异,而在24h,评分结果为：6.600±0.548(p<0.001)与治疗前相比有显著统计学意义,72h后评分结果为：3.600±0.548(p<0.001)与治疗前和治疗后24h均有显著性差异。
病理学结果显示：肿瘤组织在治疗前可见肿瘤细胞分布呈团状或片状,排列紧密,细胞大小不一,核大,深染,核仁明显,肿瘤组织内可见较丰富的微小血管,血管官腔结构相对较完整,组织间未见红细胞渗出。肿瘤超声微泡治疗后即刻：肿瘤血管不完整,血管内皮连续性中断,组织间有大量红细胞渗出,肿瘤细胞并没有大量坏死。肿瘤细胞超声治疗24小时后,肿瘤治疗区域未见有血管结构,组织间渗出的红细胞崩解吸收,并出现大量肿瘤细胞的坏死,主要表现为细胞核碎裂、溶解。72小时后,肿瘤组织坏死面积更大。
(三)微泡破坏对正常组织及肿瘤组织的不同影响及其机制
CEU检查结果：彩色编码图像显示,肿瘤组织在超声破坏后超声显影明显减弱,骨骼肌组织在治疗后显影声强度虽然也有减弱,但是不太显著。
免疫组化：肿瘤组织在治疗前,血管的形态结构完整,内皮细胞排列比较紧密。而在超声微泡破坏后即刻,血管官腔结构遭到严重破坏,血管内皮细胞部分缺失,呈弥散性分布；骨骼肌组织在治疗前,血管的形态结构完整,内皮细胞排列比较紧密,治疗后,骨骼肌的血管形态仍然相对完好,内皮细胞排列仍然紧密。
测量治疗前后肿瘤微血管密度结果显示：在肿瘤治疗前,肿瘤组织有大量的微血管存在,肿瘤治疗后即刻微血管密度明显减少(p<0.001)和治疗前相比较,有明显的统计学差异；骨骼肌治疗前微血管密度为：34.400±3.847,治疗后微血管密度是：27.000±3.162,有显著性差异,但是与肿瘤治疗相比较,其下降幅度明显减少。
免疫荧光：结果显示：治疗前,在肿瘤组织中发育成熟血管在微血管中占很小的比例,约为31.907±3.911%,在骨骼肌血管中,发育成熟的血管所占比例较大,约为87.869±3.061%(p<0.001),有显著性差异。在治疗后,肿瘤组织中,成熟血管在微血管中所占的比例明显增高,约为87.851±1.755%,与治疗前相比较,有显著统计学差异(p<0.001),骨骼肌组织中,成熟血管所占的比例也有所增高,为90.444±4.298,与治疗前比较没有显著统计学差异(p=0.307)。
结论：
1.超声微泡破坏使肿瘤的血流明显减少,持续时间达72小时以上；超声微泡破坏使肿瘤的生长得到了明显的抑制,使肿瘤小鼠的生存率得到了显著的提高。
2.超声微泡通过直接破坏肿瘤微血管使血流灌注明显减少,导致肿瘤细胞大面积的缺血坏死,在这过程中,中性粒细胞的激活起了很重要的作用。
3.肿瘤组织微血管较正常组织微血管的发育更为幼稚,结构更为不完整,导致超声微泡破坏对正常组织微血管的破坏程度轻,而对肿瘤血管的破坏程度重,保证了治疗的安全性。
Background
Malignant tumor is one of the major diseases which threaten human health and life, even as the leading cause of death in some countries. The common strategies of the current treatment include surgery, radiotherapy and chemotherapy which is mainly to remove tumor tissue, kill tumor cells or induce cell apoptosis. However, these methods are all failed in inhibiting the formation of tumor vessels, inhibiting the growth of tumors and preventing metastasis, while the chemotherapy lacks of specific target and has side effects for not only be able to kill tumor cells but also kill a large number of bone marrow cells and proliferation cells at the same time,they are limited in treating the tumors.
Mammalian cells require oxygen and nutrients for growth and metastasis and are therefore located within microvessels-the diffusion limit for oxygen. Without blood vessels, tumors cannot grow beyond a critical size or metastasize to another organ. It is important significance if we intervene in some ofthe factors and key steps of angiogenesis to cut off the blood supply for the growth and metastasis. However, these antiangiogenic drugs have some limitations in clinical application because different tumor or the same tumor has different reactions to drugs in the process of the treatment. The clinical benefit is very limited when just use the single anticancer drugs even though combined with the chemotherapy drugs. So it is urgent to look for an impressive therapeutic method which is safe and effective.
Recent years, with the development of ultrasonic, ultrasound and contrast agent has become more and more widely and important in clinical application. They not only have important scientific significance in diagnosis but also show attractive prospects in the treatment field.
The basic theory of the mechanism in the treatment is the ultrasonic cavitation effect, which is endogenous or exogenous microbubble cavitation nuclei process a series of dynamic process such as oscillation, expansion, contraction, implosion and so on.it is peculiar to the ultrasonic propagation in the liquid. In previous studies it shows that Microbubble destruction during ultrasound exposure caused injury of microvessels (mostly capillaries) and the production of nonviable cells in adjacent tissue. Microbubbles can be destroyed by ultrasound, resulting in a bioeffect that could be used for tumor destruction. The next series of researches show that Ultrasound microbubble destruction can lead to the blood perfusion of the tumor significantly reduced by damaging the blood vessel of the tumor. The histopathologic studies showed that the effects of insonation were predominantly on the vascular structures within the tumor and correlated with the findings in the contrast enhanced power Doppler ultrasonographic images. There was disruption of the walls of the tumor blood vessels with associated hemorrhage, vascularcongestion and subsequent thrombosis; edema was also present.But it is not known how long the the cavitation effect which reduced blood perfusion last? Whether it can inhibit the growth of the tumor? Whether it can improve survival rates of the tumor animals? In our preliminary experiments it shows that there is no thrombosis in histopathologic studies when the tumor received1minute treatment which is the same as other scholars. So The question remains as to what were the underlying histopathological changes leading to the observed loss of contrast enhanced power Doppler signal in the treated neoplasms?
We have known that it is obviously different between the normal and tumor blood vessels in structure and function. Normal vessel are organized with arteries, veins and capillaries, but the tumor vessels do not display the recognizable features of arterioles, capillaries or venules.The tumor Blood vessels are leaky, tortuous, dilated, and saccular and have a haphazard pattern of interconnection. The endothelial cells lining these vessels have aberrant morphology, pericytes are loosely attached or absent, and the basement membrane is often abnormal—unusually thick at times, entirely absent at others.
Based on the above theory, the tumor blood vessels may be more sensitive to mechanical damage of the cavitation effect. It just damages the tumor angiogenesis not normal blood vessels which suppress tumor growth. This therapy can bring us important clinical value of as it guard the safety and effective effect in the treatment. but it is not reported by now, and it is lack the mechanism explanation to a high degree.
Objective
(1) How long the the cavitation effect which reduced blood perfusion last? Whether it can inhibit the growth of the tumor? Whether it can improve survival rates of the tumor animals?
(2) What histopathological changes lead to the observed loss of contrast enhanced power Doppler signal in the treated neoplasms?
(3) To observe whether there are some differences in ultrasound microbubble treatment response between normal and tumor blood vessels, if they are different, what's the mechanism?
Materials and Methods
1. Materials
Principal Experimental instruments:(1) small, pulsed, focused ultrasonic cavitation treatment apparatus, offered by ultrasound department of the Third Military Medical University. With a treatment probe frequency of0.94MHz, an emission duty of0.1%, a peak negative pressure of1500kPa, time=lmin, Pulse repetition frequency10HZ.(2) Doppler ultrasonic machine with ultrasonic contrasting mode (Sequoia, Siemens Medical Systems, Mountain View, Calif). A17L5transducer, probe frequency of7-14MHz, the machine with contrast pulse sequencing technology.
Experimental reagents,"perflutren protein-type microbubbles developed by Department of Pharmacology, Nanfang Hospital, Southern Medical University, with perfluoropropane as core gas, at approximately2.0-4.25X109microbubbles/ml. A mean of2.07±1.13um in diameter.
2. Method
(1) The research on microbubble destruction inhibits growth of tumor
Mice were randomly divided into treated group (n=36) and control group (n=29) each anaesthetized pentobarbital sodium60mg/kg ip) mouse were imaged before and after tumor insonation and histology was performed in a selected number, a tail vein was catheterized before insonation. In the treated group, an initial B-mode study was followed by the intravenous injection of an ultrasound contrast agent (0.02ml perflutren protein-type microbubbles) and contrast-enhanced power Doppler observations were made. After the contrast agent was no longer detectable in these initial observations, a further0.1ml microbubbles was injected and the physiotherapy transducer (acoustic pressures=1500KPa; f=0.94MHZ; duty factor=0.1%;) was applied to the tumor for1min (transducer worked at an intermittent mode of2s on and10s off), after the procedure the B-mode and contrast-enhanced power Doppler studies were repeated at Oh,24h and72h post-treatment in the identical anatomical plane. In the control group, a similar study to treated group was performed, however, the physiotherapy transducer was applied to the tumor was not switched on after a further0.1ml microbubbles was injected.
Tumor CEU imaging:All experimental mice were performed with CEU respectively by using MBa before treatment with the low mechanic index (MI=0.18), the intravenous injection of0.1ml microbubbles and subsequent post-treatment(Oh,24h and72h) images were acquired at the same mechanical index. To identify the signal from microbubbles retained in the tumor tissue, several post-treatment contrast frames representing the signal from circulating microbubbles were averaged and digitally subtracted from several averaged frames which acquired at pre-treatment, Oh, and24h and72h post-treatment with use of software (Yabko MCE2.7; University of Virginia, Charlottesville, Va), transmission frequency of7.0MHz and receiving frequency of14.0MHz.
B-mode:Ultrasound measurements of the growth of the tumor continued every5-7days with the low mechanic index (MI=0.57). In each of the B-mode images, the length (L), width (W) of the tumor was measured and its volume (mm3) was calculated as:Tumor volume=length×(width)2/2.
Contrast-enhanced Ultrasound imaging analysis:To identify the signal from microbubbles retained in the tumor tissue, several post-treatment contrast frames representing the signal from circulating microbubbles were averaged and digitally subtracted from several averaged frames which acquired at pre-treatment,0h, and24h and72h post-treatment with use of software (Yabko MCE2.7; University of Virginia, Charlottesville, Va) and then color coded. The background-subtracted video intensity (VI) was measured in a region of interest fit the boundary of the tumor. CEUS of the muscle was obtained at the same method.
(2)The mechanism of ultrasound mediated microbubble disruption inhibits the tumor growth
20mice were randomly divided into pre-treatment group (n=5), post-treatment Oh group (n=5), post-treatment24h group (n=5) and72h group (n=5).0.1ml microbubbles was injected and the physiotherapy transducer (acoustic pressures=1500KPa; f=0.94MHZ; duty factor=0.1%) was applied to the tumor for1min (transducer worked at an intermittent mode of2s on and10s off).
Histology and Immunohistochemistry:For Immunohistochemistry and Histology, the tissue of the tumor was taken out at the time of pre-treatment, Oh, and24h and72h post-treatment, to observe the expression of the anti-mouse CD31antibody and anti-MPO antibody.
Microvessel density measurement:The area of highest neovascularization was identified; individual microvessels were counted first on a100x field and then on a200×field.Any brown-staining endothelial cell or endothelial-cell cluster that was clearly separate from adjacent microvessels, tumor cells, and other connective-tissue elements were considered a single, countable microvessel. Vessel lumens, although usually present, were not necessary for a structure to be defined as a microvessel.
MPO assessment:The tissue sections were scanned entirely to assign the scores.The staining intensity was scored as0(negative),1(weak),2(medium), or3(s trong). The extent of staining was scored as0(0%),1(1-25%),2(26-50%),3(51-75%), or4(76-100%), According to the percentages of positively stained areas in relation to the whole version field. The sum of the staining intensity and extent scores was used as the final staining scores (0-7) for MPO.
(3) The mechanism and different action to ultrasound microbubble destruction between normal and tumor blood vessels.
Mice were randomly divided into tumor group (n=5), muscle group (n=5),0.1ml microbubbles was injected and the physiotherapy transducer (acoustic pressures=1500KPa; f=0.94MHZ; duty factor=0.1%;) was applied to the tumor for1min (transducer worked at an intermittent mode of2s on and10s off).
Histology, Immunohistochemistry and Immunofluorescent:For Histology, Immunohistochemistry and Immunofluorescent, the tissue of the tumor and muscle were taken out at the time of pre-treatment and post-treatment Oh.
Assessing the maturity of the vessels:To assess the maturity of the vessels, a double-labeling immun technique was used to simultaneously stain endothelial cells (CD31) and mural cells/vascular Muscle Smooth cell (a-SMA).Immature vessels are CD31-positive vessels but lacking a-SMA-positive periendothelial cells. Mature vessels are CD31-positive vessels and a-SMA-positive periendothelial cells.
3. Results
(1)The research on microbubble destruction inhibits growth of tumor
Results for CEU imaging:Significant ultrasound imaging was observed both in the tumor treatment group and control group. While there was no significant changes of the CEU imaging after injecting the microbubble in the control group, CEU signal reduced obviously in the treated group after treatment. The signal was a little recovery at the time of post-treatment24h and72h, but there is significant difference compare to pre-treatment.
Results for CEU-derived VI:CEU image were acquired at the same of pre-treatment, Oh, and24h and72h post-treatment with the MCE software and then color coded. There was significant reduced of VI at the time of post-treatment Oh (16.677±3.297),24h (26.524±2.193) and72h (33.225±19.952) compared with pre-treatment (66.012±4.129)(P<0.001).
Tumor volume:the growth of the tumor in the treated group is significantly inhibited, while the growth was not in the control group. at7days after implantation, the tumor volume were0.315±0.080cm3and0.364±0.135cm3between the control and treated group respectively, which is no difference (p=0.098).At11,18and25days after implantation, the mean tumor volume were1.112±0.340cm3,2.526±0.483cm3and5.718±1.078cm3in the control group which is significantly larger than the treat group that the volume were0.662±0.186cm3,1.856±0.349cm3and3.290±0.772cm3respectively(p<0.001)
Survival of the tumor mouse:The mean survival time of tumor mice was50.931±8.426days in the control group, while the survival time is significantly extend in the treated group that is72.722±10.870days (p<0.001).
(2)The mechanism of ultrasound mediated microbubble disruption inhibits the tumor growth
Immunohistochemistry result:The expression of the anti-mouse CD31antibody which represents Endothelium, the structure of the vessels is integrated,the endothelial cells lines very closed before the treatment.but immediately after treatment, the structure of the vessel is Severe and diffused disruption, the endothelial cells lines very loose, some parts of which has missed. at the time of post-treatment24h and72h, the structure of the vessels have not recovered, in the edge of the tumor, a small amount of vessels formed.
Microvascular density measurement:Before the treatment, there are a number of vessels in the tumor filed, the number of which was decreased significantly at the time of post-treatment Oh,24h and72h(p<0.001).
Expression of MPO which represents enhanced neutrophil infiltration: pre-treatment and immediately post-treatment, the expression of myeloperoxidase is little, the expression is increased significantly at post-treatment24h(p<0.001), and it is declined at post-treatment72h, but which is also significantly increased compared to pre-treatment(p<0.001).
Scores of MPO:There was no significant increase the scores of MPO at neither pre-treatment (1.400±0.548) nor immediately post-treatment (1.600±0.548)(p=0.572). As compared to pre-treatment, the scores are significant increased at post-treatment24h (6.600±0.548) and post-treatment72h (3.600±0.548)(p<0.001).
Results for examination of pathology:Before treatment, the tumor cell lines compact solidlike pattern with a tight intercellular space, the neoplastic cells varied considerably in size and shape with relatively large, deep-blue dyed nuclei. There are abundant of vessels in the tumor, the structure of the vessel is intact, and the endothelial cells lines very closed. There was no remarkable hemorrhage. At immediately post-treatment, the structure of the tumor is Severe and diffused disruption, the continuity of the endothelial cell is interrupted, hemorrhage was present in large part of the tumor, there are a lot of red blood cells. At the time of post-treatment24h, the red blood cells have been absorbed, massive tumor cell necrosis was observed in the tumor, and the area was increased at post-treatment72h.
(3) The mechanism and different action to ultrasound microbubble destruction between normal and tumor blood vessels
Results for CEU imaging:Significant ultrasound imaging was observed in the tumor group before treatment the signal was reduced obviously after treatment(p<0.001); Significant ultrasound imaging was also observed in the muscle group before treatment, but the signal was not significant reduced after treatment (p=0.134).
Immunohistochemistry result:The expression of the anti-mouse CD31antibody which represents Endothelium, the structure of the vessels is integrated, the endothelial cells lines very closed before the treatment. But immediately after treatment, the structure of the vessel is Severe and diffused disruption, the endothelial cells lines very loose, some parts of which has missed. In the muscle group, the structure of the vessels is also integrated, the endothelial cells lines very closed before the treatment, but there is not much change of the structure of the vessels.
Microvascular density measurement:Before the treatment, there are a number of vessels in the tumor filed (66.600±2.408), the number of which was decreased significantly after treatment (12.800±1.923)(p<0.001), in the muscle group, the microvascular density is34.400±3.847before treatment, the number of the vessel was also decreased (27.000±3.162), but compare to decrease of the tumor, the degree is slight.
Immunofluorescence result:It shows that mature blood vessels take up a small part of proportion which is31.907±3.911%in the tumor group, the proportion is87.869±3.061%in the muscle group, it is significant differences (p<0.001) before treatment. After treatment, the percent of the mature blood vessels is significantly improved which is87.851±1.755%(p<0.001) in the tumor group, the proportion is also improved which is90.444±4.298%, but there is no significant statistical difference (p=0.307).
4. Conclusions
(1) Ultrasound microbubble by directly destroy the tumor microvascular blood perfusion decreased significantly, leading to tumor cells in the ischemic necrosis of large area, in this process, the activation of neutrophils play an important role.
(2) Ultrasound microbubble destruction leading to the blood perfusion of the tumor significantly reduced by disruption the blood vessels directly, which lead to massive tumor necrosis. The activation of neutrophils play an important role in this process.
(3) The tumor blood vessels are far more serious damage to ultrasound microbubble destruction than the normal blood vessels which guard the safety and effective effect in the treatment because the tumor vessels are immature than the normal.
恶性肿瘤是目前威胁人类健康的主要疾病之一,在某些国家甚至成为首要的死亡原因。现在恶性肿瘤主要的治疗手段有手术、放疗和化疗,其作用的主要机制在于直接切除肿瘤组织,杀灭肿瘤细胞和诱导细胞凋亡。但是手术、放疗未能从根本上解决抑制肿瘤新生血管形成,控制肿瘤的生长,阻止肿瘤细胞侵袭和转移等问题。而目前用于肿瘤治疗的化疗药物大多缺乏肿瘤细胞的靶向性并且具有较大的毒副作用,在杀伤肿瘤细胞的同时,也杀伤了大量的骨髓细胞及其他增殖旺盛的正常细胞,因此以上治疗在临床应用中有很大的局限性。
有研究表明,实体肿瘤的生长依赖于持续和大量的血管生成,血管生成为肿瘤组织提供营养物质和氧气,同时血管又是肿瘤细胞向远处转移的主要途径,因此针对血管形成的某些因子及其关键步骤进行干预,有望切断肿瘤血供及其转移途径,对肿瘤的治疗和防止肿瘤向远处转移有重要意义。近年来,抗血管治疗成为肿瘤治疗领域的研究热点,但是,目前的抗血管新生药物在临床的应用中有很大的局限性,不同肿瘤或者不同患者的同一肿瘤在抗血管生成的治疗中,对药物的反应不同；同时,单一抗肿瘤药物必须和化疗药物联合使用才能起到抗肿瘤的作用,即便如此肿瘤患者的临床获益也非常有限。因此,寻求一种新的安全有效的抗肿瘤血管治疗方法便显得尤为必要。
近些年来,随着超声学的发展,超声及微泡造影剂在临床中应用的更加广泛,他们不仅在诊断方面具有极其重要的科学意义和临床价值,而且随着对超声领域的深入研究,其在治疗方面也显示出比较诱人的前景。
超声微泡在疾病治疗中的主要作用机制是超声空化效应,所谓超声空化效应就是液体中内源性或外源性的微气泡空化核在超声作用下发生振荡、膨胀、收缩及内爆等一系列的动力学过程,它是超声在液态物质中传播所特有的现象。既往研究表明微泡破坏产生的空化效应可以损伤微血管内皮,并对周围的组织细胞产生影响,而这种作用有可能为肿瘤的治疗提供一种新的方法。接下来的一系列研究显示：超声微泡破坏可以损伤肿瘤血管,从而使肿瘤区域的血流灌注得到明显减少,其病理变化主要表现为出血、血管扩张、组织间水肿、血栓形成。但是这样的空化效应使血流灌注明显减少能持续多长时间,是否能使肿瘤的生长得到抑制,以及是否可以明显改善荷瘤动物的生存率,至今仍未见报道。同时,我们预实验显示,超声微泡破坏治疗1分钟后的病理变化中未发现明显的血栓形成,Bunte等在其研究中也显示同样的实验结果。那么,什么样的病理变化使肿瘤在治疗后血流灌注明显减少,目前还不太清楚。
目前有大量文献表明,肿瘤血管在形态结构和功能上与正常血管明显不同。正常的微血管有动脉、静脉和毛细血管之分,而实体瘤的血管没有动静脉的区别,其形态结构主要表现在扩张、分叉、迂曲没有规律,管壁直径不均匀,基底膜缺陷,血管外周细胞不完整等特点。基于以上理论依据,我们假设肿瘤血管对空化效应的机械性损伤可能更为敏感,超声微泡破坏有可能更容易损伤肿瘤血管,在达到抑制肿瘤生长的同时而不损伤正常组织。以上假设可以使我们在保证疗效的同时更确保其安全性,这些都可以给肿瘤的治疗带来重要的临床价值,但类似的设想和研究鲜见报道,更缺乏实验验证和机理解释。
研究目的
(1)通过超声微泡空化效应,观察微泡破坏能使肿瘤血流灌注减少持续多长时间；观察微泡破坏是否抑制肿瘤的生长；观察肿瘤小鼠的生存率是否得到明显提高。(2)探索超声微泡破坏使肿瘤血流灌注减少的原因及其机制(3)观察正常组织血管和肿瘤血管对超声微泡破坏反应的差异及其机制。从而为肿瘤的治疗提供一种安全有效的治疗方法及理论依据。
材料和方法
1.实验材料
主要实验仪器：(1)小型脉冲式聚焦超声空化治疗仪：由第三军医大学新桥医院超声科提供,治疗探头频率：0.94MHZ,超声发射占空比0.1%,声压：1500MPa, t=1min。脉冲重复频率(PRF)10HZ。(2)超声诊断仪(Sequoia512,德国Siemens公司)；15L8w探头,频率7.0～14.0MHz,配有增强脉冲序列(contrast pulse sequencing, CPS)造影成像技术。主要实验试剂：白蛋白微泡造影剂：由南方医科大学药学院自行研制,其成膜材料为白蛋白,核心气体为全氟丙烷,外观呈乳白色凝乳状MBc平均粒径为2.07±1.13um,浓度约为2.00-4.25x109个/ml。
2.实验方法：
(一)超声微泡破坏抑制肿瘤生长疗效的研究
实验分组：将建成的肿瘤模型随机分为两组,即超声治疗组(MB+US组)36只,单纯微泡组29只。超声微泡组采用超声照射联合经尾静脉微泡注射；直接抽取“白蛋白”微泡悬液0.02ml,经尾静脉直接注入,进行对比增强超声(CEU)和二维超声成像,待对比增强超声成像信号完全消失后,再次经尾静脉注入0.1ml的白蛋白微泡,在肿瘤区域行超声破坏治疗,治疗后再次注入0.02ml的白蛋白微泡,再次进行对比增强超声(CEU)和二维超声成像。单纯微泡组(MB)：操作步骤同上,但超声探头不发射超声脉冲波。
肿瘤对比增强超声检查：对所有肿瘤模型组经尾静脉随机注射MB,处理后对肿瘤组织行对比超声检查。动物模型制备后,用自制支架固定超声探头(17L5)于肿瘤上,调整探头位置获得良好肿瘤显像后保持探头位置在整个实验过程中不变,仪器的各项参数在整个实验过程中保持不变。CEU采用经尾静脉微泡弹丸式注射法进行,经过一段时间待血池中循环微泡完全消失后,经尾静脉再次注入0.1ml造影剂,用上述超声治疗条件进行治疗,治疗后即刻、24h和72h再次注入0.02ml,对实验小鼠进行肿瘤CEU检查,获取实验肿瘤的显影图像。CEU检查采用二次谐波成像(second harmonic imaging)技术进行,探头发射频率(transmission frequency)和接收频率分(receiving frequency)别为7.0MHz和14MHz,机械指数(Mechanical Index, MI)为0.18。
肿瘤二维超声检查：每隔5-7天对肿瘤进行B-型超声检查,每次测量肿瘤最大切面的长径和短径,肿瘤体积V=1/2ab2(a：长径,b：短径)探头发射频率15MHz,机械指数(Mechanical Index, MI)为0.57。全部声学图像存于MO盘,以备脱机分析。
对比超声图像的分析：应用MCE软件对超声图像进行分析,在两组中随机选择5只小鼠测量治疗前、治疗后即刻、24h、72h显影的声强度(video intensity,Ⅵ)。
(二)微泡破坏抑制肿瘤生长作用机制的研究
实验分组：20只S180肿瘤模型随即分为治疗前组、治疗后即刻组、治疗后24h组和治疗后72h组。动物模型制备后,经尾静脉注入0.1ml的白蛋白微泡,在肿瘤区域行超声破坏治疗。
免疫组化及组织病理学检测：分别在治疗前、治疗后即刻、24h、72h等四个时间点将肿瘤取出,行免疫组化、HE染色。观察MPO、CD31表达情况。
微血管密度测定方法：采用CD31抗体染色,将与邻近肿瘤细胞和结缔组织分离的被染成棕色的内皮细胞、内皮细胞簇或分支状血管结构视为一个微血管,每个样本先在低倍镜(×100)下观察阳性染色的微血管,择染色最多的区域(即“热点”),然后选择5个不重复高倍镜视野,最后计算单位视野(×200)的平均微血管数,即MVD。
MPO半定量评估：组织免疫组化采用以下得分方法评估,免疫染色的强度分为以下四个等级：0(无)、1(弱)、2(中等)、3(强)；染色范围得分可分为以下四个等级：0(0%)、1(10%-25%)、2(26%-50%)、3(51%-75%)、4(76%-100%),两者评分结果相加即为最终得分。
(三)微泡破坏对正常组织及肿瘤组织的不同影响及其机制
实验分组：实验分为肿瘤治疗组和正常组织治疗组小鼠各5只,分别在小鼠的肿瘤区域和腿部骨骼肌行相同条件超声空化治疗,实验步骤及条件同前。
免疫组化组织病理学检测：分别在治疗前、治疗后即刻、时间点将肿瘤取出,行免疫组化、HE染色、免疫荧光检测。
血管成熟程度评估：用cd31抗体特异性标记血管内皮细胞,用α-sma特异性标记外周细胞/平滑肌细胞。血管如果同时标记两种抗体,表明血管发育较为成熟,如果只标记cd31抗体,表明血管发育较为幼稚。
结果
1.超声微泡破坏抑制肿瘤生长疗效的研究
CEU检查结果：CEU图像显示肿瘤治疗组和对照组均可见显著的超声显影,而肿瘤对照组单纯给予微泡造影剂后,超声显影无明显的变化,而超声治疗组在超声破坏后即刻超声显影明显减弱,治疗后24h和72h时间点造影图虽可见肿瘤内血流灌注信号部分恢复,但与治疗前比较,仍具有显著性差异。
CEU图像分析及统计学分析结果：应用CEU软件对图像进行分析,测量实验组肿瘤组织不同时间点显影的声强度(rideo intensity,Ⅵ),并采用彩色编码技术制作显影的彩色编码图像。治疗前肿瘤区域的Ⅵ值较高,为66.012±4.129,应用超声微泡治疗后即刻、24h、72h与治疗前的VI值相比较明显减少,分别是16.677±3.297、26.524±2.193和33.225±19.952(p<0.001),有显著性差异。
二维超声检查：实验结果显示,单纯超声微泡组肿瘤的生长没有得到抑制,而肿瘤治疗组在超声微泡治疗后,显著抑制了肿瘤生长。在肿瘤种植第七天,两组肿瘤的体积分别为0.315±0.080cm3和0.364±0.135cm3(p=0.098),两者无明显的统计学差异,在单纯微泡组,肿瘤种植第11、17、25天,肿瘤的平均体积分别为1.112±0.340cm3、2.526±0.483cm3和5.718±1.078cm3,而在超声治疗组,肿瘤种植第11、17、25天,肿瘤的平均体积分别为0.662±0.186cm3、1.856±0.349cm3和3.290±0.772cm3(p<0.001),与单纯微泡组相比较,明显抑制了肿瘤的生长。
肿瘤小鼠的生存状况：与单纯的微泡组相比较,超声微泡破坏显著延长了肿瘤小鼠的生存时间。单纯微泡组小鼠的平均生存时间是50.931±8.426天,而肿瘤治疗组小鼠的平均生存时间是72.722±10.870天(p<0.001),与单纯微泡组相比较,有显著性差异。
2.微泡破坏抑制肿瘤生长作用机制的研究
免疫组化结果：免疫组化CD31表达：肿瘤组织在治疗前,血管的形态结构完整,内皮细胞排列比较紧密。而在超声微泡破坏后即刻,血管管腔结构遭到严重破坏,血管内皮细胞部分缺失,呈弥散性分布,超声治疗后24小时、72小时,仍然看到大量的血管没有管腔结构,血管内皮细胞连续性中断,呈弥散性分布,但在肿瘤的周边区域,有少量的新生血管。
测量治疗前后肿瘤微血管密度结果显示：在肿瘤治疗前,肿瘤组织有大量的微血管存在,肿瘤治疗后即刻、24h、72h后微血管密度明显减少,和治疗前相比较,有明显的统计学差异(p<0.001)
免疫组化MPO表达：微泡破坏前,肿瘤组织髓过氧化物酶(MPO)的表达并不明显,中性粒细胞的侵润并不严重,在肿瘤治疗后即刻,髓过氧化物酶的表达并没有明显增加,但治疗24h,髓过氧化物酶的表达明显增加,到72小时后,其表达有所下降,但与治疗前相比,仍然是明显增加。
半定量分析显示：肿瘤治疗前,MPO表达不明显,染色评分为：1.400±0.548,治疗后即刻,评分结果为：1.600±0.548(p=0.572),没有显著性差异,而在24h,评分结果为：6.600±0.548(p<0.001)与治疗前相比有显著统计学意义,72h后评分结果为：3.600±0.548(p<0.001)与治疗前和治疗后24h均有显著性差异。
病理学结果显示：肿瘤组织在治疗前可见肿瘤细胞分布呈团状或片状,排列紧密,细胞大小不一,核大,深染,核仁明显,肿瘤组织内可见较丰富的微小血管,血管官腔结构相对较完整,组织间未见红细胞渗出。肿瘤超声微泡治疗后即刻：肿瘤血管不完整,血管内皮连续性中断,组织间有大量红细胞渗出,肿瘤细胞并没有大量坏死。肿瘤细胞超声治疗24小时后,肿瘤治疗区域未见有血管结构,组织间渗出的红细胞崩解吸收,并出现大量肿瘤细胞的坏死,主要表现为细胞核碎裂、溶解。72小时后,肿瘤组织坏死面积更大。
(三)微泡破坏对正常组织及肿瘤组织的不同影响及其机制
CEU检查结果：彩色编码图像显示,肿瘤组织在超声破坏后超声显影明显减弱,骨骼肌组织在治疗后显影声强度虽然也有减弱,但是不太显著。
免疫组化：肿瘤组织在治疗前,血管的形态结构完整,内皮细胞排列比较紧密。而在超声微泡破坏后即刻,血管官腔结构遭到严重破坏,血管内皮细胞部分缺失,呈弥散性分布；骨骼肌组织在治疗前,血管的形态结构完整,内皮细胞排列比较紧密,治疗后,骨骼肌的血管形态仍然相对完好,内皮细胞排列仍然紧密。
测量治疗前后肿瘤微血管密度结果显示：在肿瘤治疗前,肿瘤组织有大量的微血管存在,肿瘤治疗后即刻微血管密度明显减少(p<0.001)和治疗前相比较,有明显的统计学差异；骨骼肌治疗前微血管密度为：34.400±3.847,治疗后微血管密度是：27.000±3.162,有显著性差异,但是与肿瘤治疗相比较,其下降幅度明显减少。
免疫荧光：结果显示：治疗前,在肿瘤组织中发育成熟血管在微血管中占很小的比例,约为31.907±3.911%,在骨骼肌血管中,发育成熟的血管所占比例较大,约为87.869±3.061%(p<0.001),有显著性差异。在治疗后,肿瘤组织中,成熟血管在微血管中所占的比例明显增高,约为87.851±1.755%,与治疗前相比较,有显著统计学差异(p<0.001),骨骼肌组织中,成熟血管所占的比例也有所增高,为90.444±4.298,与治疗前比较没有显著统计学差异(p=0.307)。
结论：
1.超声微泡破坏使肿瘤的血流明显减少,持续时间达72小时以上；超声微泡破坏使肿瘤的生长得到了明显的抑制,使肿瘤小鼠的生存率得到了显著的提高。
2.超声微泡通过直接破坏肿瘤微血管使血流灌注明显减少,导致肿瘤细胞大面积的缺血坏死,在这过程中,中性粒细胞的激活起了很重要的作用。
3.肿瘤组织微血管较正常组织微血管的发育更为幼稚,结构更为不完整,导致超声微泡破坏对正常组织微血管的破坏程度轻,而对肿瘤血管的破坏程度重,保证了治疗的安全性。
Background
Malignant tumor is one of the major diseases which threaten human health and life, even as the leading cause of death in some countries. The common strategies of the current treatment include surgery, radiotherapy and chemotherapy which is mainly to remove tumor tissue, kill tumor cells or induce cell apoptosis. However, these methods are all failed in inhibiting the formation of tumor vessels, inhibiting the growth of tumors and preventing metastasis, while the chemotherapy lacks of specific target and has side effects for not only be able to kill tumor cells but also kill a large number of bone marrow cells and proliferation cells at the same time,they are limited in treating the tumors.
Mammalian cells require oxygen and nutrients for growth and metastasis and are therefore located within microvessels-the diffusion limit for oxygen. Without blood vessels, tumors cannot grow beyond a critical size or metastasize to another organ. It is important significance if we intervene in some ofthe factors and key steps of angiogenesis to cut off the blood supply for the growth and metastasis. However, these antiangiogenic drugs have some limitations in clinical application because different tumor or the same tumor has different reactions to drugs in the process of the treatment. The clinical benefit is very limited when just use the single anticancer drugs even though combined with the chemotherapy drugs. So it is urgent to look for an impressive therapeutic method which is safe and effective.
Recent years, with the development of ultrasonic, ultrasound and contrast agent has become more and more widely and important in clinical application. They not only have important scientific significance in diagnosis but also show attractive prospects in the treatment field.
The basic theory of the mechanism in the treatment is the ultrasonic cavitation effect, which is endogenous or exogenous microbubble cavitation nuclei process a series of dynamic process such as oscillation, expansion, contraction, implosion and so on.it is peculiar to the ultrasonic propagation in the liquid. In previous studies it shows that Microbubble destruction during ultrasound exposure caused injury of microvessels (mostly capillaries) and the production of nonviable cells in adjacent tissue. Microbubbles can be destroyed by ultrasound, resulting in a bioeffect that could be used for tumor destruction. The next series of researches show that Ultrasound microbubble destruction can lead to the blood perfusion of the tumor significantly reduced by damaging the blood vessel of the tumor. The histopathologic studies showed that the effects of insonation were predominantly on the vascular structures within the tumor and correlated with the findings in the contrast enhanced power Doppler ultrasonographic images. There was disruption of the walls of the tumor blood vessels with associated hemorrhage, vascularcongestion and subsequent thrombosis; edema was also present.But it is not known how long the the cavitation effect which reduced blood perfusion last? Whether it can inhibit the growth of the tumor? Whether it can improve survival rates of the tumor animals? In our preliminary experiments it shows that there is no thrombosis in histopathologic studies when the tumor received1minute treatment which is the same as other scholars. So The question remains as to what were the underlying histopathological changes leading to the observed loss of contrast enhanced power Doppler signal in the treated neoplasms?
We have known that it is obviously different between the normal and tumor blood vessels in structure and function. Normal vessel are organized with arteries, veins and capillaries, but the tumor vessels do not display the recognizable features of arterioles, capillaries or venules.The tumor Blood vessels are leaky, tortuous, dilated, and saccular and have a haphazard pattern of interconnection. The endothelial cells lining these vessels have aberrant morphology, pericytes are loosely attached or absent, and the basement membrane is often abnormal—unusually thick at times, entirely absent at others.
Based on the above theory, the tumor blood vessels may be more sensitive to mechanical damage of the cavitation effect. It just damages the tumor angiogenesis not normal blood vessels which suppress tumor growth. This therapy can bring us important clinical value of as it guard the safety and effective effect in the treatment. but it is not reported by now, and it is lack the mechanism explanation to a high degree.
Objective
(1) How long the the cavitation effect which reduced blood perfusion last? Whether it can inhibit the growth of the tumor? Whether it can improve survival rates of the tumor animals?
(2) What histopathological changes lead to the observed loss of contrast enhanced power Doppler signal in the treated neoplasms?
(3) To observe whether there are some differences in ultrasound microbubble treatment response between normal and tumor blood vessels, if they are different, what's the mechanism?
Materials and Methods
1. Materials
Principal Experimental instruments:(1) small, pulsed, focused ultrasonic cavitation treatment apparatus, offered by ultrasound department of the Third Military Medical University. With a treatment probe frequency of0.94MHz, an emission duty of0.1%, a peak negative pressure of1500kPa, time=lmin, Pulse repetition frequency10HZ.(2) Doppler ultrasonic machine with ultrasonic contrasting mode (Sequoia, Siemens Medical Systems, Mountain View, Calif). A17L5transducer, probe frequency of7-14MHz, the machine with contrast pulse sequencing technology.
Experimental reagents,"perflutren protein-type microbubbles developed by Department of Pharmacology, Nanfang Hospital, Southern Medical University, with perfluoropropane as core gas, at approximately2.0-4.25X109microbubbles/ml. A mean of2.07±1.13um in diameter.
2. Method
(1) The research on microbubble destruction inhibits growth of tumor
Mice were randomly divided into treated group (n=36) and control group (n=29) each anaesthetized pentobarbital sodium60mg/kg ip) mouse were imaged before and after tumor insonation and histology was performed in a selected number, a tail vein was catheterized before insonation. In the treated group, an initial B-mode study was followed by the intravenous injection of an ultrasound contrast agent (0.02ml perflutren protein-type microbubbles) and contrast-enhanced power Doppler observations were made. After the contrast agent was no longer detectable in these initial observations, a further0.1ml microbubbles was injected and the physiotherapy transducer (acoustic pressures=1500KPa; f=0.94MHZ; duty factor=0.1%;) was applied to the tumor for1min (transducer worked at an intermittent mode of2s on and10s off), after the procedure the B-mode and contrast-enhanced power Doppler studies were repeated at Oh,24h and72h post-treatment in the identical anatomical plane. In the control group, a similar study to treated group was performed, however, the physiotherapy transducer was applied to the tumor was not switched on after a further0.1ml microbubbles was injected.
Tumor CEU imaging:All experimental mice were performed with CEU respectively by using MBa before treatment with the low mechanic index (MI=0.18), the intravenous injection of0.1ml microbubbles and subsequent post-treatment(Oh,24h and72h) images were acquired at the same mechanical index. To identify the signal from microbubbles retained in the tumor tissue, several post-treatment contrast frames representing the signal from circulating microbubbles were averaged and digitally subtracted from several averaged frames which acquired at pre-treatment, Oh, and24h and72h post-treatment with use of software (Yabko MCE2.7; University of Virginia, Charlottesville, Va), transmission frequency of7.0MHz and receiving frequency of14.0MHz.
B-mode:Ultrasound measurements of the growth of the tumor continued every5-7days with the low mechanic index (MI=0.57). In each of the B-mode images, the length (L), width (W) of the tumor was measured and its volume (mm3) was calculated as:Tumor volume=length×(width)2/2.
Contrast-enhanced Ultrasound imaging analysis:To identify the signal from microbubbles retained in the tumor tissue, several post-treatment contrast frames representing the signal from circulating microbubbles were averaged and digitally subtracted from several averaged frames which acquired at pre-treatment,0h, and24h and72h post-treatment with use of software (Yabko MCE2.7; University of Virginia, Charlottesville, Va) and then color coded. The background-subtracted video intensity (VI) was measured in a region of interest fit the boundary of the tumor. CEUS of the muscle was obtained at the same method.
(2)The mechanism of ultrasound mediated microbubble disruption inhibits the tumor growth
20mice were randomly divided into pre-treatment group (n=5), post-treatment Oh group (n=5), post-treatment24h group (n=5) and72h group (n=5).0.1ml microbubbles was injected and the physiotherapy transducer (acoustic pressures=1500KPa; f=0.94MHZ; duty factor=0.1%) was applied to the tumor for1min (transducer worked at an intermittent mode of2s on and10s off).
Histology and Immunohistochemistry:For Immunohistochemistry and Histology, the tissue of the tumor was taken out at the time of pre-treatment, Oh, and24h and72h post-treatment, to observe the expression of the anti-mouse CD31antibody and anti-MPO antibody.
Microvessel density measurement:The area of highest neovascularization was identified; individual microvessels were counted first on a100x field and then on a200×field.Any brown-staining endothelial cell or endothelial-cell cluster that was clearly separate from adjacent microvessels, tumor cells, and other connective-tissue elements were considered a single, countable microvessel. Vessel lumens, although usually present, were not necessary for a structure to be defined as a microvessel.
MPO assessment:The tissue sections were scanned entirely to assign the scores.The staining intensity was scored as0(negative),1(weak),2(medium), or3(s trong). The extent of staining was scored as0(0%),1(1-25%),2(26-50%),3(51-75%), or4(76-100%), According to the percentages of positively stained areas in relation to the whole version field. The sum of the staining intensity and extent scores was used as the final staining scores (0-7) for MPO.
(3) The mechanism and different action to ultrasound microbubble destruction between normal and tumor blood vessels.
Mice were randomly divided into tumor group (n=5), muscle group (n=5),0.1ml microbubbles was injected and the physiotherapy transducer (acoustic pressures=1500KPa; f=0.94MHZ; duty factor=0.1%;) was applied to the tumor for1min (transducer worked at an intermittent mode of2s on and10s off).
Histology, Immunohistochemistry and Immunofluorescent:For Histology, Immunohistochemistry and Immunofluorescent, the tissue of the tumor and muscle were taken out at the time of pre-treatment and post-treatment Oh.
Assessing the maturity of the vessels:To assess the maturity of the vessels, a double-labeling immun technique was used to simultaneously stain endothelial cells (CD31) and mural cells/vascular Muscle Smooth cell (a-SMA).Immature vessels are CD31-positive vessels but lacking a-SMA-positive periendothelial cells. Mature vessels are CD31-positive vessels and a-SMA-positive periendothelial cells.
3. Results
(1)The research on microbubble destruction inhibits growth of tumor
Results for CEU imaging:Significant ultrasound imaging was observed both in the tumor treatment group and control group. While there was no significant changes of the CEU imaging after injecting the microbubble in the control group, CEU signal reduced obviously in the treated group after treatment. The signal was a little recovery at the time of post-treatment24h and72h, but there is significant difference compare to pre-treatment.
Results for CEU-derived VI:CEU image were acquired at the same of pre-treatment, Oh, and24h and72h post-treatment with the MCE software and then color coded. There was significant reduced of VI at the time of post-treatment Oh (16.677±3.297),24h (26.524±2.193) and72h (33.225±19.952) compared with pre-treatment (66.012±4.129)(P<0.001).
Tumor volume:the growth of the tumor in the treated group is significantly inhibited, while the growth was not in the control group. at7days after implantation, the tumor volume were0.315±0.080cm3and0.364±0.135cm3between the control and treated group respectively, which is no difference (p=0.098).At11,18and25days after implantation, the mean tumor volume were1.112±0.340cm3,2.526±0.483cm3and5.718±1.078cm3in the control group which is significantly larger than the treat group that the volume were0.662±0.186cm3,1.856±0.349cm3and3.290±0.772cm3respectively(p<0.001)
Survival of the tumor mouse:The mean survival time of tumor mice was50.931±8.426days in the control group, while the survival time is significantly extend in the treated group that is72.722±10.870days (p<0.001).
(2)The mechanism of ultrasound mediated microbubble disruption inhibits the tumor growth
Immunohistochemistry result:The expression of the anti-mouse CD31antibody which represents Endothelium, the structure of the vessels is integrated,the endothelial cells lines very closed before the treatment.but immediately after treatment, the structure of the vessel is Severe and diffused disruption, the endothelial cells lines very loose, some parts of which has missed. at the time of post-treatment24h and72h, the structure of the vessels have not recovered, in the edge of the tumor, a small amount of vessels formed.
Microvascular density measurement:Before the treatment, there are a number of vessels in the tumor filed, the number of which was decreased significantly at the time of post-treatment Oh,24h and72h(p<0.001).
Expression of MPO which represents enhanced neutrophil infiltration: pre-treatment and immediately post-treatment, the expression of myeloperoxidase is little, the expression is increased significantly at post-treatment24h(p<0.001), and it is declined at post-treatment72h, but which is also significantly increased compared to pre-treatment(p<0.001).
Scores of MPO:There was no significant increase the scores of MPO at neither pre-treatment (1.400±0.548) nor immediately post-treatment (1.600±0.548)(p=0.572). As compared to pre-treatment, the scores are significant increased at post-treatment24h (6.600±0.548) and post-treatment72h (3.600±0.548)(p<0.001).
Results for examination of pathology:Before treatment, the tumor cell lines compact solidlike pattern with a tight intercellular space, the neoplastic cells varied considerably in size and shape with relatively large, deep-blue dyed nuclei. There are abundant of vessels in the tumor, the structure of the vessel is intact, and the endothelial cells lines very closed. There was no remarkable hemorrhage. At immediately post-treatment, the structure of the tumor is Severe and diffused disruption, the continuity of the endothelial cell is interrupted, hemorrhage was present in large part of the tumor, there are a lot of red blood cells. At the time of post-treatment24h, the red blood cells have been absorbed, massive tumor cell necrosis was observed in the tumor, and the area was increased at post-treatment72h.
(3) The mechanism and different action to ultrasound microbubble destruction between normal and tumor blood vessels
Results for CEU imaging:Significant ultrasound imaging was observed in the tumor group before treatment the signal was reduced obviously after treatment(p<0.001); Significant ultrasound imaging was also observed in the muscle group before treatment, but the signal was not significant reduced after treatment (p=0.134).
Immunohistochemistry result:The expression of the anti-mouse CD31antibody which represents Endothelium, the structure of the vessels is integrated, the endothelial cells lines very closed before the treatment. But immediately after treatment, the structure of the vessel is Severe and diffused disruption, the endothelial cells lines very loose, some parts of which has missed. In the muscle group, the structure of the vessels is also integrated, the endothelial cells lines very closed before the treatment, but there is not much change of the structure of the vessels.
Microvascular density measurement:Before the treatment, there are a number of vessels in the tumor filed (66.600±2.408), the number of which was decreased significantly after treatment (12.800±1.923)(p<0.001), in the muscle group, the microvascular density is34.400±3.847before treatment, the number of the vessel was also decreased (27.000±3.162), but compare to decrease of the tumor, the degree is slight.
Immunofluorescence result:It shows that mature blood vessels take up a small part of proportion which is31.907±3.911%in the tumor group, the proportion is87.869±3.061%in the muscle group, it is significant differences (p<0.001) before treatment. After treatment, the percent of the mature blood vessels is significantly improved which is87.851±1.755%(p<0.001) in the tumor group, the proportion is also improved which is90.444±4.298%, but there is no significant statistical difference (p=0.307).
4. Conclusions
(1) Ultrasound microbubble by directly destroy the tumor microvascular blood perfusion decreased significantly, leading to tumor cells in the ischemic necrosis of large area, in this process, the activation of neutrophils play an important role.
(2) Ultrasound microbubble destruction leading to the blood perfusion of the tumor significantly reduced by disruption the blood vessels directly, which lead to massive tumor necrosis. The activation of neutrophils play an important role in this process.
(3) The tumor blood vessels are far more serious damage to ultrasound microbubble destruction than the normal blood vessels which guard the safety and effective effect in the treatment because the tumor vessels are immature than the normal.
引文
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