靶向VEGFR-3的多肽TMTP2在恶性肿瘤靶向治疗中的应用研究
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摘要
研究背景与目的
近年来,VEGF-C/VEGFR-3信号通路在多项研究中被证实介导实体瘤周及瘤内新生淋巴管生成过程中起重要的作用,该信号通路是肿瘤淋巴管生成的关键环节,同时对肿瘤细胞的移动、浸润以及转移也起到了非常重要的作用。大量的临床病理研究表明肿瘤细胞中VEGF-C或VEGF-D的表达与多种肿瘤的转移直接相关。最近有研究表明,在小鼠黑色素瘤模型中利用重组腺相关病毒的基因治疗方法表达可溶性VEGFR-3可阻断淋巴结的转移。这些研究结果提示,阻断VEGFR-3信号通路能有效抑制淋巴结转移,也有可能降低远处器官转移的发生率。所以,基于该通路在肿瘤转移以及淋巴管生成过程的重要性,阻断该信号途径的相关研究有望成为抗肿瘤淋巴转移治疗新的有效手段。
在前期的研究中应用新型细菌鞭毛展示随机肽库技术对重组活性蛋白VEGFR-3的胞外区进行筛选,经体外验证测序获得一段5肽序列,命名为"TMTP2";本研究拟选择肿瘤淋巴管及淋巴管生成为研究靶点,通过建立体内外淋巴管生成及肿瘤相关模型来探讨验证TMTP2与淋巴管内皮细胞的靶向识别效应,以及对淋巴管生成及肿瘤转移的靶向阻遏效应；旨在为多肽载体的应用带来新的理论依据和实践经验,如能进而利用该多肽载体携带化疗药物进行靶向肿瘤淋巴管生成与转移的治疗,则有望提供一个早期诊断转移和控制肿瘤转移和复发的多肽药物,为肿瘤的分子治疗带来新的契机和希望。
方法
通过体外三维培养的方法建立淋巴管体外成管模型,再通过相关的软件分析体外形成的管样结构进行分析；在C57BL/6小鼠角膜微袋中移植TC-1细胞相应肿瘤,通过HE染色及免疫荧光的方法检测肿瘤及肿瘤淋巴管的新生；在BALB/c小鼠乳腺皮下脂肪垫处注射小鼠来源的乳腺癌4T1细胞,建立自发性肺及其他器官的转移模型,2-3周分别取肺组织于体视显微镜下观察,并且通过HE染色进一步验证；在BALB/c裸鼠的乳腺原位脂肪垫皮下注射人类乳腺癌细胞系MDA-MB-231建立乳腺癌异种移植模型,通过染色观察肿瘤内部淋巴管的生成及转移情况。通过尾静脉注射或者显微注射的方法将带有荧光标记多肽注射到小鼠或者斑马鱼的体内,不同时间点取下组织,利用免疫荧光的方法检测多肽的分布及靶向情况。通过建立乳腺癌原位移植模型、角膜微袋肿瘤生长模型及模式生物-斑马鱼,检测TMTP2在新生淋巴管生成过程中的靶向识别作用,通过尾静脉注射或者显微注射的方法将带有荧光标记多肽注射到小鼠或者斑马鱼的体内,不同时间点取下组织,利用免疫荧光的方法检测多肽的分别及靶向情况。利用上述模型体内验证多肽TMTP2-DKK对上述模型的肿瘤及淋巴管新生的靶向阻遏作用。体外先对4T1细胞进行不同浓度多肽TMTP2-DKK的处理,再通过Transwell试验验证多肽TMTP2-DKK处理后的细胞对迁移侵袭的影响；在构建的乳腺癌转移模型中验证多肽TMTP2-DKK在体内对转移的抑制效应；显微镜观察计算多肽处理后的小鼠肺部的转移灶,HE染色进一步验证其转移灶的组织结构。
结果
与空白对照组对比,HLEC体外三维培养6h后,VEGF-C融合蛋白组中形成更多的管腔样结构,随着培养时间的增加,最后形成复杂的闭合三维淋巴管网；角膜微袋肿瘤移植的小鼠从第5天出现肉眼可见的血管新生的变化,由7天直至14天血管生长更加明显；在BALB/C小鼠4T1乳腺癌转移模型中,在肿瘤细胞移植4周后所有被种植的小鼠均发生较大的原发肿瘤,同时在肺部可见多个转移结节；小鼠乳腺癌原位异种移植模型也可见皮下瘤不同程度的生长；TMTP2对LEC细胞有很好的亲和效应,在实验组的细胞胞浆内可见很强的绿色荧光,也有部分细胞核内也有较强的荧光,而在对照肽组只有蓝色的核染色之外,没有其他的荧光；在小鼠的乳腺癌及全角膜的切片中均可见较强的多肽表达荧光,而在错序对照肽无相关的荧光表达；同样在斑马鱼的胸导管内也有绿色荧光的分布；在体外成管试验中,未处理组与添加了VEGFC组都有明显的管状结构形成,而TMTP2-DKK及TMTP2-DKK+VEGFC处理组成管均明显减少,错序肽以及TMTP2则对成管没有抑制作用；在乳腺癌的荷瘤小鼠中,TMTP2-DKK处理组的肿瘤体积明显小,瘤体重量明显较对照组轻,且内部淋巴管的数目也较对照组少；在TMTP2-DKK处理组的角膜成瘤整体要比对照组小,且组化结果淋巴管的数目明显少于对照组；在斑马鱼的试验中,与对照组相比,TMTP2-DKK处理后发生明显的心包水肿及胸导管分离,高倍视野下观察到斑马鱼胸导管缺失；在体外Transwell试验中,与对照组相比,TMTP2-DKK处理组的细胞穿过率明显减少；TMTP2-DKK处理组的小鼠肿瘤体积及重量明显小于对照组,肺转移率也较对照组降低；HE染色对可见肺局部有大量的转移灶；
结论
本研究成功建立了体外三维淋巴管成管模型、体内的角膜微袋肿瘤模型、乳腺癌转移模型及裸鼠原位异种移植瘤模型,为淋巴管新生及肿瘤淋巴管生产及相关信号通路的研究提供了理想的动物模型；多肽TMTP2对表达VEGFR-3的LEC及在体内肿瘤及相应的淋巴管均有很好的靶向识别效应；偶联DKK后,可以抑制淋巴管的发生,且可抑制肿瘤的转移；多肽TMTP2如能与抗肿瘤药物如阿霉素等偶联,将大大提高肿瘤早期治疗的靶向性和高效性,可能成为临床上靶向阻遏肿瘤淋巴管的生成与抑制肿瘤转移的一种全新的诊断和治疗工具；
Background and Objective In recent years, VEGF-C/VEGFR-3 signaling pathway have been confirmed play an important role in mediated solid tumors and intratumoral lymphangiogenesis process in many studies. The signaling pathway is pay a very important role in lymphangiogenesis and tumor cell mobile,invasion and metastasis.
The clinical and pathological studies indicate that the expression of VEGF-C or VEGF-D in tumor cells is directly related to a variety of tumor metastasis. Recent studies have shown that the transfer of soluble VEGFR-3 can be blocked metastasis to the lymph nodes by using recombinant adeno-associated virus gene therapy in mice model of melanoma. These findings suggest that blocking VEGFR-3 signaling pathway can inhibit lymph node metastasis effectively, and may reduce the incidence of distant metastasis. Therefore, based on the importance of this pathway in process of tumor metastasis and lymphangiogenesis. These will be expected to become an effective means of anti-tumor lymph node metastasis by blocking the signaling pathways in cancer therapy. We obtained a period of five peptide sequences TMTP2 via using the extracellular domain of recombinant active human protein VEGFR-3 by display random peptide library screening in the previous study.
The lymphatic vessel and lymphangiogenesis was selected as targets in this study. Establishment of the model of lymphangiogenesis and tumor-related model to explore the the specific binding capacities of peptide TMTP2 and lymphatic endothelial cells (LECs. The inhibit effect of peptide TMTP2-DKK on cancer metastases were estimated in breast cancer metastasis model in vivo. The aim was to bring the new theoretical basis and practical experience in the application of the peptide carrier. The peptide carrier may carry the chemotherapy drug in targeting tumor lymphangiogenesis and metastasis therapy and expected to provide an early diagnosis and control in tumor metastasis. And bring new opportunities and hope for the molecular therapy of tumors.
Methods We establish the tube formation lymphatic vessels by the method of three-dimensional culture in vitro, and analysis those tube-like structure by related software. We also transplant TC-1 cells in C57BL/6 mouse cornea micropocket by the correspondding tumor, tumors and tumor lymphatic were detected through the HE and immunofluorescence staining. The establishment of the transfer model of spontaneous lung and other organs through injected the 4T1 cells in BALB/c mouse mammary subcutaneous fat pad. After transfer 2-3 weeks, the lung tissue observed under the stereo microscope, and further validation by HE staining respectively. The breast cancer xenograft model in BALB/c-nu mice are inject human breast cancer cell line MDA-MB-231in situ mammary fat pad subcutaneous, the inside slice of tumor lymphangiogenesis and metastasis by further staining. The fluorescently labeled peptide injected into mice or zebrafish via the tail vein injection or microinjection in vivo. After inject the organization of mouse were removed at different time and the effect of target was tested by using immunofluorescence assay. The effect of target of peptides in tumor and lymphatic were dectect in further validation. tumors and tumor lymphatic were detected through the HE and immunofluorescence staining. The targeting effect of peptide was validated in the process of lymphangiogenesis by use breast cancer orthotopic transplantation model, corneal micropocket tumor model and the zebrafish model in vivo. We injected peptide with fluorescent labeled through the tail vein or microinjection into mice or zebrafish in vivo. And further to test the expression of peptide at different time points by immunofluorescence. we used those model to validate the inhibit effect of peptide TMTP2-DKK in vivo and in vitro. The 4T1 cells treated with different concentrations of TMTP2-DKK in vitro, and the biological behavior changes of cells after TMTP2-DKK treatment were assessed by migration and invasion through transwell assay. The inhibit effect of peptide TMTP2-DKK on cancer metastases were estimated in breast cancer metastasis model in vivo. The mouse metastatic tumor of lungs were calculated using microscopy, and the structure of tissue is further validation by HE staining.
Results The human lymphatic endothelial cells culture in three-dimensional after 6h, compare with blank control group, the group of VEGF-C protein have more tube-like structure formation. The complex three-dimensional tube final form closed into lymphatic network with increasing incubation time. The corneal micropocket mice transplant with a small piece of tumor tissue appear visible changes of angiogenesis from five days, and more from 7 days until 14 days growth of vessels. After 4 weeks transplanted with 4T1 tumor cells, all mice grown and occurrence of large primary tumors and visible multiple metastatic nodules in the lungs of BALB/C mouse in 4T1 breast cancer metastasis model. The tumor growth in different degrees after transplant in mouse breast cancer orthotopic xenograft models.
The synthesized TMTP2 could specific binding to the high expression of VEGFR-3 LECs by immunofluorescence assay.It were seen a strong green fluorescence In the cytoplasm of the experimental group, also part of the nucleus. whereas it could not recognize fluorescence in the cytoplasm except visible blue nuclear in control peptide group. The tissue of slices in mouse breast cancer and whole corneal showed strong fluorescence, yet there is not expression of fluorescent in the group control peptide. After 8 hours injected TMTP2 in the yolk sac of zebrafish, the distribution of fluorescence was observed under the microscope in the thoracic duct.
Compare with untreated group and VEGFC group, tubular structure formation reduce in groups of TMTP2-DKK and TMTP2-DKK+VEGFC in vitro. The differences between those groups with specific statistical significance(P<0.05), but the control and TMTP2 peptide did not inhibit LEC tubular structure formation.Compared with the control group, tumor volume and weight were significantly smaller in the group of TMTP2-DKK treated in breast cancer tumor-bearing mice and in corneal tumor formation, and the number of internal lymphatic vessels were less than the control group(p<0.05). Moreover, compared with the control group, the pericardial edema and thoracic duct separation after treated with TMTP2-DKK in zebrafish experiment. The zebrafish lack of thoracic duct was observed under high power field in the treat group(P<0.05).
In transwell assays, compared with the control group, the number of cells accross into the bottom clozet was decreased in TMTP2-DKK treated cells (P<0.05). The volume and weight of tumor in TMTP2-DKK treated mice is smaller than the control group, and the incidence of lung metastasis also lower than in the control group(P<0.05). There are a large number of visible metastases in lung by HE staining.
Conclusion This study established the three-dimensional lymphangiogenesis model in vitro, and the corneal micro-pocket tumor model, breast cancer metastasis model and orthotopic xenograft tumor model in vivo. This study will provide the ideal animal model for lymphangiogenesis and lymphatic pathway and for studying this complex biological process. The peptide TMTP2 was acquired by a random 12-mer cyclic peptide display library could specific binding to the high expression of VEGFR-3 LECs. The peptide TMTP2 with DKK can inhibit lymphangiogenesis in vitro and tumor growth and tumor intratumoral lymphatic vessels in vivo. The peptide TMTP2 will greatly improve the targeting and efficiency in early tumor treatment if conjugated with anticancer drugs such as doxorubicin. It also may become a significant tool for the diagnostic and therapeutic in targeting repressor lymphatic formation and inhibition of tumor metastasis in clinical.
近年来,VEGF-C/VEGFR-3信号通路在多项研究中被证实介导实体瘤周及瘤内新生淋巴管生成过程中起重要的作用,该信号通路是肿瘤淋巴管生成的关键环节,同时对肿瘤细胞的移动、浸润以及转移也起到了非常重要的作用。大量的临床病理研究表明肿瘤细胞中VEGF-C或VEGF-D的表达与多种肿瘤的转移直接相关。最近有研究表明,在小鼠黑色素瘤模型中利用重组腺相关病毒的基因治疗方法表达可溶性VEGFR-3可阻断淋巴结的转移。这些研究结果提示,阻断VEGFR-3信号通路能有效抑制淋巴结转移,也有可能降低远处器官转移的发生率。所以,基于该通路在肿瘤转移以及淋巴管生成过程的重要性,阻断该信号途径的相关研究有望成为抗肿瘤淋巴转移治疗新的有效手段。
在前期的研究中应用新型细菌鞭毛展示随机肽库技术对重组活性蛋白VEGFR-3的胞外区进行筛选,经体外验证测序获得一段5肽序列,命名为"TMTP2";本研究拟选择肿瘤淋巴管及淋巴管生成为研究靶点,通过建立体内外淋巴管生成及肿瘤相关模型来探讨验证TMTP2与淋巴管内皮细胞的靶向识别效应,以及对淋巴管生成及肿瘤转移的靶向阻遏效应；旨在为多肽载体的应用带来新的理论依据和实践经验,如能进而利用该多肽载体携带化疗药物进行靶向肿瘤淋巴管生成与转移的治疗,则有望提供一个早期诊断转移和控制肿瘤转移和复发的多肽药物,为肿瘤的分子治疗带来新的契机和希望。
方法
通过体外三维培养的方法建立淋巴管体外成管模型,再通过相关的软件分析体外形成的管样结构进行分析；在C57BL/6小鼠角膜微袋中移植TC-1细胞相应肿瘤,通过HE染色及免疫荧光的方法检测肿瘤及肿瘤淋巴管的新生；在BALB/c小鼠乳腺皮下脂肪垫处注射小鼠来源的乳腺癌4T1细胞,建立自发性肺及其他器官的转移模型,2-3周分别取肺组织于体视显微镜下观察,并且通过HE染色进一步验证；在BALB/c裸鼠的乳腺原位脂肪垫皮下注射人类乳腺癌细胞系MDA-MB-231建立乳腺癌异种移植模型,通过染色观察肿瘤内部淋巴管的生成及转移情况。通过尾静脉注射或者显微注射的方法将带有荧光标记多肽注射到小鼠或者斑马鱼的体内,不同时间点取下组织,利用免疫荧光的方法检测多肽的分布及靶向情况。通过建立乳腺癌原位移植模型、角膜微袋肿瘤生长模型及模式生物-斑马鱼,检测TMTP2在新生淋巴管生成过程中的靶向识别作用,通过尾静脉注射或者显微注射的方法将带有荧光标记多肽注射到小鼠或者斑马鱼的体内,不同时间点取下组织,利用免疫荧光的方法检测多肽的分别及靶向情况。利用上述模型体内验证多肽TMTP2-DKK对上述模型的肿瘤及淋巴管新生的靶向阻遏作用。体外先对4T1细胞进行不同浓度多肽TMTP2-DKK的处理,再通过Transwell试验验证多肽TMTP2-DKK处理后的细胞对迁移侵袭的影响；在构建的乳腺癌转移模型中验证多肽TMTP2-DKK在体内对转移的抑制效应；显微镜观察计算多肽处理后的小鼠肺部的转移灶,HE染色进一步验证其转移灶的组织结构。
结果
与空白对照组对比,HLEC体外三维培养6h后,VEGF-C融合蛋白组中形成更多的管腔样结构,随着培养时间的增加,最后形成复杂的闭合三维淋巴管网；角膜微袋肿瘤移植的小鼠从第5天出现肉眼可见的血管新生的变化,由7天直至14天血管生长更加明显；在BALB/C小鼠4T1乳腺癌转移模型中,在肿瘤细胞移植4周后所有被种植的小鼠均发生较大的原发肿瘤,同时在肺部可见多个转移结节；小鼠乳腺癌原位异种移植模型也可见皮下瘤不同程度的生长；TMTP2对LEC细胞有很好的亲和效应,在实验组的细胞胞浆内可见很强的绿色荧光,也有部分细胞核内也有较强的荧光,而在对照肽组只有蓝色的核染色之外,没有其他的荧光；在小鼠的乳腺癌及全角膜的切片中均可见较强的多肽表达荧光,而在错序对照肽无相关的荧光表达；同样在斑马鱼的胸导管内也有绿色荧光的分布；在体外成管试验中,未处理组与添加了VEGFC组都有明显的管状结构形成,而TMTP2-DKK及TMTP2-DKK+VEGFC处理组成管均明显减少,错序肽以及TMTP2则对成管没有抑制作用；在乳腺癌的荷瘤小鼠中,TMTP2-DKK处理组的肿瘤体积明显小,瘤体重量明显较对照组轻,且内部淋巴管的数目也较对照组少；在TMTP2-DKK处理组的角膜成瘤整体要比对照组小,且组化结果淋巴管的数目明显少于对照组；在斑马鱼的试验中,与对照组相比,TMTP2-DKK处理后发生明显的心包水肿及胸导管分离,高倍视野下观察到斑马鱼胸导管缺失；在体外Transwell试验中,与对照组相比,TMTP2-DKK处理组的细胞穿过率明显减少；TMTP2-DKK处理组的小鼠肿瘤体积及重量明显小于对照组,肺转移率也较对照组降低；HE染色对可见肺局部有大量的转移灶；
结论
本研究成功建立了体外三维淋巴管成管模型、体内的角膜微袋肿瘤模型、乳腺癌转移模型及裸鼠原位异种移植瘤模型,为淋巴管新生及肿瘤淋巴管生产及相关信号通路的研究提供了理想的动物模型；多肽TMTP2对表达VEGFR-3的LEC及在体内肿瘤及相应的淋巴管均有很好的靶向识别效应；偶联DKK后,可以抑制淋巴管的发生,且可抑制肿瘤的转移；多肽TMTP2如能与抗肿瘤药物如阿霉素等偶联,将大大提高肿瘤早期治疗的靶向性和高效性,可能成为临床上靶向阻遏肿瘤淋巴管的生成与抑制肿瘤转移的一种全新的诊断和治疗工具；
Background and Objective In recent years, VEGF-C/VEGFR-3 signaling pathway have been confirmed play an important role in mediated solid tumors and intratumoral lymphangiogenesis process in many studies. The signaling pathway is pay a very important role in lymphangiogenesis and tumor cell mobile,invasion and metastasis.
The clinical and pathological studies indicate that the expression of VEGF-C or VEGF-D in tumor cells is directly related to a variety of tumor metastasis. Recent studies have shown that the transfer of soluble VEGFR-3 can be blocked metastasis to the lymph nodes by using recombinant adeno-associated virus gene therapy in mice model of melanoma. These findings suggest that blocking VEGFR-3 signaling pathway can inhibit lymph node metastasis effectively, and may reduce the incidence of distant metastasis. Therefore, based on the importance of this pathway in process of tumor metastasis and lymphangiogenesis. These will be expected to become an effective means of anti-tumor lymph node metastasis by blocking the signaling pathways in cancer therapy. We obtained a period of five peptide sequences TMTP2 via using the extracellular domain of recombinant active human protein VEGFR-3 by display random peptide library screening in the previous study.
The lymphatic vessel and lymphangiogenesis was selected as targets in this study. Establishment of the model of lymphangiogenesis and tumor-related model to explore the the specific binding capacities of peptide TMTP2 and lymphatic endothelial cells (LECs. The inhibit effect of peptide TMTP2-DKK on cancer metastases were estimated in breast cancer metastasis model in vivo. The aim was to bring the new theoretical basis and practical experience in the application of the peptide carrier. The peptide carrier may carry the chemotherapy drug in targeting tumor lymphangiogenesis and metastasis therapy and expected to provide an early diagnosis and control in tumor metastasis. And bring new opportunities and hope for the molecular therapy of tumors.
Methods We establish the tube formation lymphatic vessels by the method of three-dimensional culture in vitro, and analysis those tube-like structure by related software. We also transplant TC-1 cells in C57BL/6 mouse cornea micropocket by the correspondding tumor, tumors and tumor lymphatic were detected through the HE and immunofluorescence staining. The establishment of the transfer model of spontaneous lung and other organs through injected the 4T1 cells in BALB/c mouse mammary subcutaneous fat pad. After transfer 2-3 weeks, the lung tissue observed under the stereo microscope, and further validation by HE staining respectively. The breast cancer xenograft model in BALB/c-nu mice are inject human breast cancer cell line MDA-MB-231in situ mammary fat pad subcutaneous, the inside slice of tumor lymphangiogenesis and metastasis by further staining. The fluorescently labeled peptide injected into mice or zebrafish via the tail vein injection or microinjection in vivo. After inject the organization of mouse were removed at different time and the effect of target was tested by using immunofluorescence assay. The effect of target of peptides in tumor and lymphatic were dectect in further validation. tumors and tumor lymphatic were detected through the HE and immunofluorescence staining. The targeting effect of peptide was validated in the process of lymphangiogenesis by use breast cancer orthotopic transplantation model, corneal micropocket tumor model and the zebrafish model in vivo. We injected peptide with fluorescent labeled through the tail vein or microinjection into mice or zebrafish in vivo. And further to test the expression of peptide at different time points by immunofluorescence. we used those model to validate the inhibit effect of peptide TMTP2-DKK in vivo and in vitro. The 4T1 cells treated with different concentrations of TMTP2-DKK in vitro, and the biological behavior changes of cells after TMTP2-DKK treatment were assessed by migration and invasion through transwell assay. The inhibit effect of peptide TMTP2-DKK on cancer metastases were estimated in breast cancer metastasis model in vivo. The mouse metastatic tumor of lungs were calculated using microscopy, and the structure of tissue is further validation by HE staining.
Results The human lymphatic endothelial cells culture in three-dimensional after 6h, compare with blank control group, the group of VEGF-C protein have more tube-like structure formation. The complex three-dimensional tube final form closed into lymphatic network with increasing incubation time. The corneal micropocket mice transplant with a small piece of tumor tissue appear visible changes of angiogenesis from five days, and more from 7 days until 14 days growth of vessels. After 4 weeks transplanted with 4T1 tumor cells, all mice grown and occurrence of large primary tumors and visible multiple metastatic nodules in the lungs of BALB/C mouse in 4T1 breast cancer metastasis model. The tumor growth in different degrees after transplant in mouse breast cancer orthotopic xenograft models.
The synthesized TMTP2 could specific binding to the high expression of VEGFR-3 LECs by immunofluorescence assay.It were seen a strong green fluorescence In the cytoplasm of the experimental group, also part of the nucleus. whereas it could not recognize fluorescence in the cytoplasm except visible blue nuclear in control peptide group. The tissue of slices in mouse breast cancer and whole corneal showed strong fluorescence, yet there is not expression of fluorescent in the group control peptide. After 8 hours injected TMTP2 in the yolk sac of zebrafish, the distribution of fluorescence was observed under the microscope in the thoracic duct.
Compare with untreated group and VEGFC group, tubular structure formation reduce in groups of TMTP2-DKK and TMTP2-DKK+VEGFC in vitro. The differences between those groups with specific statistical significance(P<0.05), but the control and TMTP2 peptide did not inhibit LEC tubular structure formation.Compared with the control group, tumor volume and weight were significantly smaller in the group of TMTP2-DKK treated in breast cancer tumor-bearing mice and in corneal tumor formation, and the number of internal lymphatic vessels were less than the control group(p<0.05). Moreover, compared with the control group, the pericardial edema and thoracic duct separation after treated with TMTP2-DKK in zebrafish experiment. The zebrafish lack of thoracic duct was observed under high power field in the treat group(P<0.05).
In transwell assays, compared with the control group, the number of cells accross into the bottom clozet was decreased in TMTP2-DKK treated cells (P<0.05). The volume and weight of tumor in TMTP2-DKK treated mice is smaller than the control group, and the incidence of lung metastasis also lower than in the control group(P<0.05). There are a large number of visible metastases in lung by HE staining.
Conclusion This study established the three-dimensional lymphangiogenesis model in vitro, and the corneal micro-pocket tumor model, breast cancer metastasis model and orthotopic xenograft tumor model in vivo. This study will provide the ideal animal model for lymphangiogenesis and lymphatic pathway and for studying this complex biological process. The peptide TMTP2 was acquired by a random 12-mer cyclic peptide display library could specific binding to the high expression of VEGFR-3 LECs. The peptide TMTP2 with DKK can inhibit lymphangiogenesis in vitro and tumor growth and tumor intratumoral lymphatic vessels in vivo. The peptide TMTP2 will greatly improve the targeting and efficiency in early tumor treatment if conjugated with anticancer drugs such as doxorubicin. It also may become a significant tool for the diagnostic and therapeutic in targeting repressor lymphatic formation and inhibition of tumor metastasis in clinical.
引文
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