1、Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination,invasion,and metastasis in a zebrafish tumor modelHypoxiaVEGFAngiogenesisMetastasis缺氧导致的病理性血管再生与肿瘤迁移1概要概要lMechanisms underlying pathological angiogenesis in relation to hypoxia in tumor invasion and metastasis remain elusive
2、.Tumor microvascular networks possess several unique pathological features including extremely high densities of leaky,tortuous,and primitive microvessels that usually lack pericyte coverage,base-ment membrane,and arteriole-venule distinctions.Although hypoxia often results in necrosis of the centra
3、l core of a fast-growing tumor,it could potentially persuade tumor cells to invade neighboring healthy vasculatures for survival,eventually leading to metastasis,which is one of the hallmarks for cancer therapy.缺氧导致的病理性血管再生与肿瘤迁移2A clinical detectable metastatic mass often represents an ultimate cons
4、equence of several distinctive steps of the metastatic cascade,including dissemination of malignant cells from the primary site,transport of tumor cells via the circulation or lymphatic system,adhesion of tumor cells in distal tissues/organs,and re-growth of tumor cells into a detectable mass.Clinic
5、al detection of a metastasis does not reveal early events of tumor cell dissemination and intimate interactions between tumor cells and microvessels.So the the cancer therapy is difficult.缺氧导致的病理性血管再生与肿瘤迁移3lWe have developed a zebrafish tumor model that allows us to study the role of pathological an
6、giogenesis under normoxia and hypoxia in arbitrating early events of the metastatic cascade at the single cell level.lHypoxia and VEGF signaling pathway significantly contribute to early events of the metastatic cascade.The findings also shed light on molecular mechanisms of beneficial effects of cl
7、inically available anti-VEGF drugs for cancer therapy.缺氧导致的病理性血管再生与肿瘤迁移4缺氧导致的病理性血管再生与肿瘤迁移5方法和结果方法和结果1 Hypoxic Metastasis Model in ZebrafishlThe Tg(fli1:EGFP)zebrafish embryos Murine T241 tumor cells were labeled with DiI dye in vitro and labeled cells were injected into the perivitelline cavity of 4
8、8 h post-fertilization embryos.lTumor-bearing zebrafish embryos were placed in either normoxic or hypoxic water(7.5%air saturation),and monitor daily in living zebrafish embryos.缺氧导致的病理性血管再生与肿瘤迁移6At day 3 after implantation,murine T241 tumor cells under hypoxia were significantly disseminated away f
9、rom primary sites.the sizes of primary tumors in both groups remained similar.The border of primary tumors under hypoxia was irregular and invasive fronts were often present.A substantial number of tumor cells under hypoxia were disseminated to distal parts of the fish body,including the head and ta
10、il regions.Consistent with increase of tumor cell dissemination,hypoxia significantly stimulated neovascularization and tortuosity of the tumor vasculature.缺氧导致的病理性血管再生与肿瘤迁移7lwe also studied hypoxia induced tumor cell dissemination and metastasis in another murine tumor cell line,Lewis lung carcinom
11、a(LLC).Increasing of tumor dissemination was well correlated with enhanced tumor neovascularization缺氧导致的病理性血管再生与肿瘤迁移82 Dissemination and Metastasis of Human Tumor Cellsl we selected highly metastatic MDAMB-231 breast cancer and lowly metastaticOVCAR8 ovarian cancer cell lines.Implantation of MDA MB-
12、231breast cancer cells in zebrafish embryos resulted in widespread tumor cell dissemination and metastasis at day 6 post-injection.all MDA MB-231 tumor cells were spread away from the primary site.缺氧导致的病理性血管再生与肿瘤迁移9lThe difference of metastatic potentials between these two cell lines is due to their
13、 different capacities of dissemination.lThus,this zebrafish model might be used to discriminate highly and lowly metastatic potentials of human cancers and to predict prognosis.缺氧导致的病理性血管再生与肿瘤迁移103 Tumor-Derived VEGF Facilitates Dissemination and MetastasislT241 tumor cells were stably transfected t
14、o express VEGF at a high level.Total numbers of disseminated tumor cells and the maximal distance of metastasis in the T241-VEGF group were significantly greater than those in controls.Microvessel density in T241-VEGF tumors was significantly higher relative to control tumors.At day 2 after implanta
15、tion,a significant number of tumor cells invaded the neighboring tissues around primary tumors.At day 4,increasing numbers of invasive tumor cells were detected and were disseminated to distal parts of the fish body.缺氧导致的病理性血管再生与肿瘤迁移11lTo generalize these findings to other tumor types,murine LLC wer
16、e also stably transfected with VEGF.Similar to T241 fibrosarcoma,implantation of LLC-VEGF tumors resulted in marked increase of tumor cell dissemination,invasion and distal metastasis compared with vector controls.缺氧导致的病理性血管再生与肿瘤迁移124 VEGF Blockade Inhibits VEGF-Tumor Cell Invasion,Dissemination,and
17、 MetastasislT241-VEGF tumor-bearing embryos were treated with sunitinib,a known VEGFR blockadeIt showed that tumor cell dissemination,metastasis and vessel density in primary tumors were significantly inhibited by sunitinib in a dose-dependent manner.the average size of primary tumors was also signi
18、ficantly reduced by sunitinib treatment.lsunitinib effectively inhibited tumor cell dissemination and metastasis in LLC tumors缺氧导致的病理性血管再生与肿瘤迁移135 Inhibition of Tumor Cell Invasion,Dissemination,and Metastasis by VEGFR-2 MorpholinoslA specific morpholino against VEGFR-2,a functional receptor for VEG
19、F-induced angiogenesis,was microinjected into developing zebrafish embryos.Administration of high dosages of VEGFR-2 morpholinos resulted in early death of zebrafish embryos owing to the essential role of VEGF in development of embryonicvasculatures as seen in mice.At a relatively low concentration(
20、0.1 mM),the VEGFR-2 morpholino selectively blocked tumor angiogenesis without significantly impairing development of physiological vascular networks,suggesting that the tumor vasculature was more susceptible to VEGF inhibition.缺氧导致的病理性血管再生与肿瘤迁移14缺氧导致的病理性血管再生与肿瘤迁移156 VEGFR Blockade Effectively Blocks
21、 Hypoxia-Induced Tumor Angiogenesis and Metastasisl Sunitinib virtually completely blocked the hypoxia-induced T241 tumor cell invasion,dissemination and metastasis,inhibited tumor neovascularizationlHypoxia-induced malignant cell invasion,dissemination,and metastasis are mediated by VEGF-induced pa
22、thological blood vessels.缺氧导致的病理性血管再生与肿瘤迁移16讨论讨论lEarly detection of malignant tumors is essential for curative therapy.lHowever,cancer metastasis can occur at the very early stage of malignant diseases and in a substantial number of cases metastatic disease is the first sign of cancer whereas primar
23、y tumors remain undetectablelMolecular mechanisms underlying the early onset of cancer metastasis are complex缺氧导致的病理性血管再生与肿瘤迁移17lThe role of angiogenesis in tumor cell dissemination has not been well-described in mammalian tumor models.lTo recapitulate early steps of clinical metastasis,we develop a
24、 zebrafish metastatic model that allows us to monitor dissemination of single tumor cells from primary sites in the living body.The transparent nature of zebrafish embryos allows us to monitor tumor development and dissemination in vivo without any invasive operation;缺氧导致的病理性血管再生与肿瘤迁移18EGFP positive
25、 vasculature in fli1:EGFPzebrafish embryos further permit us to study tumor cell dissemination in relation to development of angiogenic vessels;Zebrafish could easily be placed in hypoxic water to study the role of tissue hypoxia in facilitating tumor cell dissemination and metastasis;Assessment of
26、anti-metastatic effects of orally active drugs;Morpholino for reverse genetics by silencing host gene functions involving angiogenesis and metastasis缺氧导致的病理性血管再生与肿瘤迁移19lHow does VEGF-induced angiogenesis facilitate tumor cell invasion and metastasis?One possibility is that VEGF induces disorganized,
27、leaky and tortuous vasculatures,which are susceptible for malignant cell invasion.In support of this hypothesis,increases of vascular density and tortuosity have been observed in our zebrafish model.The other possible mechanism is that outgrowth of blood vessels in tumors promotes intimate interacti
28、ons between malignant and endothelial cells and the latter provides niches for tumor cell invasion and metastasis.Additionally,perfusion of VEGF-induced vessels could act as a chemoattractant for tumor cell migration and eventually lead to tumor cell invasion along the vascular system.缺氧导致的病理性血管再生与肿
29、瘤迁移20l Inhibition of tumor angiogenesis might prevent metastasis.Inversely,recent reports show that antiangiogenic drugs for treatment of established animal tumors lead to invasion and metastasis.The difference between these findings and our data could be due to the sizes of primary tumors.In an est
30、ablished tumor,anti-VEGF drugs could generate tissue hypoxia,leading to an invasive and metastatic phenotype.In zebrafish tumor model,primary tumors remain in situ as tiny nodules and antiangiogenic drugs would not result in significant hypoxia as in a well-established tumor.lThus,tumor sizes might
31、be a key determinant for antiangiogenic drug-produced therapeutic benefits or tumor cell invasion and metastasis.缺氧导致的病理性血管再生与肿瘤迁移21lA substantial number of patients are diagnosed for malignant diseases owing to metastasis as the first sign of cancer.Primary tumors remain undetectable using conventional techniques,suggesting that dissemination of tumor cells occurs at the very early stage.Thus,our results shed light on early metastatic processes by visualizing single cell invasion and metastasis in the living body without invasive procedures.缺氧导致的病理性血管再生与肿瘤迁移22
