Novel Mechanism Of Tumor Invasion, Metastasis And Intravasation Revealed In Zebrafish Models

A single cell-based zebrafish cancer model reveals a novel mechanism of macrophage dependent cancer metastasisBACKGROUDThe incidence and mortality of malignant tumor are now increasingly high in the worldwide, and most of the cancer patients died of metastasis. What is the reason for tumor metastasis? Increasing evidence points to the tumor microenvironment. The tumor microenvironment is a complicated complex mixed by a variety of cells and components, which including tumor cells, macrophages, fibroblasts, vascular endothelial cells, pericytes and so on. Tumor microenvironment can provide support for the growth and metastasis of cancers. In all kinds of cells recruited by tumor cells, macrophages have a huge number and appear in every stage of tumor progression. Under normal circumstances, macrophages are considered to be able to swallow pathogens and kill tumor cells, and play the role of scavenger. However many clinical and basic researches show that macrophages can promote tumor growth and metastasis. In primary tumors, macrophages can induce angiogenesis; promote the migration, invasion and intravasation of tumor cells. In the process of tumor metastasis, macrophage can promote tumor cells to localize and survive in the metastatic site. Macrophages have many subtypes. The most commonly used classification is M1 and M2. M1, also known as the "classical activated" macrophages, responses to pathogens associated with the Thl cells. M2, also known as the "alternatively activated" macrophages, is correlated with Th2 cells. Studies have shown that tumor infiltrating macrophages transformed from M1 subtype to M2 subtype. Although many researches suggest that the tumor associated macrophages can promote metastasis, it is still controversial. So far, The tumor metastasis model mainly based on mouse model, which usually can only observe the metastasis of the late stage tumor. But the problem is that a lot of patients with cancer are initially diagnosed because of metastasis, and their primary tumors are still not detectable. So how to study metastasis in the early stage of tumor development needs another new animal model.Because of the similarity between zebrafish genes and human genes is nearly 87%, Zebrafish has been widely used in the field of biological research in recent years. As an experimental animal zebrafish has the advantages of rapid reproduction, low cost and short experimental period. In the 1960’s, researchers already began to use zebrafish model in the study of development genetics. But zebrafish hadn’t been used as tumor model until 1982, when a study found that long-term exposure to carcinogens could cause tumor formation in zebrafish. Compared with the mice tumor model, zebrafish model has two main advantages. First, for zebrafish, each experimental group can easily use hundreds of embryos. But for mice it will spend tremendous energy and financial resources if each group has more than 10 recipients. Secondly, imaging of transplanted tumor cells in mice needs fluorescence protein or bioluminescence with low resolution. But in the transparent zebrafish single cells can be detected by using high-resolution fluorescence confocal microscopy. We recently established a new tumor model by implanting the fluorescent dye labeled tumor cells into the perivitelline space of zebrafish embryos, the transparent characteristics of the zebrafish embryo allow us to imaging tumor growth and metastasis from the single cell level in the living body. The results showed that hypoxia could promote the dissemination of tumor cells. In present study, we aim to combine different cellular components in the tumor microenvironment and establish a brand new zebrafish tumor model.OBJECTIVESWe aim to study the role of tumor-associated macrophages (TAMs) in facilitating cancer metastasis at the single cell level using a zebrafish tumor model to recapitulate the microenvironment of clinical primary tumors.METHODS1. Use different cytokines (IL6, TNFa, TGFβ, IL4, IL10, LPS, IFNy) to stimulate mouse or human macrophages, and then get Ml and M2 subtype macrophages.2. Isolation of TAMs (tumor associated macrophages) by MACS separation from mouse or human tumor tissues.3. Tumor cells were injected into the perivitelline space (PVS) of zebrafish embryos which the transgenic strain expressing enhanced green fluorescent protein under the flil promoter (Fli1:EGFP).4. Injected embryos were checked 4 days from injection to investigate cancer cells dissemination and metastasis by using a fluorescent microscope.RESULTS1. We successfully developed the tumor cells-macrophages co-injection zebrafish tumor model.2. Co-implantation of tumor cells with TAMs that isolated from wt tumors led to a significant but modest effect of tumor cells dissemination. On the contrary, co-implantation of tumor cells with TAMs that isolated from TNFα-, IL6-expressing tumors resulted in obvious tumor cells dissemination and invasion. On the other hand, co-implantation of TNFα- and IL6-activated macrophages also remarkably increased the capability of tumor invasion and metastasis.3. Co-implantation of CD86+ and PDL1+ Ml macrophages did not change the metastatic capacity of tumor cells. On the contrary, the IL10-IL4-TGFβ-stimulated M2 macrophages significantly enhanced tumor metastasis, resulting in wide spreading of tumor cells in the fish body. Similarly, co-implantation of tumor cells with PDL1+F4/80+M1-TAMs did not led to metastasis. On the contrary, CD206+F4/80+M2-TAMs remarkably facilitated tumor metastasis, resulting in the extensive disseminating of tumor cells in fish body.4. In a non-metastatic human endometrial cancer sample, the isolated TAMs did not significantly increase the metastatic ability of OVCAR8 ovarian tumor cells. On the other hand, co-implantation of TAMs that isolated from a metastatic endometrial cancer sample led to extensive spreading of tumor cells in zebrafish body. Similar positive correlations also seen in other types of cancer.CONCLUSIONS1. We successfully developed the tumor cells and macrophages co-implantation zebrafish tumor model.2. IL6- and TNFα-stimulated macrophages significantly promoted cancer metastasis.3. The M2 subtype of mouse and human macrophages markedly facilitated cancer metastasis. In contrast, M1 macrophages lacked the ability to promote metastasis in this model.4. There is a positive correlation between TAMs-mediated metastasis in our zebrafish model and tumor metastasis in human patients. TAMs often hijacked tumor cells for intravasation and carried them to distal organs. The macrophage-dependent tumor metastasis occurred at the early stage of primary tumor development.A new orthotopic zebrafish tumor model shows Invasiveness and metastasis of retinoblastomaPhD candidate:Jian Wang Supervisor:Prof. Yuping SunBACKGROUDRetinoblastoma is one of the most common malignant tumors in children and also is the most common intraocular tumor. It is usually found in the nuclear layer of the retina and mostly occurred in children under the age of 5. This malignant disease can be monocular or binocular and extremely easy to metastasis. The most common way of invasive spreading is follow the optic nerve to the brain, where tumor cells can further metastasize to other places. The mortality of retinoblastoma is very high. So early detection, early diagnosis and early treatment is the key to improve survival rate and reduce mortality. Operation is the most important approach for retinoblastoma therapy. Other treatments include chemotherapy, radiation therapy, cryotherapy, photocoagulation treatment etc.Inactivation of both alleles in the retinoblastoma gene (Rb) is responsible for the pathogeny due to dysfunction of Rb tumor suppressor gene. It is a highly genetically related malignant tumor. Approximately 40% of patients are autosomal dominant inheritance and about 50% Rb gene carriers’offspring will suffer. Recently, retinoblastoma tumor models have been widely used in the biology research. Initially researchers only injected the retinoblastoma cell lines such as Y79 cells into immunodeficiency mice to make xenograft models or implanted the Y79 cells into the vitreous cavity of rats’eyes to establish orthotopic tumor models. Since then, a large number of transgenic mice models have been applied to basic research owing to the rapid development of transgenic technology. For example, The LHBETATAG retinoblastoma model is genetically manipulated to overexpress oncogene SV40-T antigen. These transgenic mice models provide convenience for the mechanism study of retinoblastoma and evaluation of new anti-tumor drugs. But it is still in lack of an optimal animal model to reproduce the development of retinoblastoma at the early age and thus recapitulate the pediatric situation in human patients.OBJECTIVESWe aim to develop an orthotopic zebrafish model that retinoblastoma invasiveness and metastasis could be detected at the single cell level. Interactions between surrounding vasculatures and retinoblastoma cell could be studied by using a transgenic zebrafish strain Flil:EGFP that showed green fluorescent colors in the blood vessels. We also try to assess therapeutic efficacies of antiangiogenic drug or morpholino that block retinoblastoma invasion.METHODS1. Intravitreal injection of retinoblastoma cells into zebrafish embryos.2. Vegf-αα morpholino and standard scrambled control morpholino were prepared at 3mM for stocks and diluted in the nuclease-free water. About lnL morpholino was injected into the yolk sac of each embryo at the 1-2 cell stage. After injection, the embryos were transferred to fresh Danieu’s medium, followed by injecting retinoblastoma cells at 48hpf.3. After retinoblastoma tumor cells implantation, sunitinib was directly added to the aquaria water to get a final concentration of μM.4 days after treatment, Tumor cells dissemination and invasion were examined by using a fluorescent microscopy.RESULTS1. We successfully developed an orthotopic zebrafish retinoblastoma model.2. The tumor cells began to disseminate from the primary sites at 2 days after RB355 and SJmRBL-8 tumor cells injection. On the contrary, injection of non-invasive WERI-Rbl tumor cells did not lead to metastasis in this zebrafish model.3. Treatment of tumor-bearing zebrafish embryos with 1μM. sunitinib significantly inhibited human and mouse retinoblastoma cells dissemination and metastasis. Delivery of Vegf-αα morpholino to retinoblastoma-bearing embryos also effectively attenuated tumor cells dissemination to distal tissues.CONCLUSIONS1. The invasive retinoblastoma tumor cells disseminated to blood vessels and neighboring regions when primary tumors were still at the microscopic sizes. However, the non- invasive tumor cells maintain their non-metastatic features in our zebrafish model. It proves this zebrafish model has high clinical relevance.2. Antiangiogenic drugs for example sunitinib and Vegf morpholino could effectively inhibit retinoblastoma invasion and metastasis.