Gla-domain mediated targeting of externalized phosphatidylserine for intracellular delivery.

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging, and targeted delivery of therapeutic agents. While an array of PS-binding molecules exist, their utility has been limited by their inability to internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS-binding motif comprised of a carboxylated glutamic acid (GLA) residue domain that both recognizes and binds cell surface-exposed PS, and then unlike other PS-binding molecules is internalized into these cells. Internalization is independent of the traditional endosomal-lysosomal pathway, directly entering the cytosol of the target cell rapidly. We demonstrate that this PS recognition extends to stem cells and that GLA-domain-conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offer new opportunities for specifically targeting cells with surface-exposed PS for imaging and delivery of therapeutics.

Phosphatidylserine-Exposing Annexin A1-Positive Extracellular Vesicles: Potential Cancer Biomarkers.

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1+) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

Cord Blood Plasma and Placental Mesenchymal Stem Cells-Derived Exosomes Increase Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells While Maintaining Their Stemness.

BACKGROUND: Mesenchymal stem cells (MSCs) have been used for ex vivo expansion of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) to maintain their primitive characters and long-term reconstitution abilities during transplantation. Therapeutic effects of MSCs mainly rely on paracrine mechanisms, including secretion of exosomes (Exos). The objective of this study was to examine the effect of cord blood plasma (CBP)-derived Exos (CBP Exos) and Placental MSCs-derived Exos (MSCs Exos) on the expansion of UCB HSCs to increase their numbers and keep their primitive characteristics. METHODS: CD34+ cells were isolated from UCB, cultured for 10 days, and the expanded HSCs were sub-cultured in semisolid methylcellulose media for primitive colony forming units (CFUs) assay. MSCs were cultured from placental chorionic plates. RESULTS: CBP Exos and MSCs Exos compared with the control group significantly increased the number of total nucleated cells (TNCs), invitro expansion of CD34+ cells, primitive subpopulations of CD34+38+ and CD34+38-Lin- cells (p < 0.001). The expanded cells showed a significantly higher number of total CFUs in the Exos groups (p < 0.01). CONCLUSION: CBP- and placental-derived exosomes are associated with significant ex vivo expansion of UCB HSCs, while maintaining their primitive characters and may eliminate the need for transplantation of an additional unit of UCB.

Listeria monocytogenes Infection Alters the Content and Function of Extracellular Vesicles Produced by Trophoblast Stem Cells.

Placental immunity is critical for fetal health during pregnancy, as invading pathogens spread from the parental blood to the fetus through this organ. However, inflammatory responses in the placenta can adversely affect both the fetus and the pregnant person, and the balance between protective placental immune response and detrimental inflammation is poorly understood. Extracellular vesicles (EVs) are membrane-enclosed vesicles that play a critical role in placental immunity. EVs produced by placental trophoblasts mediate immune tolerance to the fetus and to the placenta itself, but these EVs can also activate detrimental inflammatory responses. The regulation of these effects is not well characterized, and the role of trophoblast EVs (tEVs) in the response to infection has yet to be defined. The Gram-positive bacterial pathogen Listeria monocytogenes infects the placenta, serving as a model to study tEV function in this context. We investigated the effect of L. monocytogenes infection on the production and function of tEVs, using a trophoblast stem cell (TSC) model. We found that tEVs from infected TSCs can induce the production of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) in recipient cells. Surprisingly, this tEV treatment could confer increased susceptibility to subsequent L. monocytogenes infection, which has not been reported previously as an effect of EVs. Proteomic analysis and RNA sequencing revealed that tEVs from infected TSCs had altered cargo compared with those from uninfected TSCs. However, no L. monocytogenes proteins were detected in tEVs from infected TSCs. Together, these results suggest an immunomodulatory role for tEVs during prenatal infection.

BRET Sensors for Imaging Membrane Integrity of Microfluidically Generated Extracellular Vesicles.

Extracellular vesicles (EVs) derived from various cell lines have been extensively used as natural nanodelivery vehicles for drug, protein, and nucleic acid deliveries in therapeutic applications for cancer. Recently, we developed a microfluidic-based reconstruction strategy as a novel method to generate microRNA-loaded membrane vesicles for cancer therapy in vivo. We used EVs and cell membranes isolated from different source of cells for this reconstruction process. The microfluidic system produced reconstructed vesicles of uniform sizes with high microRNA loading efficiency independent of input membrane sources (EVs or cell membranes). To address the functional integrity of the membrane structure and of proteins in the reconstructed EVs, we introduce a membrane-insertable bioluminescence resonance energy transfer (BRET) sensor system. This sensor, with its membrane-insertable palmitoylation signal peptide sequence derived from a growth-associated protein 43 (GAP43), helps in trafficking the fusion protein to the cell membrane upon its expression in cells and allows for imaging reconstructed membrane vesicles using optical imaging. In this chapter, we detail the stepwise methods used for the engineering of cells using this sensor, isolation of EVs from the engineered cells, preparation of reconstructed EVs by microfluidic processing, and BRET imaging of reconstructed EVs for membrane integrity evaluation.

Exosome-mediated remodeling of the tumor microenvironment: From local to distant intercellular communication.

Extracellular vesicles (EVs) are membrane-enveloped nanoscale particles that carry various bioactive signaling molecules secreted by cells. Their biological roles depend on the original cell type from which they are derived and their inclusions. Exosomes, a class of EVs, are released by almost all eukaryotic cell types, including tumor cells. Tumor cell-derived exosomes mediate signal transduction between tumor cells and surrounding non-tumor cells. This intercellular communication actively contributes to the remodeling of the tumor microenvironment (TME) to enable tumor growth, invasion, and metastasis. This review summarizes the latest progress in the exploration of exosome-mediated cell-cell communication implicated in TME remodeling and underlying mechanisms. We focus on the role of cell-cell interactions mediated by tumor cell-derived exosomes in promoting invasion and metastasis, and their potential as a therapeutic intervention target against distant metastasis. We also discuss the clinical translational significance of tumor-derived exosomes for early diagnosis, efficacy and progression evaluations.

A Dual-Reporter Platform for Screening Tumor-Targeted Extracellular Vesicles.

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor-homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.

In Vitro and In Vivo Analysis of Extracellular Vesicle-Mediated Metastasis Using a Bright, Red-Shifted Bioluminescent Reporter Protein.

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. This study focuses on a novel method to image EV subtypes and their biodistribution in vivo. A red-shifted bioluminescence resonance energy transfer (BRET) EV reporter is developed, called PalmReNL, which allows for highly sensitive EV tracking in vitro and in vivo. PalmReNL enables the authors to study the common surface molecules across EV subtypes that determine EV organotropism and their functional differences in cancer progression. Regardless of injection routes, whether retro-orbital or intraperitoneal, PalmReNL positive EVs, isolated from murine mammary carcinoma cells, localized to the lungs. The early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple intraperitoneal injections of the medium and large EV (m/lEV)-enriched fraction derived from mammary carcinoma cells is demonstrated. In addition, the results presented here show that tumor cell-derived m/lEVs act on distant tissues through upregulating LC3 expression within the lung.

Camouflaged Hybrid Cancer Cell-Platelet Fusion Membrane Nanovesicles Deliver Therapeutic MicroRNAs to Presensitize Triple-Negative Breast Cancer to Doxorubicin.

Camouflaged cell-membrane-based nanoparticles have been gaining increasing attention owing to their improved biocompatibility and immunomodulatory properties. Using nanoparticles prepared from the membranes of specific cell types, or fusions derived from different cells membranes, can improve their functional performance in several aspects. Here, we used cell membranes extracted from breast cancer cells and platelets to fabricate a hybrid-membrane vesicle fusion (cancer cell-platelet-fusion-membrane vesicle, CPMV) in which we loaded therapeutic microRNAs (miRNAs) for the treatment of triple-negative breast cancer (TNBC). We used a clinically scalable microfluidic platform for the fusion of cell membranes. The reconstitution process during synthesis allows for efficient loading of miRNAs into CPMVs. We systematically optimized the conditions for preparation of miRNA-loaded CPMVs and demonstrated their property of homing to source cells using in vitro experiments, and by therapeutic evaluation in vivo. In vitro, the CPMVs exhibited significant recognition of their source cells and avoided engulfment by macrophages. After systemic delivery in mice, the CPMVs showed a prolonged circulation time and site-specific accumulation at implanted TNBC-xenografts. The delivered antimiRNAs sensitized TNBCs to doxorubicin, resulting in an improved therapeutic response and survival rate. This strategy has considerable potential for clinical translation to improve personalized therapy for breast cancer and other malignancies.

Minicircles for a two-step blood biomarker and PET imaging early cancer detection strategy.

Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection reagent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection reagent. Secreted GLuc correlated with HSV-1 sr39TK expression with R2 = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.

Visualizing Extracellular Vesicles and Their Function in 3D Tumor Microenvironment Models.

Extracellular vesicles (EVs) are cell-derived nanostructures that mediate intercellular communication by delivering complex signals in normal tissues and cancer. The cellular coordination required for tumor development and maintenance is mediated, in part, through EV transport of molecular cargo to resident and distant cells. Most studies on EV-mediated signaling have been performed in two-dimensional (2D) monolayer cell cultures, largely because of their simplicity and high-throughput screening capacity. Three-dimensional (3D) cell cultures can be used to study cell-to-cell and cell-to-matrix interactions, enabling the study of EV-mediated cellular communication. 3D cultures may best model the role of EVs in formation of the tumor microenvironment (TME) and cancer cell-stromal interactions that sustain tumor growth. In this review, we discuss EV biology in 3D culture correlates of the TME. This includes EV communication between cell types of the TME, differences in EV biogenesis and signaling associated with differing scaffold choices and in scaffold-free 3D cultures and cultivation of the premetastatic niche. An understanding of EV biogenesis and signaling within a 3D TME will improve culture correlates of oncogenesis, enable molecular control of the TME and aid development of drug delivery tools based on EV-mediated signaling.

Microvesicle-Mediated Delivery of Minicircle DNA Results in Effective Gene-Directed Enzyme Prodrug Cancer Therapy.

An emerging approach for cancer treatment employs the use of extracellular vesicles, specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of codelivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.

The Exosome Total Isolation Chip.

Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4-1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the device's broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10-100 µL) of blood.

Imaging of Tumor-Associated Macrophages in a Transgenic Mouse Model of Orthotopic Ovarian Cancer.

PURPOSE: Tumor-associated macrophages (TAMs) are often associated with a poor prognosis in cancer. To gain a better understanding of cellular recruitment and dynamics of TAM biology during cancer progression, we established a novel transgenic mouse model for in vivo imaging of luciferase-expressing macrophages. PROCEDURES: B6.129P2-Lyz2tm1(cre)Ifo/J mice, which express Cre recombinase under the control of the lysozyme M promoter (LysM) were crossed to Cre-lox Luc reporter mice (RLG), to produce LysM-LG mice whose macrophages express luciferase. Cell-type-specific luciferase expression in these mice was verified by flow cytometry, and via in vivo bioluminescence imaging under conditions where macrophages were either stimulated with lipopolysaccharide or depleted with clodronate liposomes. The distribution of activated macrophages was longitudinally imaged in two immunocompetent LysM-LG mouse models with either B16 melanoma or ID8 ovarian cancer cells. RESULTS: In vivo imaging of LysM-LG mice showed luciferase activity was generated by macrophages. Clodronate liposome-mediated depletion of macrophages lowered overall bioluminescence while lipopolysaccharide injection increased macrophage bioluminescence in both the B16 and ID8 models. Tracking macrophages weekly in tumor-bearing animals after intraperitoneal (i.p.) or intraovarian (i.o.) injection resulted in distinct, dynamic patterns of macrophage activity. Animals with metastatic ovarian cancer after i.p. injection exhibited significantly higher peritoneal macrophage activity compared to animals after i.o. injection. CONCLUSION: The LysM-LG model allows tracking of macrophage recruitment and activation during disease initiation and progression in a noninvasive manner. This model provides a tool to visualize and monitor the benefit of pharmacological interventions targeting macrophages in preclinical models.

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals.

Functional delivery of mRNA to tissues in the body is key to implementing fundamentally new and potentially transformative strategies for vaccination, protein replacement therapy, and genome editing, collectively affecting approaches for the prevention, detection, and treatment of disease. Broadly applicable tools for the efficient delivery of mRNA into cultured cells would advance many areas of research, and effective and safe in vivo mRNA delivery could fundamentally transform clinical practice. Here we report the step-economical synthesis and evaluation of a tunable and effective class of synthetic biodegradable materials: charge-altering releasable transporters (CARTs) for mRNA delivery into cells. CARTs are structurally unique and operate through an unprecedented mechanism, serving initially as oligo(α-amino ester) cations that complex, protect, and deliver mRNA and then change physical properties through a degradative, charge-neutralizing intramolecular rearrangement, leading to intracellular release of functional mRNA and highly efficient protein translation. With demonstrated utility in both cultured cells and animals, this mRNA delivery technology should be broadly applicable to numerous research and therapeutic applications.

Signaling by Extracellular Vesicles Advances Cancer Hallmarks.

Mammalian cells secrete various extracellular vesicles (EVs; exosomes, microvesicles, and apoptotic bodies) that differ in biogenesis, composition, and function. Each vesicle type can originate from normal or cancerous cells, transfer molecular cargo to both neighboring and distant cells, and modulate cellular behaviors involved in eubiology and pathology, such as tumor development. Here, we review evidence for the role of EVs in the establishment and maintenance of cancer hallmarks, including sustaining proliferative signaling, evading growth suppression, resisting cell death, reprogramming energy metabolism, acquiring genomic instability, and remodeling the tumor microenvironment. We also discuss how EVs are implicated in the induction of angiogenesis, control of cellular invasion, initiation of premetastatic niches, maintenance of inflammation, and evasion of immune surveillance. The deeper understanding of the biology of EVs and their contribution to the development and progression of tumors is leading to new opportunities in the diagnosis and treatment of cancer.

Photodynamic Treatment of Tumor with Bacteria Expressing KillerRed.

Photodynamic therapy (PDT) is a cancer treatment modality in which a photosensitizing dye is administered and exposed to light to kill tumor cells via the production of reactive oxygen species (ROS). A fundamental obstacle for PDT is the low specificity for staining solid tumors with dyes. Recently, a tumor targeting system guided by anaerobic bacteria was proposed for tumor imaging and treatment. Here, we explore the feasibility of the genetically encoded photosensitizer KillerRed, which is expressed in Escherichia coli, to treat tumors. Using nitroblue tetrazolium (NBT), we detected a lengthy ROS diffusion from the bodies of KillerRed-expressing bacteria in vitro, which demonstrated the feasibility of using bacteria to eradicate cells in their surroundings. In nude mice, Escherichia coli (E. coli) expressing KillerRed (KR-E. coli) were subcutaneously injected into xenografts comprising CNE2 cells, a human nasopharyngeal carcinoma cell line, and HeLa cells, a human cervical carcinoma cell line. KR-E. coli seemed to proliferate rapidly in the tumors as observed under an imaging system. When the intensity of fluorescence increased and the fluorescent area became as large as the tumor one day after KR-E. coli injection, the KR-E. coli-bearing tumor was irradiated with an orange light (λ = 540-580 nm). In all cases, the tumors became necrotic the next day and were completely eliminated in a few days. No necrosis was observed after the irradiation of tumors injected with a vehicle solution or a vehicle carrying the E. coli without KillerRed. In successfully treated mice, no tumor recurrence was observed for more than two months. E. coli genetically engineered for KillerRed expression are highly promising for the diagnosis and treatment of tumors when the use of bacteria in patients is cleared for infection safety.

Blockage of LMP1-modulated store-operated Ca(2+) entry reduces metastatic potential in nasopharyngeal carcinoma cell.

Epstein-Barr virus (EBV)-encoded latent membrane proteins (LMPs) expedite progression of EBV-relevant cancers. Of the full set of LMPs, latent membrane protein 1 (LMP1) was identified to uniquely augment store-operated Ca(2+) entry (SOCE). Previously, we reported that the suppression of SOCE exhibited inhibitory effects on cell migration and the extravasation from vasculature in EBV-negative nasopharyngeal carcinoma (NPC) cells. In this follow-up study, we aimed to expand our understanding of the modulation of SOCE by LMP1 and test the possibility that blockage of LMP1-modulated SOCE affects the LMP1-promoted metastatic potential. Here we showed that suppressions of the LMP1-boosted SOCE blunted the LMP1-promoted cell migration, VEGF-mediated angiogenesis and permeabilization in vitro. Blockage of SOCE inhibited vasculature-invasion of circulating cells and distant metastatic colonization in vivo. Notably, utilizing VEGFR2-EGFP-tag zebrafish we revealed that the LMP1-expressing cells arrested in a small-caliber vessel mobilized surrounding endothelial cells to facilitate vasculature-invasion. Thus, the LMP1-boosted SOCE promotes metastatic potential of NPC cells by solidifying their collaborations with the nearby non-cancer cells through the manipulation of oncogenic Ca(2+) signaling. Our study highlights the advantage of using both conventional mammal and transgenic zebrafish for developing a novel therapeutic strategy targeting the multiple steps of invasion-metastasis cascade.