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In JoVE (1)

Other Publications (8)

Articles by Jessica S. Blackburn in JoVE

 JoVE Biology

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

1Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, 2Department of Molecular Pathology, Massachusetts General Hospital Cancer Center, Harvard Stem Cell Institute


JoVE 2790

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Other articles by Jessica S. Blackburn on PubMed

RNA Interference Inhibition of Matrix Metalloproteinase-1 Prevents Melanoma Metastasis by Reducing Tumor Collagenase Activity and Angiogenesis

Melanoma incidence is increasing worldwide, and metastatic melanoma is almost completely resistant to every known therapy. New approaches to treating melanoma are urgently needed, and a greater understanding of the biology of melanoma invasion and metastasis will aid in their creation. A high proportion of invasive melanomas have a constitutively active Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling cascade; however, the downstream effectors of ERK signaling that contribute to melanoma invasion and metastasis are unknown. ERK signaling drives the production of the interstitial collagenase matrix metalloproteinase-1 (MMP-1), which is expressed specifically by invasive melanomas. Using short hairpin RNAs (shRNA) to knock down MMP-1 expression in a human melanoma cell line, we investigated the role of MMP-1 in melanoma metastasis in a xenograft model. Knockdown of MMP-1 had no effect on primary tumor growth, but reduction of MMP-1 expression significantly decreased the ability of the melanoma to metastasize from the orthotopic site in the dermis to the lung. Mechanistically, tumor cells expressing MMP-1 shRNAs had diminished collagenase activity, which is required for tumor cell invasion. Additionally, attenuation of MMP-1 expression reduced angiogenesis. These results show, for the first time, that targeted inhibition of MMP-1, a single effector of the Raf/MEK/ERK signaling cascade, prevents the progression of melanoma from a primary to metastatic tumor and, as such, may represent a useful therapeutic tool in controlling this disease.

Matrix Metalloproteinase-1 and Thrombin Differentially Activate Gene Expression in Endothelial Cells Via PAR-1 and Promote Angiogenesis

Many tumor types express matrix metalloproteinase-1 (MMP-1); its collagenase activity facilitates both tumor cell invasion and metastasis. MMP-1 expression is also associated with increased angiogenesis; however, the exact mechanism by which this occurs is not clear. MMP-1 proteolytically activates protease activated receptor-1 (PAR-1), a thrombin receptor that is highly expressed in endothelial cells. Thrombin is also present in the tumor microenvironment, and its activation of PAR-1 is pro-angiogenic. It is currently unknown whether MMP-1 activation of PAR-1 induces angiogenesis in a similar or different manner compared with thrombin. We sought to determine the mechanism by which MMP-1 promotes angiogenesis and to compare the effects of MMP-1 with those of thrombin. Our results demonstrate that via PAR-1, MMP-1 activates mitogen-activated protein kinase signaling cascades in microvessel endothelial cells. Although thrombin activation of PAR-1 also induces signaling through these pathways, the time-course of activation appears to vary. Gene expression analysis revealed a possible consequence of these signaling differences as MMP-1 and thrombin induce expression of different subsets of pro-angiogenic genes. Furthermore, the combination of thrombin and MMP-1 is more angiogenic than either protease alone. These data demonstrate that MMP-1 acts directly on endothelial cells as a pro-angiogenic signaling molecule and also suggest that the effects of MMP-1 may complement the activity of thrombin to better facilitate angiogenesis and promote tumor progression.

Wild-type Versus Mutant MMP-8 in Melanoma: 'when You Come to a Fork in the Road, Take It'

Matrix Metalloproteinase and G Protein Coupled Receptors: Co-conspirators in the Pathogenesis of Autoimmune Disease and Cancer

Similarities in the pathologies of autoimmune diseases and cancer have been noted for at least 30 years. Inflammatory cytokines and growth factors mediate cell proliferation, and proteinases, especially the collagenase, Matrix Metalloproteinase-1 (MMP-1), contribute to disease progression by remodeling the extracellular matrix and modulating the microenvironment. This review focuses on two cancers (melanoma and breast) and on the autoimmune disorder, rheumatoid arthritis (RA), and discusses the activated stromal cells found in these diseases. MMP-1 was originally thought to function only to degrade interstitial collagens, but recent studies have revealed novel roles for MMP-1 involving the G protein-coupled receptors: the chemokine receptor, CXCR-4, and Protease Activated Receptor-1 (PAR-1). Cooperativity between MMP-1 and CXCR4/SDF-1 signaling influences the behavior of activated fibroblasts in both RA and cancer. Further, MMP-1 is a vital part of an autocrine/paracrine MMP-1/PAR-1 signal transduction axis, a function that amplifies its potential to remodel the matrix and to modify cell behavior. Finally, new therapeutic agents directed at MMP-1 and G protein-coupled receptors are emerging. Even though these agents are more specific in their targets than past therapies, these targets are often shared between RA and cancer, underscoring fundamental similarities between autoimmune disorders and some cancers.

High-throughput Cell Transplantation Establishes That Tumor-initiating Cells Are Abundant in Zebrafish T-cell Acute Lymphoblastic Leukemia

Self-renewal is a feature of cancer and can be assessed by cell transplantation into immune-compromised or immune-matched animals. However, studies in zebrafish have been severely limited by lack of these reagents. Here, Myc-induced T-cell acute lymphoblastic leukemias (T-ALLs) have been made in syngeneic, clonal zebrafish and can be transplanted into sibling animals without the need for immune suppression. These studies show that self-renewing cells are abundant in T-ALL and comprise 0.1% to 15.9% of the T-ALL mass. Large-scale single-cell transplantation experiments established that T-ALLs can be initiated from a single cell and that leukemias exhibit wide differences in tumor-initiating potential. T-ALLs also can be introduced into clonal-outcrossed animals, and T-ALLs arising in mixed genetic backgrounds can be transplanted into clonal recipients without the need for major histocompatibility complex matching. Finally, high-throughput imaging methods are described that allow large numbers of fluorescent transgenic animals to be imaged simultaneously, facilitating the rapid screening of engrafted animals. Our experiments highlight the large numbers of zebrafish that can be experimentally assessed by cell transplantation and establish new high-throughput methods to functionally interrogate gene pathways involved in cancer self-renewal.

AMAZe-ing Tools for Mosaic Analysis in Zebrafish

Selection-free Zinc-finger-nuclease Engineering by Context-dependent Assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.

High-throughput Imaging of Adult Fluorescent Zebrafish with an LED Fluorescence Macroscope

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis.

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