Fibronectin is an extracellular matrix protein found only in vertebrate organisms containing endothelium-lined vasculature and is required for cardiovascular development in fish and mice. Fibronectin ...and its splice variants containing EIIIA and EIIIB domains are highly upregulated around newly developing vasculature during embryogenesis and in pathological conditions including atherosclerosis, cardiac hypertrophy, and tumorigenesis. However, their molecular roles in these processes are not entirely understood. We review genetic studies examining functions of fibronectin and its splice variants during embryonic cardiovascular development, and discuss potential roles of fibronectin in vascular disease and tumor angiogenesis.
Metastasis is a major clinical problem whose biology is not yet fully understood. This lack of understanding is especially true for the events at the metastatic site, which include arrest, ...extravasation, and growth into macrometastases. Intravital imaging is a powerful technique that has shown great promise in increasing our understanding of these events. To date, most intravital imaging studies have been performed in mice, which has limited its adoption. Zebrafish are also a common system for the intravital imaging of metastasis. However, as imaging in embryos is technically simpler, relatively few studies have used adult zebrafish to study metastasis and none have followed individual cells at the metastatic site over time. The aim of this study was to demonstrate that adult casper zebrafish offer a convenient model system for performing intravital imaging of the metastatic site over time with single-cell resolution.
ZMEL1 zebrafish melanoma cells were injected into 6 to 10-week-old casper fish using an intravenous injection protocol. Because casper fish are transparent even as adults, they could be imaged without surgical intervention. Individual cells were followed over the course of 2 weeks as they arrested, extravasated, and formed macroscopic metastases.
Our injection method reliably delivered cells into circulation and led to the formation of tumors in multiple organs. Cells in the skin and sub-dermal muscle could be imaged at high resolution over 2 weeks using confocal microscopy. Arrest was visualized and determined to be primarily due to size restriction. Following arrest, extravasation was seen to occur between 1 and 6 days post-injection. Once outside of the vasculature, cells were observed migrating as well as forming protrusions.
Casper fish are a useful model for studying the events at the metastatic site using intravital imaging. The protocols described in this study are relatively simple. Combined with the reasonably low cost of zebrafish, they offer to increase access to intravital imaging.
The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment ...to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients.
Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling ...interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.
Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length ...HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 105infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-α and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.
The most damaging change during cancer progression is the switch from a
locally growing tumour to a metastatic killer. This switch is believed to
involve numerous alterations that allow tumour cells ...to complete the complex
series of events needed for metastasis. Relatively few genes
have been implicated in these events. Here we use
an in vivo selection scheme to select highly metastatic melanoma cells.
By analysing these cells on DNA arrays, we define a pattern of gene expression
that correlates with progression to a metastatic phenotype. In particular,
we show enhanced expression of several genes involved in extracellular matrix
assembly and of a second set of genes that regulate, either directly or indirectly,
the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances
metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis.
Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC
indicates that RhoC is important in tumour cell invasion. The genomic approach
allows us to identify families of genes involved in a process, not just single
genes, and can indicate which molecular and cellular events might be important
in complex biological processes such as metastasis.
The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare ...in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines.
The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. ...The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.
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