Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation in vivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively to invadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin), maturation (Tks5), or function (Tks4) resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process.

Nanomaterials with elongated architectures have been shown to possess differential tumor homing properties compared to their spherical counterparts. Here, we investigate whether this phenomenon is mirrored by plant viral nanoparticles that are filamentous (Potato virus X) or spherical (Cowpea mosaic virus). Our studies demonstrate that Potato virus X (PVX) and Cowpea mosaic virus (CPMV) show distinct biodistribution profiles and differ in their tumor homing and penetration efficiency. Analogous to what is seen with inorganic nanomaterials, PVX shows enhanced tumor homing and tissue penetration. Human tumor xenografts exhibit higher uptake of PEGylated filamentous PVX compared to CPMV, particularly in the core of the tumor. This is supported by immunohistochemical analysis of the tumor sections, which indicates greater penetration and accumulation of PVX within the tumor tissues. The enhanced tumor homing and retention properties of PVX along with its higher payload carrying capacity makes it a potentially superior platform for applications in cancer drug delivery and imaging applications.

Aims Vimentin, a type III intermediate filament, is upregulated during epithelial–mesenchymal transition and tumor progression. Vimentin is surface-expressed on cells involved in inflammation; the function remains unknown. We investigated the expression of surface vimentin on cancer cells and evaluated targeting nanoparticles to tumors exploiting vimentin. Materials & methods Cowpea mosaic virus nanoparticles that interact with surface vimentin were used as probes. Tumor homing was tested using the chick chorioallantoic membrane model with human tumor xenografts. Results & discussion Surface vimentin levels varied during cell cycle and among the cell lines tested. Surface vimentin expression correlated with cowpea mosaic virus uptake, underscoring the utility of cowpea mosaic virus to detect invasive cancer cells. Targeting to tumor xenografts was observed; homing was based on the enhanced permeability and retention effect. Our data provide novel insights into the role of surface vimentin in cancer and targeting nanoparticles in vivo.

Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of cpMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application.

Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [β-Ala11, Phe13, Nle14]bombesin-(7–14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further development of this viral nanoparticle platform may open the door to potential clinical noninvasive molecular imaging strategies.

Background: Panx1 is a channel-forming glycoprotein that regulates epidermal differentiation and proliferation. Results: Depletion of Panx1 in melanomas causes cell re-differentiation into a melanocytic-like phenotype and reduced tumorigenesis. Conclusion: Panx1 is up-regulated during melanoma progression promoting tumor growth and metastasis. Significance: This is the first report of Panx1 as a proto-oncogene establishing it as a potential target for melanoma treatment.

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130Cas, which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1–Rho-GTPase interaction in metastasis, we disrupted Cav1–Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130Cas phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130Cas/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.