Bojana Gligorijevic

Intravital imaging of metastatic behavior through a mammary imaging window

We report a technique to evaluate the same tumor microenvironment over multiple intravital imaging sessions in living mice. We optically marked individual tumor cells expressing photoswitchable proteins in an orthotopic mammary carcinoma and followed them for extended periods through a mammary imaging window. We found that two distinct microenvironments in the same orthotopic mammary tumor affected differently the invasion and intravasation of tumor cells.

N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors

Invadopodia are proteolytic membrane protrusions formed by highly invasive cancer cells, commonly observed on substrate(s) mimicking extracellular matrix. Although invadopodia are proposed to have roles in cancer invasion and metastasis, direct evidence has not been available. We previously reported that neural Wiskott–Aldrich syndrome protein (N-WASP), a member of WASP family proteins that regulate reorganization of the actin cytoskeleton, is an essential component of invadopodia. Here, we report that N-WASP-mediated invadopodium formation is essential in breast cancer invasion, intravasation and lung metastasis. We established stable cell lines based on MTLn3 rat mammary adenocarcinoma cells that either overexpressed a dominant-negative (DN) N-WASP construct or in which N-WASP expression was silenced by a pSuper N-WASP shRNA. Both the N-WASP shRNA and DN N-WASP cells showed a markedly decreased ability to form invadopodia and degrade extracellular matrix. In addition, formation of invadopodia in primary tumors and collagen I degradation were reduced in the areas of invasion (collagen-rich areas in the invasive edge of the tumor) and in the areas of intravasation (blood-vessel-rich areas). Our results suggest that tumor cells in vivo that have a decreased activity of N-WASP also have a reduced ability to form invadopodia, migrate, invade, intravasate and disseminate to lung compared with tumor cells with parental N-WASP levels.

Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer

We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (MenaINV) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated MenaINV increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by MenaINV is dependent on a macrophage–tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of MenaINV and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells.

Multiparametric classification links tumor microenvironments with tumor cell phenotype.

Tumor microenvironment features are established as predictors of tumor cell behavior and fate.

High-Resolution Multiphoton Imaging of Tumors In Vivo

Analysis of the individual steps in metastasis is crucial if insights at the molecular level are to be linked to the cell biology of cancer. A technical hurdle to achieving the analysis of the individual steps of metastasis is the fact that, at the gross level, tumors are heterogeneous in both animal models and patients. Human primary tumors show extensive variation in all properties ranging from growth and morphology of the tumor through tumor-cell density in the blood and formation and growth of metastases. Methods capable of the direct visualization and analysis of tumor-cell behavior at single-cell resolution in vivo have become crucial in advancing the understanding of mechanisms of metastasis, the definition of microenvironment, and the markers related to both. This article discusses the use of high-resolution multiphoton imaging of tumors (specifically breast tumors in mice) in vivo.

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

A new chemotaxis device for cell migration studies

This study presents the design and optimization for in vitro use of a new versatile chemotaxis device called the NANIVID (NANo IntraVital Imaging Device), developed using advanced nano/micro fabrication techniques. The device is fabricated using microphotolithographic techniques and two substrates are bonded together using a thin polymer layer creating a sealed device with one outlet. The main structure of the device consists of two Pyrex substrates: an etched chemoattractant reservoir and a top cover, with a final size of 0.2 × 2 × 3 mm. This reservoir contains a hydrogel blend with EGF which diffuses out through a small (∼9.10(3)μm(2)) outlet. This reservoir sustains a steady release of growth factor into the surrounding environment for several hours establishing a consistent concentration gradient from the device. The focus of this study was to design and optimize the new device for cell chemotaxis studies in breast cancer cells in cell culture. Our results show that we have created a flexible, cheap, miniature and autonomous chemotaxis device and demonstrate its usefulness in 2D and 3D cell culture. We also provide preliminary data for use of the device in vivo.

Invadopodia in context

Invadopodia are dynamic protrusions in motile tumor cells whose function is to degrade extracellular matrix so that cells can enter into new environments. Invadopodia are specifically identified by microscopy as proteolytic invasive protrusions containing TKS5 and cortactin. The increasing complexity in models for the study of invadopodia, including engineered 3D environments, explants, or animal models in vivo, entails a higher level of microenvironment complexity as well as cancer cell heterogeneity. Such experimental setups are rich in information and offer the possibility of contextualizing invadopodia and other motility-related structures. That is, they hold the promise of revealing more realistic microenvironmental conditions under which the invadopodium assembles and functions or in which tumor cells switch to a different cellular phenotype (focal adhesion, lamellipodia, proliferation, and apoptosis). For such an effort, we need a systemic approach to microscopy, which will integrate information from multiple modalities. While the individual technologies needed to achieve this are mostly available, data integration and standardization is not a trivial process. In a systems microscopy approach, microscopy is used to extract information on cell phenotypes and the microenvironment while -omics technologies assess profiles of cancer cell and microenvironment genetic, transcription, translation, and protein makeups. Data are classified and linked via in silico modeling (including statistical and mathematical models and bioinformatics). Computational considerations create predictions to be validated experimentally by perturbing the system through use of genetic manipulations and molecular biology. With such a holistic approach, a deeper understanding of function of invadopodia in vivo will be reached, opening the potential for personalized diagnostics and therapies.

Invadosomes in their natural habitat

Podosomes and invadopodia (collectively known as invadosomes) are small, F-actin-rich protrusions that are located at points of cell-ECM contacts and endow cells with invasive capabilities. So far, they have been identified in human or murine immune (myelomonocytic), vascular and cancer cells. The overarching reason for studying invadosomes is their connection to human disease. For example, macrophages and osteoclasts lacking Wiskott-Aldrich syndrome protein (WASp) are not able to form podosomes, and this leads to altered macrophage chemotaxis and defective bone resorption by osteoclasts. In contrast, the ability of cancer cells to form invadopodia is associated with high invasive and metastatic potentials. While invadosome composition, dynamics and signaling cascades leading to their assembly can be followed easily in in vitro assays, studying their contribution to pathophysiological processes in situ remains challenging. A number of recent papers have started to address this issue and describe invadosomes in situ in mouse models of cancer, cardiovascular disease and angiogenesis. In addition, in vivo invadosome homologs have been reported in developmental model systems such as C. elegans, zebrafish and sea squirt. Comparative analyses among different invasion mechanisms as they happen in their natural habitats, i.e., in situ, may provide an outline of the invadosome evolutionary history, and guide our understanding of the roles of the invasion process in pathophysiology versus development.

Visualization of Actin Polymerization in Invasive Structures of Macrophages and Carcinoma Cells Using Photoconvertible β-Actin – Dendra2 Fusion Proteins

Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin - Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.