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Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

University of Alberta, Edmonton, Alberta, Canada

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.

Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis.

Metastasis, or the spread of cancer cells from a primary tumor to distant sites, is the leading cause of cancer-associated death. Metastasis is a complex multi-step process comprised of invasion, intravasation, survival in circulation, extravasation, and formation of metastatic colonies. Currently, in vitro assays are limited in their ability to investigate these intricate processes and do not faithfully reflect metastasis as it occurs in vivo. Traditional in vivo models of metastasis are limited by their ability to visualize the seemingly sporadic behavior of where and when cancer cells spread (Reymond et al., Nat Rev Cancer 13:858-870, 2013). The avian embryo model of metastasis is a powerful platform to study many of the critical steps in the metastatic cascade including the migration, extravasation, and invasion of human cancer cells in vivo (Sung et al., Nat Commun 6:7164, 2015; Leong et al., Cell Rep 8, 1558-1570, 2014; Kain et al., Dev Dyn 243:216-28, 2014; Leong et al., Nat Protoc 5:1406-17, 2010; Zijlstra et al., Cancer Cell 13:221-234, 2008; Palmer et al., J Vis Exp 51:2815, 2011). The chicken chorioallantoic membrane (CAM) is a readily accessible and well-vascularized tissue that surrounds the developing embryo. When the chicken embryo is grown in a shell-less, ex ovo environment, the nearly transparent CAM provides an ideal environment for high-resolution fluorescent microcopy approaches. In this model, the embryonic chicken vasculature and labeled cancer cells can be visualized simultaneously to investigate specific steps in the metastatic cascade including extravasation. When combined with the proper image analysis tools, the ex ovo chicken embryo model offers a cost-effective and high-throughput platform for the quantitative analysis of tumor cell metastasis in a physiologically relevant in vivo setting. Here we discuss detailed procedures to quantify cancer cell extravasation in the shell-less chicken embryo model with advanced fluorescence microscopy techniques.

Quantitative Analysis of Human Cancer Cell Extravasation Using Intravital Imaging.

The feasibility of personalized medicine approaches will be greatly improved by the development of noninvasive methods to interrogate tumor biology. Extracellular vesicles shed by solid tumors into the bloodstream have been under recent investigation as a source of tumor-derived biomarkers such as proteins and nucleic acids. We report here an approach using submicrometer perfluorobutane nanodroplets and focused ultrasound to enhance the release of extracellular vesicles from specific locations in tumors into the blood. The released extracellular vesicles were enumerated and characterized using micro flow cytometry. Only in the presence of nanodroplets could ultrasound release appreciable levels of tumor-derived vesicles into the blood. Sonication of HT1080-GFP tumors did not increase the number of circulating tumor cells or the metastatic burden in the tumor-bearing embryos. A variety of biological molecules were successfully detected in tumor-derived extracellular vesicles, including cancer-associated proteins, mRNAs, and miRNAs. Sonication of xenograft HT1080 fibrosarcoma tumors released extracellular vesicles that contained detectable RAC1 mRNA with the highly tumorigenic N92I mutation known to exist in HT1080 cells. Deep sequencing serum samples of embryos with sonicated tumors allowed the identification of an additional 13 known heterozygous mutations in HT1080 cells. Applying ultrasound to HT1080 tumors increased tumor-derived DNA in the serum by two orders of magnitude. This work is the first demonstration of enhanced extracellular vesicle release by ultrasound stimulation and suggests that nanodroplets/ultrasound offers promise for genetic profiling of tumor phenotype and aggressiveness by stimulating the release of extracellular vesicles. Cancer Res; 77(1); 3-13. ©2016 AACR.

Enhanced Detection of Cancer Biomarkers in Blood-Borne Extracellular Vesicles Using Nanodroplets and Focused Ultrasound.

Angiogenesis is a dynamic process fundamental to the development of solid tumors. Epidermal growth factor-like domain 7 (EGFL7) is a protein whose expression is restricted to endothelial cells undergoing active remodeling that has emerged as a key mediator of this process. EGFL7 expression is associated with poor outcome in several cancers, making it a promising target for imaging or therapeutic strategies. Here, EGFL7 is explored as a molecular target for active neovascularization. Using a combinatorial peptide screening approach, we describe the discovery and characterization of a novel high affinity EGFL7-binding peptide, E7p72, that specifically targets human endothelial cells. Viral nanoparticles decorated with E7p72 peptides specifically target tumor-associated neovasculature with high specificity as assessed by intravital imaging. This work highlights the value of EGFL7 as a target for angiogenic vessels and opens the door for novel targeted therapeutic approaches.

Viral nanoparticles decorated with novel EGFL7 ligands enable intravital imaging of tumor neovasculature.

Previous studies indicate that breast cancer cells with high aldehyde dehydrogenase (ALDH) activity and CD44 expression (ALDHCD44⁺) contribute to metastasis and therapy resistance, and that ALDH1 correlates with poor outcome in breast cancer patients. The current study hypothesized that ALDH1 functionally contributes to breast cancer metastatic behavior and therapy resistance. Expression of ALDH1A1 or ALDH1A3 was knocked down in MDA-MB-468 and SUM159 human breast cancer cells using siRNA. Resulting impacts on ALDH activity (Aldefluor assay); metastatic behavior and therapy response in vitro (proliferation/adhesion/migration/colony formation/chemotherapy and radiation) and extravasation/metastasis in vivo (chick choroiallantoic membrane assay) was assessed. Knockdown of ALDH1A3 but not ALDH1A1 in breast cancer cells decreased ALDH activity, and knockdown of ALDH1A1 reduced breast cancer cell metastatic behavior and therapy resistance relative to control ( < 0.05). In contrast, knockdown of ALDH1A3 did not alter proliferation, extravasation, or therapy resistance, but increased adhesion/migration and decreased colony formation/metastasis relative to control ( < 0.05). This is the first study to systematically examine the function of ALDH1 isozymes in individual breast cancer cell behaviors that contribute to metastasis. Our novel results indicate that ALDH1 mediates breast cancer metastatic behavior and therapy resistance, and that different enzyme isoforms within the ALDH1 family differentially impact these cell behaviors.

Differential Functional Roles of ALDH1A1 and ALDH1A3 in Mediating Metastatic Behavior and Therapy Resistance of Human Breast Cancer Cells.

Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.

Quantitative in vivo whole genome motility screen reveals novel therapeutic targets to block cancer metastasis.

Nanoparticle platforms are particularly attractive for theranostic applications due to their capacity for multifunctionality and multivalency. Some of the most promising nano-scale scaffold systems have been co-opted from nature including plant viruses such as cowpea mosaic virus (CPMV). The use of plant viruses like CPMV as viral nanoparticles is advantageous for many reasons; they are non-infectious and nontoxic to humans and safe for use in intravital imaging and drug delivery. The CPMV capsid icosahedral shape allows for enhanced multifunctional group display and the ability to carry specific cargoes. The native tropism of CPMV for cell-surface displayed vimentin and the enhanced permeability and retention effect allow them to preferentially extravasate from tumor neovasculature and efficiently penetrate tumors. Furthermore, CPMVs can be engineered via several straightforward chemistries to display targeting and imaging moieties on external, addressable residues and they can be loaded internally with therapeutic drug cargoes. These qualities make them highly effective as biocompatible platforms for tumor targeting, intravital imaging and cancer therapy.

Cowpea mosaic virus nanoparticles for cancer imaging and therapy.

The Alberta Prostate Cancer Research Initiative (APCaRI) Registry and Biorepository was established in 2014 by the APCaRI to facilitate the collection of clinical and patient-reported data, biospecimen, to measure prostate cancer outcomes and to support the development and clinical translation of innovative technologies to better diagnose and predict outcomes for patients with prostate cancer.

Cohort profile: the Alberta Prostate Cancer Research Initiative (APCaRI) Registry and Biorepository facilitates technology translation to the clinic through the use of linked, longitudinal clinical and patient-reported data and biospecimens from men in Alberta, Canada.

John D. Lewis

Department of Oncology

University of Alberta, Edmonton, Alberta, Canada

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model