Use of PiggyBac Transposon System Constructed Murine Breast Cancer Model For Reporter Gene Imaging And Characterization of Metastatic Tumor Cells

The piggyBac transposon system is known to non-viral integrate exogenous genes to chromosomes of mammalian cells. For reporter gene imaging, this transposon system is believed to efficiently establish xenograft tumor model with low immunogenicity. Because tumor cells usually exhibit genomic instability, it is important to investigate if piggyBac mediated transduction of reporter genes would change tumor characteristics. In this study, reporter gene imaging mediated by the piggyBac transposon system was exploited to track the growth and dissemination of 4T1 triple-negative murine breast cancer cells in vivo, followed by ex vivo analysis of the metastatic cells expressing reporter genes. We demonstrated that several cell properties, including proliferation rate, invasion and migration rate, and mammosphere formation ability of 4T1 cells were not influenced by piggyBac transposon system. Further, we isolated the liver metastatic cells, named 4T1-3R_L cells for further analysis. Compared to parental 4T1 cells, 4T1-3R_L cells exhibited several cancer stem cells (CSC) related characteristics, including significant mammosphere formation ability, resistance to doxorubicin, high tumorigenicity potential in Balb/C mice and expression of CD44 CSC marker. We also found that 4T1-3R_L cells exhibited stronger migrated and invasive abilities, by wound healing assay and in vitro invasion assay, respectively. The cell adhesive ability of 4T1-3R_L cells was also lower than that of 4T1 cells. The microarray assay showed that several epithelial-mesenchymal transition (EMT) promoting markers, including vimentin, N-cadherin, Twist1, and Snail were up-regulated, and anti-EMT marker E-cadherin was down-regulated in 4T1-3R_L cells. Current data suggest that the piggyBac transposon system is a reliable and biocompatible tool to engineer cancer cells for tacking and characterizing tumor development in vivo and in vitro.

The Role of Extracellular Vesicles in the Progression of Tumors towards Metastasis

Extracellular vesicles (EVs) are cell-derived lipid membrane bound vesicles that serve as mediators of intercellular communication. EVs have been found to regulate a wide range of cellular processes through the transference of genetic, protein and lipid messages from the host cell to the recipient cell. Unsurprisingly, this major mode of intracellular communication would be abrogated in cancer. Ever increasing evidence points towards a key role of EVs in promoting tumor development and in contributing to the various stages of metastasis. Tumor released EVs have been shown to facilitate the transference of oncogenic proteins and nucleic acids to other tumor cells and to the surrounding stromal cells, thereby setting up a tumor permissive microenvironment. EVs released from tumor cells have been shown to promote extracellular matrix (ECM) remodeling through the modulation of neighboring tumor cells and stromal cells. EVs released from disseminated tumor cells have been reported to attract circulating tumor cells (CTCs) via chemotaxis and induce the production of specific extracellular matrix components from neighboring stromal cells so as to support the growth of metastatic cells at the secondary tumor site. Circulating levels of tumor derived EVs of patients have been correlated with incidence of metastasis and disease relapse.

Targeting Oxidative Phosphorylation-Proteasome Activity in Extracellular Detached Cells Promotes Anoikis and Inhibits Metastasis

Metastasis arises owing to tumor cells’ capacity to evade pro-apoptotic signals. Anoikis—the apoptosis of detached cells (from the extracellular matrix (ECM)) is often circumvented by metastatic cells as a result of biochemical and molecular transformations. These facilitate cells’ ability to survive, invade and reattach to secondary sites. Here, we identified deregulated glucose metabolism, oxidative phosphorylation, and proteasome in anchorage-independent cells compared to adherent cells. Metformin an anti-diabetic drug that reduces blood glucose (also known to inhibit mitochondrial Complex I), and proteasome inhibitors were employed to target these changes. Metformin or proteasome inhibitors alone increased misfolded protein accumulation, sensitized tumor cells to anoikis, and impaired pulmonary metastasis in the B16F10 melanoma model. Mechanistically, metformin reduced cellular ATP production, activated AMPK to foster pro-apoptotic unfolded protein response (UPR) through enhanced expression of CHOP in ECM detached cells. Furthermore, AMPK inhibition reduced misfolded protein accumulation, thus highlight relevance of AMPK activation in facilitating metformin-induced stress and UPR cell death. Our findings provide insights into the molecular biology of anoikis resistance and identified metformin and proteasome inhibitors as potential therapeutic options for tumor metastasis.

Oral Cancer Theranostic Application of FeAu Bimetallic Nanoparticles Conjugated with MMP-1 Antibody

Metastatic oral squamous cell carcinoma (SCC) displays a poor disease prognosis with a 5-year survival rate of 39%. Chemotherapy has emerged as the mainstream treatment against small clusters of cancer cells but poses more risks than benefits for metastatic cells due to the non-specificity and cytotoxicity. To overcome these obstacles, we conjugated antibodies specific for matrix metalloproteinase-1 (MMP-1), a prognostic biomarker of SCC, to iron–gold bimetallic nanoparticles (FeAu NPs) and explored the capability of this complex to target and limit SSC cell growth via magnetic field-induced hyperthermia. Our results showed that 4.32 ± 0.79 nm sized FeAu NPs were superparamagnetic in nature with a saturation magnetization (Ms) of 5.8 emu/g and elevated the media temperature to 45 °C, confirming the prospect to deliver hyperthermia. Furthermore, conjugation with MMP-1 antibodies resulted in a 3.07-fold higher uptake in HSC-3 (human tongue squamous cell carcinoma) cells as compared to L929 (fibroblast) cells, which translated to a 5-fold decrease in cell viability, confirming SCC targeting. Finally, upon magnetic stimulation, MMP-1-FeAu NPs conjugate triggered 89% HSC-3 cellular death, confirming the efficacy of antibody-conjugated nanoparticles in limiting SCC growth. The synergistic effect of biomarker-specific antibodies and magnetic nanoparticle-induced hyperthermia may open new doors towards SCC targeting for improved disease prognosis.

PARK7/DJ-1 inhibition decreases invasion and proliferation of uveal melanoma cells

Introduction: PARK7/DJ-1 is an oncogene that is associated with tumorigenesis in many cancers. Recent studies have demonstrated the importance of DJ-1 in the origin and development of uveal melanoma (UM). We present an analysis of the role of the DJ-1 protein in UM cells, especially in its effect on proliferation and migration. Methods: UM cells from a primary tumor, Mel 270, and its liver metastasis, OMM2.5, were transfected with lentiviral-delivered shRNA against PARK7/DJ-1. Evaluation of cell migration and proliferation was performed using the xCELLigence real-time cell analyzer (RTCA). The effect of DJ-1 inhibition on the PTEN-Akt signaling pathway was also studied by immunoblotting. Results: The silencing of PARK7/DJ-1 oncoprotein expression produced a significant decrease of phosphorylated Akt (S473) in Mel270 and in metastatic OMM2.5 UM cells with no alteration on tumor suppressor PTEN expression. The diminution of PARK7/DJ-1 expression significantly inhibited real-time proliferation and invasion of Mel270 and OMM2.5 and the invasion potential of the metastatic cells. Conclusion: DJ-1 appears to play a key role on the PTEN/Akt pathway in UM. DJ-1 inhibition appears to have a negative effect on proliferation and invasion of UM cells. This suggests DJ-1 as a potential therapeutic target in UM.

Zebrafish xenografts to isolate unique human breast cancer metastatic cell populations

Cancer metastasis is a critical culprit frequently blamed for treatment failure, drug resistance, poor prognosis, and high mortality rate among all human cancers. Laboratory efforts to isolate metastatic cell populations have typically been confined to mouse models, which are time-consuming and expensive. Here, we present a model system based on xenografting zebrafish embryos to select for cells that are predisposed to progress through the early stages of metastasis. This model requires only 3-5 days to achieve distinct intravasation to the zebrafish circulatory system. The metastatic cells are easily tracked in real-time as they migrate, as well as isolated and propagated in vitro. Once expanded, molecular characterization of the serially derived invasive cell populations from the tails of the zebrafish accurately predicts genes, signaling pathways, protein-protein interactions, and differential splicing products that are important for an invasive phenotype. This zebrafish model therefore offers a high-throughput and robust method for identifying gene targets critical for cancer metastasis.

Confined Cell Migration and Asymmetric Hydraulic Environments to Evaluate The Metastatic Potential of Cancer Cells

Metastasis, a hallmark of cancer development, is also the leading reason for most cancer-related deaths. Furthermore, cancer cells are highly adaptable to microenvironments and can migrate along pre-existing channel-like tracks of anatomical structures. However, more representative three-dimensional models are required to reproduce the heterogeneity of metastatic cell migration in vivo to further understand the metastasis mechanism and develop novel therapeutic strategies against it. Here, we designed and fabricated different microfluidic-based devices that recreate confined migration and diverse environments with asymmetric hydraulic resistances. Our results show different migratory potential between metastatic and nonmetastatic cancer cells in confined environments. Moreover, although nonmetastatic cells have not been tested against barotaxis due to their low migration capacity, metastatic cells present an enhanced preference to migrate through the lowest resistance path, being sensitive to barotaxis. This device, approaching the study of metastasis capability based on confined cell migration and barotactic cell decisions, may pave the way for the implementation of such technology to determine and screen the metastatic potential of certain cancer cells.

The Relevance of Ultrastructural Studies of Metastatic Cells from Women with Breast Cancer History

The interaction between cancer cells and the surrounding microenvironment is determinant for metastasis success. In this study, the ultrastructural relevance of cells in the malignant pleural effusion (MPE) of women with breast cancer history was investigated. In MPE, it is possible to observe single cells and clusters. Women whose MPE presents carcinomas in aggregates have a better prognosis when compared to cases in which metastatic single cells are found. Samples were collected via fine-needle aspiration puncture (US-FNA). Subsequent to the material preparation and ultrathin cuts, they were observed using light and transmission electron microscopy (LM/TEM). LM and TEM images served as a basis for the creation of a digital sculpture using ZBrush® software. Clusters exhibited structural stability, en route vesicles allowing exocytosis of electron-dense fibrous elements, and cytoplasmic protrusions contributing to migratory and invasive skills. Single cells presented different necrotic phenotypes and many displayed leukocyte-like characteristics. Cluster cooperative relationships seem to be related to a long-term permanence in MPE. The absence of a collaborative network presumably triggers a more aggressive behavior of single cells. Its putative fusion with leukocytes can maximize the efficiency for transendothelial migration, increasing chances of metastatic success and, unfortunately, reducing survival of women with recidivism.

MVP Expression Facilitates Tumor Cell Proliferation and Migration Supporting the Metastasis of Colorectal Cancer Cells

Cancer cells show significant dysregulation of genes expression, which may favor their survival in the tumor environment. In this study, the cellular vault’s components MVP (major vault protein), TEP1 (telomerase-associated protein 1) and vPARP (vault poly(ADP-ribose) polymerase) were transiently or completely inhibited in U2OS cells (human bone osteosarcoma epithelial cells) to evaluate their impact on the cell proliferative and migratory capacity as well as on the development of their resistance to the drug vinorelbine. Comparative analysis of MVP protein expression level in normal colon tissue, primary colorectal tumor, and metastasis showed that the expression of this protein does not increase significantly in the primary tumor, but its expression increases in metastatic cells. Further comparative molecular analysis using the whole transcriptome microarrays for MVP-positive and MVP-negative cells showed that MVP is involved in regulating proliferation and migration of cancer cells. MVP may facilitate metastasis of colon cancer due to its impact on cell migration. Moreover, two vault proteins, MVP and TEP1, contribute the resistance to vinorelbine, while vPARP does not.