Nonadherent Cells Research Articles

Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization, Direct RBC Contact, and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC-derived ATP. These studies incorporate on-chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC-EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum-black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8 ± 0.2 nM NO per endothelial cell (average ± SEM, n = 8). Pretreatment of RBCs with glybenclamide inhibited treprostinil-induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92 ± 0.1 nM NO per endothelial cell, n = 7). In the future, this device can be used to study interactions between many other cell types (both adherent and non-adherent cell lines) and incorporate other detection schemes.

Expandable Bioreactor for the culture of immune cells

Background & Aim With the recent successes of autologous immunotherapy, with 2 therapeutic products approved by the Food & Drugs Administration (FDA) since 2017 and more in the clinical pipeline, there is a need for cell and gene therapy companies to transition from lab scale production to mass manufacture. Traditionally, bioreactors have been routinely used in process development and scale up manufacturing of biologics. However, such bioreactor systems are not suitable for immunotherapy production as, beyond standard external factors such as shear stresses, immune cells are also sensitive to factors such as density and require cell-to-cell contact to proliferate well. Methods, Results & Conclusion A novel single use bioreactor for non-adherent cell culture has been developed in SIMTech (Patent Application WO2018/182533 A1) that allows for culturing cells at constant density while increasing volume of culture media to support cell proliferation. The bioreactor features two different chambers, one for media reserves and the other for cells. The two chambers are separated by a membrane that allow for fluidic exchange between these chambers. The cell chamber has a built-in expansion mechanism that can be adjusted as cells proliferate, thus allowing the user to adjust at any time point and maintain consistent culture density. The bioreactor has been validated with Jurkat cell line and T cells derived from commercial PBMCs with promising results; it was observed that the cultured cells exhibited high viability and selectivity. Jurkat cells had a 240-fold increase in cell number after 14 days in culture while T cells (from commercial PBMCs), had a 47-fold increase in cell number after 21 days in culture. A direct comparison study (1 bioreactor vs multiple 24-well plates for the same increase in cell number) also demonstrated a significant reduction in protocol steps and culture handling time. The bioreactor is currently being tested on donor PBMCs and other cell types, in collaborations with clinicians and cell therapy laboratories, in order to develop a GMP workflow for clinical use.

Stimulation with Porphyromonas gingivalis enhances malignancy and initiates anoikis resistance in immortalized oral keratinocytes.

The aim of this study was to get new insights into molecular processes involved in tumor propagation of immortalized oral keratinocytes induced by the keystone pathogen Porphyromonas gingivalis. Cell culture experiments with immortalized OKF6 cells were performed to analyze cellular effects caused by bacterial stimulation focusing on altered gene expression, signaling pathways, proliferation rate, cell viability, migration and invasion behavior, and on the development of antiapoptotic pathways. Gene and protein expression were analyzed using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blot, and protein arrays. Trypan blue staining was used to analyze proliferation and viability, transwell assays for cellular migration, Matrigel assays for invasion, and anoikis-assays for evaluating anoikis resistance. Stimulation of OKF6 cells with Porphyromonas gingivalis led to an alteration in the molecular repertoire of proteins which are involved in cell proliferation, epithelial-mesenchymal transition, stem cell formation, migration, invasion, and anoikis resistance. Higher proliferation rates were detected in conjunction with an activation of PI3K/Akt signaling and the mTOR-pathway. Additionally, inhibition of glycogen-synthase-kinase3-β led to stabilization of β-catenin and Snail, which resulted in a switch from predominant E-cadherin to N-cadherin expression and increased expression of the stem cell markers Oct3/4, Sox2, and Nanog. Enhanced biosynthesis and enzyme activity of matrix metalloproteinase-9 was accompanied by elevated invasion behavior. Finally, anoikis resistance was detected in stimulated keratinocytes by decreased apoptosis of nonadherent cells and elevated expression of epidermal growth factor receptor and c-Met. Hence, Porphyromonas gingivalis is able to induce a more aggressive tumor-like phenotype in immortalized oral keratinocytes, thus contributing to enhanced tumor features.

The biophysics and mechanics of blood from a materials perspective

Cells actively interact with their microenvironment, constantly sensing and modulating biochemical and biophysical signals. Blood comprises a variety of non-adherent cells that interact with each other and with endothelial and vascular smooth muscle cells of the blood vessel walls. Blood cells are further experiencing a range of external forces by the hemodynamic environment and they also exert forces to remodel their local environment. Therefore, the biophysics and material properties of blood cells and blood play an important role in determining blood behaviour in health and disease. In this Review, we discuss blood cells and tissues from a materials perspective, considering the mechanical properties and biophysics of individual blood cells and endothelial cells as well as blood cell collectives. We highlight how blood vessels provide a mechanosensitive barrier between blood and tissues and how changes in vessel stiffness and flow shear stress can be correlated to plaque formation and exploited for the design of vascular grafts. We discuss the effect of the properties of fibrin on blood clotting, and investigate how forces exerted by platelets are correlated to disease. Finally, we hypothesize that blood and vascular cells are constantly establishing a mechanical homeostasis, which, when imbalanced, can lead to hematologic and vascular diseases.

CADM1 is a TWIST1-regulated suppressor of invasion and survival

Metastatic cancer remains a clinical challenge; however, patients diagnosed prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion steps within the metastatic cascade, but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome of melanoma cells. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Increased expression of CADM1 resulted in significant inhibition of motility and invasiveness of melanoma cells. In addition, elevated CADM1 elicited caspase-independent cell death in non-adherent conditions. Expression array analysis suggests that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Importantly, tissue microarray analysis and clinical data from TCGA indicate that CADM1 expression is inversely associated with melanoma progression and positively correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be considered a biomarker for favorable prognosis.

Characterisation of multipotent stem cells from human peripheral blood using an improved protocol

BackgroundA promising approach of bone repair is to use stem cells, such as mesenchymal stem cells (MSCs). Seeking available source of MSCs still remains a great challenge in tissue engineering and cell therapy. Peripheral blood (PB) emerges as an alternative source of MSCs which can be easily acquired with minimal invasiveness. This study was undertaken to evaluate the multipotency of PB-MSCs and effects of human PB-MSCs transplantation on ectopic bone regeneration in nude mice. MethodsHuman venous blood collected was mixed with heparin and then red blood cells were removed using red blood cell lysis buffer. Cell suspension was cultured in normoxia-culture and hypoxia-culture conditions, respectively. The non-adherent cells were removed by half changing culture media every three days. Cells were selected due to plastic adherence. The adherent cells were then passaged and subjected to multi-differentiation induction assays in vitro and in vivo ectopic bone formation assay. ResultsCharacterization assays indicated that cells cultured under hypoxia possessed potent multi-lineage differentiation capacity and expressed Nanog and Lgr5, as well as a series of MSC surface antigens (including CD29, CD90, CD105, and CD73). Additionally, regenerated bone tissues by transplantation of human PB-MSCs in vivo were confirmed by histological examinations of ectopic osteogenesis assay. A purified population of MSCs can be obtained within a short period of time using this protocol with a successful rate of 60%. ConclusionWe reported an effective and reliable method to harvest highly purified MSCs with potent multi-differentiation potential from human peripheral blood. Lgr5 may be a potential biomarker for identification of a subpopulation of PB-MSCs. The translational potential of this articlePB-MSCs is an alternative cell source for cell therapy, which may be harvested, culture expanded and PB-MSCs loaded with β-tricalcium phosphate (β-TCP) may be used to promote bone repair.

Regulation of cell migration by α4 and α9 integrins.

Integrins are heterodimeric transmembrane receptors that play an essential role in enabling cells to sense and bind to extracellular ligands. Activation and clustering of integrins leads to the formation of focal adhesions at the plasma membrane that subsequently initiate signalling pathways to control a broad range of functional endpoints including cell migration, proliferation and survival. The α4 and α9 integrins form a small sub-family of receptors that share some specific ligands and binding partners. Although relatively poorly studied compared with other integrin family members, emerging evidence suggests that despite restricted cell and tissue expression profiles, these integrins play a key role in the regulation of signalling pathways controlling cytoskeletal remodelling and migration in both adherent and non-adherent cell types. This review summarises the known shared and specific roles for α4 and α9 integrins and highlights the importance of these receptors in controlling cell migration within both homeostatic and disease settings.

Natural killer activity of the spleen cells of Ehrlich tumor-bearing mice treated with Copaifera multijuga extract

AIMS: Copaifera multijuga Hayne oleoresin is commonly used in traditional medicine owing to its anti-inflammatory, antiseptic, antitumor, and antibacterial properties. However, little is known about the effect of the compounds from the bark of this plant. In this study, the immunomodulatory effect of the ethanolic extract of C. multijuga bark via natural killer activity of non-adherent spleen cells of Ehrlich tumor-bearing mice was evaluated.METHODS: Male Swiss mice were inoculated subcutaneously with 1×106 Ehrlich tumor cells (Ehrlich and Ehrlich/C. multijuga group) or phosphate buffered saline solution (control group and C. multijuga group) and treated orally daily with C. multijuga extract (200 mg kg-1, 0.1 mL per mouse, for the Ehrlich/C. multijuga and C. multijuga groups) or phosphate buffered saline solution (control group and Ehrlich group). The four experimental groups consisted in eight mice each and were organized in two sets, one treated for seven days and another treated for 14 days, totalizing 64 mice throughout the experiment. Twenty-four hours after the last oral administration, the mice were euthanized and the spleen tissue was isolated to prepare a non-adherent spleen cell suspension and to evaluate natural killer activity. Data are presented as the cell lysis percentage of Yac.1 target cells by non-adherent spleen cells.RESULTS: Treatment for seven days increased natural killer activity in the Ehrlich/C. multijuga group (21.20±8.89, p<0.05) compared to the control group (3.14±2.71, p<0.05); however, this effect was not maintained in the groups treated for 14 days (Control: 6.02±6.98, Ehrlich: 4.82±7.72, C. multijuga: 2.07±2.10, Ehrlich/C. multijuga: 2.01±1.63, p>0.05).CONCLUSIONS: Treatment for seven days with an ethanolic extract of C. multijuga bark enhanced the natural killer activity of non-adherent spleen cells from Ehrlich tumor-bearing mice.

Phenotypic differences in early outgrowth angiogenic cells based on in vitro cultivation.

Bone marrow-derived early outgrowth cells play an important role in endothelial repair. In vitro isolation techniques have identified two distinct morphological early outgrowth cell populations, but it is still unknown whether they present some functional phenotypic differences. Accordingly, the aim of the present study was to determine whether there are phenotypic differences in cellular function between two putative early outgrowth cells in culture. Peripheral blood samples were collected from 18 healthy adults. Thereafter, mononuclear cells were isolated by Ficoll density-gradient centrifugation and plated on 6-well plates coated with human fibronectin. After 2 and 7days, respectively, non-adherent cells (NAC) and adherent cells (AC) underwent functional assays in order to measure the migratory capacity (Boyden chamber), angiogenic growth factor release (ELISA) and apoptosis (TUNEL). Migration to both VEGF (517 ± 74 vs. 273 ± 74AU) and SDF-1 (517 ± 68 vs. 232 ± 68AU) were approximately twofold higher (P < 0.05) in the NAC when compared to AC. Release of angiogenic factors, granulocyte colony-stimulating and hepatocyte growth factor, were not different between cell types. Apoptotic response to staurosporine was significantly lower in NAC (20 ± 32 vs. 125 ± 32%). In summary, NAC and AC demonstrated functional phenotypic differences in migratory capacity and apoptotic susceptibility, which makes it difficult to compare these two early outgrowth cell populations in literature.

Microfluidic platforms for cell cultures and investigations

This review covers several aspects of microfluidic devices used for culturing and monitoring of both adherent and non-adherent cells, including a multitude of applications. A comparison of available platforms with high throughput analysis, automation capability, interface to sensors and integration, is reported. Aspects, such as operational versatility of the devices, are scrutinized in terms of their analytical efficacy. It is found that due to multi-functionality capability of modern microfluidics, there is big amount of experimental data obtainable from a single device, allowing complex experimental control and efficient data correlation, particularly important when biomedical studies are considered. Hence several examples on cell culture and monitoring are given in this review, including details on design of microfluidic devices with their distinctive technological peculiarities.

Optical sensor revealed abnormal nuclease spatial activity on cancer cell membrane.

Nucleases are important enzymes that cleave nucleic acids and play critical roles in DNA repair, immune defense and potentially in cancer invasion. However, their spatial dynamics at subcellular level is much less studied. Here, we developed a surface‐tethered nuclease sensor (SNS) which directly converts membrane‐bound nuclease (MN) activity to fluorescent signal, therefore, mapping MN activity on cell adhesion sites with high resolution and sensitivity. With SNS, we studied MN activity on the ventral membrane of cancer cells, where MN activity initially occurs in punctate regions and advances in a coral‐shaped pattern. In six tested cell‐lines, the MN activity levels in cancer cells are significantly higher than those in non‐cancer cells. We then tested SNS as a sensitive approach to detect cancer cells at single cell level. Single breast cancer cells were successfully detected from thousands of adherent non‐cancer cells and from millions of non‐adherent blood cells.

Reversible and Photoresponsive Immobilization of Nonadherent Cells by Spiropyran-Conjugated PEG-Lipids.

Spatiotemporal control of cell-material interactions contributes to our understanding of cell biology and the development of cell engineering. Here, we first report the reversible and spatio-selective immobilization of nonadherent cells through the use of photoswitchable polymeric materials. The substrate coated with spiropyran-conjugated poly(ethylene glycol) (PEG) lipids, which bind to cell membranes via the lipid moiety only in their merocyanine form, enabled rapid cell immobilization and release in an on-off manner by irradiation with ultraviolet and visible light, respectively. Our work has the potential to improve the performance of cell manipulations on chips and to enable rapid cell arrangement/sorting on various surfaces.

A microfluidic platform utilizing anchored water-in-oil-in-water double emulsions to create a niche for analyzing single non-adherent cells.

Non-adherent cells play key roles in various biological processes. Studies on this type of cell, especially at single-cell resolution, help reveal molecular mechanisms underlying many biological and pathological processes. The emerging microfluidics technology has developed effective methods for analyzing cells. However, it remains challenging to treat and monitor single live non-adherent cells in an in situ, long-term, and real-time manner. Herein, a microfluidic platform was set up to generate and anchor cell-laden water-in-oil-in-water (W/O/W) double emulsions (DEs) to investigate these cells. Within the device, W/O/W DEs encapsulating non-adherent cells were generated through two adjacent flow-focusing structures and subsequently anchored in an array of microchambers. These droplets maintained the W/O/W structure and the anchorage status in the continuous perfusion fluid for at least one week. The mass transfer of different molecules with suitable molecular weights and partition coefficients between the interior and exterior of W/O/W DEs could be regulated by perfusion fluid flow rates. These features endow this platform with potential to continuously supply encapsulated non-adherent cells with nutrients or small-molecule stimuli/drugs through fluid perfusion. Meanwhile, the confinement of cells in the anchored DEs favored long-term monitoring of cellular dynamic behaviors and responses. As a proof of concept, fluorescein diacetate (FDA) was employed to visualize the cellular uptake and biochemical metabolism of TF-1 human erythroleukemia cells. We believe that this W/O/W DE anchorage and perfusion platform would benefit single-cell-level studies as well as small-molecule drug discovery requiring live non-adherent cells.

Quantification of Mouse Hematopoietic Progenitors' Formation Using Time-lapse Microscopy and Image Analysis.

In vitro differentiation of mouse embryonic stem cells (mESCs) towards blood cells constitutes a well-established system to study the endothelial-to-hematopoietic transition (EHT) at the onset of blood development. Assessing the emergence of small non-adherent round blood cells in the culture without disturbing it is essential to evaluate the progression of EHT and also to test conditions potentially enhancing or repressing this process. Here, we describe how to quantify the formation of mouse hematopoietic progenitors during EHT in normal conditions or following over-expression of eight essential transcription factors using time-lapse microscopy and image analysis.

CHARACTERISTICS OF CULTURES OF CELLS OBTAINED FROM HUMAN LIMB

Limbal epithelial stem cells are localized in the limb, which determine the regeneration of the corneal epithelium, both physiological and reparative. The aim of the study is to characterize the culture of cells isolated by different methods. Materials and methods. Limbal stem cells were isolated from the limbus region of enucleated human eyes. Cell isolation was produced by the enzymatic and explantation methods. Cell cultivation was performed on the amniotic membrane and without it, in DMEM/F12 growth media (Dullbecco’s Modified Eagle Medium/Nutrient F12 Ham) and CECBM (Corneal Epithelial Cell Basal Medium). The morphological assessment and immunophenotyping of cell cultures were performed. Results and its discussion. The obtained culture was distinguished by cell heterogeneity at the enzymatic method of isolating cells from the limbus. The presence of fibroblast-like cells was revealed in the suspension of non-adherent cells, which resembled stromal cells in appearance and growth pattern. Immunophenotyping revealed that most cells carry markers of multipotent mesenchymal stromal cells (CD90+/CD73+/CD105+/CD34–/CD45–) and a small number – markers of epithelial cells (p63α+, ABCG2 +, CK19+). Explant culture was more uniform. The cells had a rounded, epithelial-like shape, adhered well to the surface of the amniotic membrane and grew on it. The results of phenotyping indicated that cells are represented mainly by epithelial cells (p63α+, ABCG2 +, CK19+). Conclusion . The culture is represented mainly by MMSC at the enzymatic method of isolating cells from the limbal graft and cultivating them without feeder in the growth medium DMEM/F12. Isolation of limb cells by the explantation method and their cultivation in the selective medium CECBM allow us to obtain mainly the culture of epithelial stem cells.

Microfluidic device for expedited tumor growth towards drug evaluation.

Patient derived organoids have emerged as robust preclinical models for screening anti-cancer therapeutics. Current 2D culturing methods do not provide physiological responses to therapeutics, therefore 3D models are being developed to better reproduce physiological responses. 3D culturing however often requires large initial cell populations and one week to one month to grow tumors ready for therapeutic testing. As a solution a 3D culturing system has been developed capable of producing physiologically relevant tumors in an expedited fashion while only requiring a small number of initial cancer cells. A bi-layer microfluidic system capable of facilitating active convective nutrient supply to populations of cancer cells facilitates expedited growth of cancer cells when starting with populations as small as 8 cells. The system has been shown to function well with adherent and non-adherent cell types by expediting cell growth by a factor ranging from 1.27 to 4.76 greater than growth under static conditions. Utilizing such an approach has enable to formation of tumors ready for therapeutic screening within 3 days and the ability to perform therapeutic screening within the microfluidic system is demonstrated. A mathematical model has been developed which allows for adjustments to be made to the dynamic delivery of nutrients in order to efficiently use culture media without excessive waste. We believe this work to be the first attempt to grow cancers in an expedited fashion utilizing only a convective nutrient supply within a microfluidic system which also facilitates on-device therapeutic screening. The developed microfluidic system and cancer growth method have the potential to offer improved drug screening for patients in clinical settings.

Photo-responsive materials with strong cell trapping ability for light-guided manipulation of nonadherent cells.

We report a photo-cleavable material for tight trapping of nonadherent cells to substrate surfaces. Model immunocytes were selectively trapped in a non-irradiated area as single cells after the projection of a light pattern and withstood high-speed laminar flow, achieving light-guided cell release from the substrates.

A single-cell micro-trench platform for automatic monitoring of cell division and apoptosis after chemotherapeutic drug administration

Cells vary in their dynamic response to external stimuli, due to stochastic fluctuations and non-uniform progression through the cell cycle. Hence, single-cell studies are required to reveal the range of heterogeneity in their responses to defined perturbations, which provides detailed insight into signaling processes. Here, we present a time-lapse study using arrays of micro-trenches to monitor the timing of cell division and apoptosis in non-adherent cells at the single-cell level. By employing automated cell tracking and division detection, we precisely determine cell cycle duration and sister-cell correlations for hundreds of individual cells in parallel. As a model application we study the response of leukemia cells to the chemostatic drug vincristine as a function of cell cycle phase. The time-to-death after drug addition is found to depend both on drug concentration and cell cycle phase. The resulting timing and dose-response distributions were reproduced in control experiments using synchronized cell populations. Interestingly, in non-synchronized cells, the time-to-death intervals for sister cells appear to be correlated. Our study demonstrates the practical benefits of micro-trench arrays as a platform for high-throughput, single-cell time-lapse studies on cell cycle dependence, correlations and cell fate decisions in general.

Effect of Radiation on self-renewality of prostate cancer stem cells.

Introduction: CSCs have been identified in prostate cancer (PCa), one of the most diagnosed malignancies in men over the world, for which radiation resistance is a major problem in the treatment of advanced stages. Cancer stem cells (CSCs) have the ability to self-renew and differentiate to give rise to heterogeneous phenotype of the tumor cells. It is believed that CSCs are involved in metastasis, recurrence and therapy resistance in various cancers. Recently CSCs have been identified in prostate cancer. In vitro one of the main properties used to enriched cancer stem cells is their capacity to form spheres in non-adherent culture conditions. Materials and Methods: Human prostate carcinoma cell line (DU145) were plated in serum-free suspension culture system allowed for tumorsphere forming that enriched cancer stem cells. To identify prostate CSCs in adherent cells and nonadherent sphere cells of prostate cancer cell lines, the expression of the CD133 and CD44 cell-surface markers were assessed by flowcytometry. To evaluate of different doses of radiation (electron 6 MeV) on the self-renewal and proliferation of the Du145, sphere-formation assay (SFA) and plating efficiency (colony formation assay (CFA)) were assessed. The results were comparing with the parental cells. SFA is based on the self-renewing properties unique to CSCs. CFA is used as a measure of the proliferative potential of cells. Results: The results show that both SF (CFA) and SF (SFA) declined with the increase of radiation dose in cells treated as monolayer or sphere culture. However, the extent of reduction in cells treated as monolayer culture was significantly higher compared to cells treated as spheroids, suggesting higher resistance of spheroids to radiation. According to these results, tumorspheres from PCa showed more radioresistant than bulk of tumor. Conclusion: Due to the relative resistance of CSCs to radiotherapy, determination and characterization of radioresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.

BST-2 promotes survival in circulation and pulmonary metastatic seeding of breast cancer cells

Bone marrow stromal antigen 2 (BST-2) mediates various facets of cancer progression and metastasis. Here, we show that BST-2 is linked to poor survival in invasive breast cancer patients as its expression positively correlates with disease severity. However, the mechanisms that drive the pro‐metastatic functions of BST-2 are not fully understood. Correlation of BST-2 expression and tumor aggressiveness was analyzed in human tissue samples. Migration, invasion, and competitive experimental metastasis assays were used to measure the cellular responses after silencing BST-2 expression. Using a mouse model of breast cancer, we show that BST-2 promotes metastasis independent of the primary tumor. Additional experiments show that suppression of BST-2 renders non-adherent cancer cells non-viable by sensitizing cells to anoikis. Embedment of cancer cells in basement membrane matrix reveals that silencing BTS-2 expression inhibits invadopodia formation, extracellular matrix degradation, and subsequent cell invasion. Competitive experimental pulmonary metastasis shows that silencing BST-2 reduces the numbers of viable circulating tumor cells (CTCs) and decreases the efficiency of lung colonization. Our data define a previously unknown function for BST-2 in the i) formation of invadopodia, ii) degradation of extracellular matrix, and iii) protection of CTCs from hemodynamic stress. We believe that physical (tractional forces) and biochemical (ECM type/composition) cues may control BST-2’s role in cell survival and invadopodia formation. Collectively, our findings highlight BST-2 as a key factor that allows cancer cells to invade, survive in circulation, and at the metastatic site.