Extravasation Of Breast Cancer Cells Research Articles

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling.

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also showthat brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

Invasion/chemotaxis- and extravasation-chip models for breast cancer bone metastasis.

Bone is one of the most frequently targeted organs in metastatic cancers including the breast. Breast cancer bone metastasis often results in devastating outcomes as limited treatment options are currently available. Therefore, innovative methods are needed to provide earlier detection and thus better treatment and prognosis. Here, we present a new approach to model bone-like microenvironments to detect invasion and extravasation of breast cancer cells using invasion/chemotaxis (IC-) and extravasation (EX-) chips, respectively. Our results show that the behaviors of MDA-MB-231 breast cancer cells on IC- and EX-chip models correlate with their in vivo metastatic potential. Our culture model constitutes cell lines representing osteoblasts, bone marrow stromal cells, and monocytes embedded in three-dimensional (3D) collagen I-based extracellular matrices of varying composition and stiffness. We show that collagen I offers a better bone-like environment for bone cells and matrix composition and stiffness regulate the invasion of breast cancer cells. Using in situ contactless rheological measurements under cell culture conditions, we show that the presence of cells increased the stiffness values of the matrices up to 1200 Pa when monitored for five days. This suggests that the cellular composition has a significant effect on regulating matrix mechanical properties, which in turn contribute to the invasiveness. The platforms we present here enable the investigation of the underlying molecular mechanisms in breast cancer bone metastasis and provide the groundwork of developing preclinical tools for the prediction of bone metastasis risk.

Macrophages Promote Tumor Cell Extravasation across an Endothelial Barrier through Thin Membranous Connections.

Macrophages are important players involved in the progression of breast cancer, including in seeding the metastatic niche. However, the mechanism by which macrophages in the lung parenchyma interact with tumor cells in the vasculature to promote tumor cell extravasation at metastatic sites is not clear. To mimic macrophage-driven tumor cell extravasation, we used an in vitro assay (eTEM) in which an endothelial monolayer and a matrigel-coated filter separated tumor cells and macrophages from each other. The presence of macrophages promoted tumor cell extravasation, while macrophage conditioned media was insufficient to stimulate tumor cell extravasation in vitro. This finding is consistent with a requirement for direct contact between macrophages and tumor cells. We observed the presence of Thin Membranous Connections (TMCs) resembling similar structures formed between macrophages and tumor cells called tunneling nanotubes, which we previously demonstrated to be important in tumor cell invasion in vitro and in vivo. To determine if TMCs are important for tumor cell extravasation, we used macrophages with reduced levels of endogenous M-Sec (TNFAIP2), which causes a defect in tunneling nanotube formation. As predicted, these macrophages showed reduced macrophage-tumor cell TMCs. In both, human and murine breast cancer cell lines, there was also a concomitant reduction in tumor cell extravasation in vitro when co-cultured with M-Sec deficient macrophages compared to control macrophages. We also detected TMCs formed between macrophages and tumor cells through the endothelial layer in the eTEM assay. Furthermore, tumor cells were more frequently found in pores under the endothelium that contain macrophage protrusions. To determine the role of macrophage-tumor cell TMCs in vivo, we generated an M-Sec deficient mouse. Using an in vivo model of experimental metastasis, we detected a significant reduction in the number of metastatic lesions in M-Sec deficient mice compared to wild type mice. There was no difference in the size of the metastases, consistent with a defect specific to tumor cell extravasation and not metastatic outgrowth. Additionally, with an examination of time-lapse intravital-imaging (IVI) data sets of breast cancer cell extravasation in the lungs, we could detect the presence of TMCs between extravascular macrophages and vascular tumor cells. Overall, our data indicate that macrophage TMCs play an important role in promoting the extravasation of circulating tumor cells in the lungs.

Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial–mesenchymal transition of breast cancer cells

Ubiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative splicing of USP19 results in the expression of two major encoded variants that are localized to the endoplasmic reticulum (ER) (USP19-ER) and cytoplasm (USP19-CY). The importance of alternative splicing for the function of USP19 remains unclear. Here, we demonstrated that USP19-CY promotes TGF-β signaling by directly interacting with TGF-β type I receptor (TβRI) and protecting it from degradation at the plasma membrane. In contrast, USP19-ER binds to and sequesters TβRI in the ER. By decreasing cell surface TβRI levels, USP19-ER inhibits TGF-β/SMAD signaling in a deubiquitination-independent manner. Moreover, USP19-ER inhibits TGF-β-induced epithelial–mesenchymal transition (EMT), whereas USP19-CY enhances EMT, as well as the migration and extravasation of breast cancer cells. Furthermore, USP19-CY expression is correlated with poor prognosis and is higher in breast cancer tissues than in adjacent normal tissues. Notably, the splicing modulator herboxidiene inhibits USP19-CY, increases USP19-ER expression and suppresses breast cancer cell migration. Targeting USP19 splicing or its deubiquitinating activity may have potential therapeutic effects on breast cancer.

Polarized NHE1 and SWELL1 regulate migration direction, efficiency and metastasis

Cell migration regulates diverse (patho)physiological processes, including cancer metastasis. According to the Osmotic Engine Model, polarization of NHE1 at the leading edge of confined cells facilitates water uptake, cell protrusion and motility. The physiological relevance of the Osmotic Engine Model and the identity of molecules mediating cell rear shrinkage remain elusive. Here, we demonstrate that NHE1 and SWELL1 preferentially polarize at the cell leading and trailing edges, respectively, mediate cell volume regulation, cell dissemination from spheroids and confined migration. SWELL1 polarization confers migration direction and efficiency, as predicted mathematically and determined experimentally via optogenetic spatiotemporal regulation. Optogenetic RhoA activation at the cell front triggers SWELL1 re-distribution and migration direction reversal in SWELL1-expressing, but not SWELL1-knockdown, cells. Efficient cell reversal also requires Cdc42, which controls NHE1 repolarization. Dual NHE1/SWELL1 knockdown inhibits breast cancer cell extravasation and metastasis in vivo, thereby illustrating the physiological significance of the Osmotic Engine Model.

Mechanism of Ginsenoside Rh1 in Regulating Breast Cancer Cell Extravasation Based on CCL20-CCR6

Objective: To explore the regulatory mechanism of ginsenoside Rh1 on breast cancer cell extravasation based on CCL20-CCR6. Methods: In 2021, a total of 34 patients with breast cancer were treated in Baoding First Central Hospital. During hospitalization, pathological examinations were performed, and all the patients were diagnosed with invasive ductal carcinoma. Out of the 34 cases, 16 cases were found to have CCR6 expression in breast cancer tissues, in which they were recorded as the CCR6 expression group, whereas 18 cases did not have CCR6 expression; these cases were recorded as the CCR6 non-expression group. During the same period, 21 normal patients were selected as the control group. The peripheral blood CCL20 level and the expression of CCR6 on the surface of CD3+ T lymphocytes were analyzed. The extracts of cancer cells were collected, purified, and cultured, and the effect of ginsenoside Rh1 on the invasion and metastasis of breast cancer cells was analyzed. Results: The peripheral blood CCL20 level in the CCR6 expression group was significantly higher than that in the CCR6 non-expression group and the control group, in which p < 0.05, indicating that the difference was statistically significant; at 12, 24, and 48 hours, the cell survival rate of each dose group was significantly higher than that of the blank control group and the dimethyl sulfoxide (DMSO) group (p < 0.05). At 48 hours, comparing the low-dose group with the high-dose group, the cell survival rate significantly decreased (p < 0.05). Compared with the blank control group and DMSO group, the invasion ability of breast cancer cells could be reduced in both, high- and medium-dose groups, where p < 0.05, indicating that the difference was statistically significant. Conclusion: CCL20 may play a role in the pathogenesis of certain breast cancers, and ginsenoside Rh1 can effectively regulate the invasion and migration of breast cancer cells.

Biomimetic Microfluidic Platforms for the Assessment of Breast Cancer Metastasis.

Of around half a million women dying of breast cancer each year, more than 90% die due to metastasis. Models necessary to understand the metastatic process, particularly breast cancer cell extravasation and colonization, are currently limited and urgently needed to develop therapeutic interventions necessary to prevent breast cancer metastasis. Microfluidic approaches aim to reconstitute functional units of organs that cannot be modeled easily in traditional cell culture or animal studies by reproducing vascular networks and parenchyma on a chip in a three-dimensional, physiologically relevant in vitro system. In recent years, microfluidics models utilizing innovative biomaterials and micro-engineering technologies have shown great potential in our effort of mechanistic understanding of the breast cancer metastasis cascade by providing 3D constructs that can mimic in vivo cellular microenvironment and the ability to visualize and monitor cellular interactions in real-time. In this review, we will provide readers with a detailed discussion on the application of the most up-to-date, state-of-the-art microfluidics-based breast cancer models, with a special focus on their application in the engineering approaches to recapitulate the metastasis process, including invasion, intravasation, extravasation, breast cancer metastasis organotropism, and metastasis niche formation.

Elucidating cancer-vascular paracrine signaling using a human organotypic breast cancer cell extravasation model

In cancer metastasis, extravasation refers to the process where tumor cells exit the bloodstream by crossing the endothelium and invade the surrounding tissue. Tumor cells engage in complex crosstalk with other active players such as the endothelium leading to changes in functional behavior that exert pro-extravasation effects. Most in vitro studies to date have only focused on the independent effects of molecular targets on the functional changes of cancer cell extravasation behavior. However, singular targets cannot combat complex interactions involved in tumor cell extravasation that affects multiple cell types and signaling pathways. In this study, we employ an organotypic microfluidic model of human vasculature to investigate the independent and combined role of multiple upregulated secreted factors resulting from cancer-vascular interactions during cancer cell extravasation. The device consists of a tubular endothelial vessel generated from induced pluripotent stem cell derived endothelial cells within a collagen-fibrinogen matrix with breast cancer cells injected through and cultured along the lumen of the vessel. Our system identified cancer-vascular crosstalk, involving invasive breast cancer cells, that results in increased levels of secreted IL-6, IL-8, and MMP-3. Our model also showed that upregulation of these secreted factors correlates with invasive/metastatic potential of breast cancer cells. We also used therapeutic inhibitors to assess the independent and combined role of multiple signaling factors on the overall changes in functional behavior of both the cancer cells and the endothelium that promote extravasation. Taken together, these results demonstrate the potential of our organotypic model in elucidating mechanisms through which cancer-vascular interactions can promote extravasation, and in conducting functional assessment of therapeutic drugs that prevent extravasation in cancer metastasis.

Correction: A microphysiological early metastatic niche on a chip reveals how heterotypic cell interactions and inhibition of integrin subunit β3 impact breast cancer cell extravasation.

Correction for 'A microphysiological early metastatic niche on a chip reveals how heterotypic cell interactions and inhibition of integrin subunit β3 impact breast cancer cell extravasation' by Martina Crippa et al., Lab Chip, 2021, DOI: .

A microphysiological early metastatic niche on a chip reveals how heterotypic cell interactions and inhibition of integrin subunit β3 impact breast cancer cell extravasation.

During metastatic progression multiple players establish competitive mechanisms, whereby cancer cells (CCs) are exposed to both pro- and anti-metastatic stimuli. The early metastatic niche (EMN) is a transient microenvironment which forms in the circulation during CC dissemination. EMN is characterized by the crosstalk among CCs, platelets, leukocytes and endothelial cells (ECs), increasing CC ability to extravasate and colonize secondary tissues. To better understand this complex crosstalk, we designed a human "EMN-on-a-chip" which involves the presence of blood cells as compared to standard metastases-on-chip models, hence providing a microenvironment more similar to the in vivo situation. We showed that CC transendothelial migration (TEM) was significantly increased in the presence of neutrophils and platelets in the EMN-on-a-chip compared to CC alone. Moreover, exploiting the EMN-on-chip in combination with multi-culture experiments, we showed that platelets increased the expression of epithelial to mesenchymal transition (EMT) markers in CCs and that the addition of a clinically approved antiplatelet drug (eptifibatide, inhibiting integrin β3) impaired platelet aggregation and decreased CC expression of EMT markers. Inhibition of integrin β3 in the co-culture system modulated the activation of the Src-FAK-VE-cadherin signaling axis and partially restored the architecture of inter-endothelial junctions by limiting VE-cadherinY658 phosphorylation and its nuclear localization. These observations correlate with the decreased CC TEM observed in the presence of integrin β3 inhibitor. Our EMN-on-a-chip can be easily implemented for drug repurposing studies and to investigate new candidate molecules counteracting CC extravasation.

Designed nanomolar small-molecule inhibitors of Ena/VASP EVH1 interaction impair invasion and extravasation of breast cancer cells

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor ([Formula: see text] Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein-protein interaction involved in actin filament processing and cell migration.

BSCI-01. ACTIVATION OF c-Met/β1-INTEGRIN COMPLEX RESULTS IN INCREASE OF MESENCHYMAL GENE EXPRESSION AND STEM CELL POPULATION IN METASTATIC BREAST CANCER TO THE BRAIN AND SPINE

INTRODUCTION: C-met and β-integrins play a central role in nearly all stages of cancer metastasis. They bind at the cell surface, driving ligand independent co-activation of downstream pathways. Greater complex is seen in metastatic tumors vs. its primary tumor counterparts in patients. The molecular, cellular, and clinical effects of complex formation in metastatic breast cancer are investigated. METHODS: Utilizing variations of the MDA-231 breast cancer cell lines (standard MDA-231, inducible complex formation MDA-231, brain seeking MDA 231, lung seeking MDA 231, and bone seeking MDA-231), in vitro and in vivo studies were performed. Clinical correlates from patient samples were studied. RESULTS: Induction of c-Met/β1 complex promotes breast cancer invasion (p< 0.001), migration (p< 0.05), circulation intravasation (p< 0.01), and adhesion (p< 0.01). These effects may be driven by the increased mesenchymal character (p< 0.05) and larger stem cell population (p< 0.001) caused by inducing c-Met/β1 complex formation. OS2966 (a therapeutic β1 integrin blocking antibody) decreases invasion (p< 0.05), intravasation (p< 0.05), and mesenchymal form factor (p< 0.001) and gene expression (p< 0.001) in MDA-MB-231 cells. Brain- and bone-seeking breast cancer cells have higher c-Met/β1 complex than parental controls and preferentially adhere to tissue-specific matrix (p< 0.01). In intracardiac metastasis models, complex formation resulted in significantly higher metastatic burden and shorter survival times (p< 0.001). qPCR data suggests that complex formation may drive exiting and colonization of cancer cells (micrometastasis) rather than tumor growth. Patient brain and bone metastases demonstrated high β1/c-Met levels. CONCLUSIONS: The c-Met/β1 complex drives intravasation and extravasation of breast cancer cells from the circulation. Preferential affinity for tissue-specific matrix enables the c-Met/β1 complex to drive formation of breast cancer metastases to the brain and bone. Pharmacological and genetic targeting of the complex with agents may provide therapeutic approaches to prevent metastases, particularly to the brain and bone.

Nanoparticles promote in vivo breast cancer cell intravasation and extravasation by inducing endothelial leakiness.

While most cancer nanomedicine is designed to eliminate cancer, the nanomaterial per se can lead to the formation of micrometre-sized gaps in the blood vessel endothelial walls. Nanomaterials-induced endothelial leakiness (NanoEL) might favour intravasation of surviving cancer cells into the surrounding vasculature and subsequently extravasation, accelerating metastasis. Here, we show that nanoparticles induce endothelial leakiness through disruption of the VE-cadherin-VE-cadherin homophilic interactions at the adherens junction. We show that intravenously injected titanium dioxide, silica and gold nanoparticles significantly accelerate both intravasation and extravasation of breast cancer cells in animal models, increasing the extent of existing metastasis and promoting the appearance of new metastatic sites. Our results add to the understanding of the behaviour of nanoparticles in complex biological systems. The potential for NanoEL needs to be taken into consideration when designing future nanomedicines, especially nanomedicine to treat cancer.

TRPV4 plays a role in breast cancer cell migration via Ca2+-dependent activation of AKT and downregulation of E-cadherin cell cortex protein

TRPV4 belongs to the ‘Transient Receptor Potential’ (TRP) superfamily. It has been identified to profoundly affect a variety of physiological processes, including nociception, heat sensation and inflammation. Unlike other TRP superfamily channels, its role in cancers are unknown until recently when we reported TRPV4 to be required for cancer cell softness that may promote breast cancer cell extravasation and metastasis. Here, we elucidated the molecular mechanisms mediated by TRPV4 in the metastatic breast cancer cells. TRPV4-mediated signaling was demonstrated to involve Ca2+-dependent activation of AKT and downregulation of E-cadherin expression, which was abolished upon TRPV4 silencing. Functionally, TRPV4-enhanced breast caner cell transendothelial migration requires AKT activity while a combination of transcriptional and post-translational regulation contributed to the TRPV4-mediated E-cadherin downregulation. Finally, mass spectrometry analysis revealed that TRPV4 is required for the expression of a network of secreted proteins involved in extracellular matrix remodeling. In conclusion, TRPV4 may regulate breast cancer metastasis by regulating cell softness through the Ca2+-dependent AKT-E-cadherin signaling axis and regulation of the expression of extracellular proteins.

LPP is a Src substrate required for invadopodia formation and efficient breast cancer lung metastasis

We have previously shown that lipoma preferred partner (LPP) mediates TGFβ-induced breast cancer cell migration and invasion. Herein, we demonstrate that diminished LPP expression reduces circulating tumour cell numbers, impairs cancer cell extravasation and diminishes lung metastasis. LPP localizes to invadopodia, along with Tks5/actin, at sites of matrix degradation and at the tips of extravasating breast cancer cells as revealed by intravital imaging of the chick chorioallantoic membrane (CAM). Invadopodia formation, breast cancer cell extravasation and metastasis require an intact LPP LIM domain and the ability of LPP to interact with α-actinin. Finally, we show that Src-mediated LPP phosphorylation at specific tyrosine residues (Y245/301/302) is critical for invadopodia formation, breast cancer cell invasion and metastasis. Together, these data define a previously unknown function for LPP in the formation of invadopodia and reveal a requirement for LPP in mediating the metastatic ability of breast cancer cells.

TRPV4 Regulates Breast Cancer Cell Extravasation, Stiffness and Actin Cortex.

Metastasis is a significant health issue. The standard mode of care is combination of chemotherapy and targeted therapeutics but the 5-year survival rate remains low. New/better drug targets that can improve outcomes of patients with metastatic disease are needed. Metastasis is a complex process, with each step conferred by a set of genetic aberrations. Mapping the molecular changes associated with metastasis improves our understanding of the etiology of this disease and contributes to the pipeline of targeted therapeutics. Here, phosphoproteomics of a xenograft-derived in vitro model comprising 4 isogenic cell lines with increasing metastatic potential implicated Transient Receptor Potential Vanilloid subtype 4 in breast cancer metastasis. TRPV4 mRNA levels in breast, gastric and ovarian cancers correlated with poor clinical outcomes, suggesting a wide role of TRPV4 in human epithelial cancers. TRPV4 was shown to be required for breast cancer cell invasion and transendothelial migration but not growth/proliferation. Knockdown of Trpv4 significantly reduced the number of metastatic nodules in mouse xenografts leaving the size unaffected. Overexpression of TRPV4 promoted breast cancer cell softness, blebbing, and actin reorganization. The findings provide new insights into the role of TRPV4 in cancer extravasation putatively by reducing cell rigidity through controlling the cytoskeleton at the cell cortex.

Abstract PR07: Dissection of cancer cells extravasation through human vascularized 3D microfluidic model: The major role of talin-1

Abstract Cancer cells spread from a primary tumor to secondary loci is responsible for more than 90% of cancer related mortality. Hematogenous metastasis is a complex process [1]. It includes a chain of events that can be summarized as follows: migration from primary tumor site and intravasation of the primary tumor cancer cells into the blood flow, dissemination through the circulation, extravasation in different organs, survival in the new microenvironment and colonization with generation of a new tumor. Recently our group presented a microfluidic 3D model reproducing the effects of the CXCL5-CXCR2 interaction between bone cells and metastatic breast cancer cells observed in vivo [2]. We further developed a human 3D microfluidic model that enables the study of human metastatic breast cancer cell extravasation within a perfusable human microvascularized bone-mimicking microenvironment [3]. Understanding the cellular and molecular events implicated in extravasation could facilitate the design of new therapeutic strategies targeting cancer cells only in order to couple these new developed therapies with already existing treatments and finally fight cancer. Talin-1 is a cytoplasmic adaptor essential for integrin-mediated adhesion to the ECM. Talin-1 links the actin cytoskeleton to integrin at the plasma membrane, regulates the focal adhesion pathway in normal cells and it is up-regulated in triple negative breast cancer cells such as MDA-MB231 and it is mutated in sarcomas. Based on the above described models, we developed a human vascularized 3D microfluidic device where human perfusable capillary like structures were embedded in fibrin matrix, characterized by mature endothelium markers (VE-cadherin, ZO-1) and physiological permeability (1.5±0.76)*10-6cm/s. Since these models can provide quantitative data on cancer cell extravasation, we focused on the role of Talin-1 in extravasation through blood vessels and in early invasion in two cell lines whose Talin-1 expression was silenced by SiRNA: triple negative metastatic breast cancer cells (MDA-MB231) and the metastatic fibro-sarcoma cell line (HT1080). Talin-1 silencing was confirmed by western blotting with specific antibodies. Adhesion to the endothelial wall, extravasation and ensuing migration out from the wall within the extracellular matrix was monitored by means of confocal high resolution real time imaging analyses. We demonstrated that silencing of Talin-1 expression dramatically and in a statistically significant way reduced both the adhesion efficiency and extravasation in both MDA-MB 231 and HT1080. Cell migration was also strongly and statistically reduced by the SiRNA treatment in both analyzed cell lines. In summary, we are the first to dissect the role of Talin-1 in each step of extravasation, demonstrating that targeting this protein could be an effective strategy to block metastasis. These data identify Talin-1 as a promising target for the development of new anti-metastatic therapies based on Talin-1 inhibition.

Abstract B165: Characterizing the role of macrophages in breast cancer cell extravasation in 3D microfluidic models

Abstract Metastasis is responsible for 90% of cancer-related deaths. It is particularly challenging to investigate because it is a complex process composed of several hidden steps that are difficult to resolve in vivo. Thus, a new generation of in vitro microfluidic models has recently been developed to study in greater detail individual metastatic steps, although few have been used to investigate the role of immune cells in cancer. In this project, we aim to develop a new microfluidic model for studying the role of macrophages in extravasation and recolonization, and to find underlying signaling pathways that could be blocked to prevent them from facilitating metastasis. Recently, metastasis associated macrophages (MAMs) have been identified in mouse studies as a specific subpopulation that increase extravasation and metastasis; we propose to exploit the high imaging capabilities of our microfluidic models combined with mouse studies to further investigate the underlying mechanisms. A previously characterized microfluidic device is used to generate microvascular networks (μVN) and replicate cancer cell extravasation in vitro. The device has 3 distinct but connected regions for containing hydrogels, all flanked by channels containing media. Briefly, endothelial cells (ECs; GFP-HUVECs) are seeded at 4×10^6/ml in fibrin (2.5 mg/ml) in the central gel region, while the two lateral gel regions are filled with fibroblast in fibrin (2.5 mg/ml) at 5×10^6/ml; following 4 days of culture in EGM media, a 3D perfusable μVN is formed. Fluorescently tagged MDA-MB-231 are perfused through the µVN and their transmigration through the vessel walls (i.e. extravasation) is imaged with a confocal microscope. To test whether macrophage increase extravasation and physically assist cancer cells during that process, immortalized bone marrow-derived macrophages (BMDM) isolated from C57BL/6 mice were directly mixed with GFP-HUVECs in the central gel region at a final concentration of 40,000/ml. BMDMs were found in the fibrin gel surrounding the microvessels, closely associated with endothelial cells. The diffusive permeability of the µVN was measured with fluorescent rhodamine dextran, and was found to be similar to values reported in vivo (2.21×10^-7 cm/s). 33.3± 20.4% of MDA-MB-231 extravasated within 6 hrs, as imaged on fixed devices stained with Phalloidin and DAPI. Extravasation was also quantified in real-time, and was shown to increase from 10% within 1 hr, to 75% after 12 hrs. Another co-culture system was used for different imaging capabilities, consisting of a monolayer of endothelial cells formed on top of a 2.5 mg/ml collagen gel seeded with or without RAW macrophages (400,000/ml) in a Matek dish. This system was used to assess if macrophages increase adhesion of cancer cells prior to extravasation. MDA-MB-231 were placed on the monolayer; 2 hrs later they were counted in three regions marked on the plate, and imaged with the confocal microscope before and after three PBS washes. Adhesion was found to be little affected by the presence of macrophages: 56±7 % adhered without RAW vs. 62±20 % in their presence (p=0.25; unpaired t-test). We also performed RT-PCR on HUVECs cultured in a transwell plate, in presence (or not) of RAW macrophages placed in a collagen gel, to test if macrophages upregulated adhesion molecules of ECs such as P-selectin, E-selectin, VCAM-1 or ICAM-1, but did not detect any significant difference. In conclusion, we successfully developed a 3D co-culture system with macrophages surrounding a network of perfusable microvessels for studying the role of macrophages in cancer cell extravasation. Our results suggest macrophages do not affect adhesion. Further studies will investigate their effect on extravasation and recolonization, and investigate pharmacological ways to block these processes in vitro and in vivo. Citation Format: Alexandra Boussommier, Roger D. Kamm. Characterizing the role of macrophages in breast cancer cell extravasation in 3D microfluidic models. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B165.

Abstract B23: Extravasation of breast cancer cells to a bone-cell conditioned microenvironment in functional 3D microvascular networks generated by vasculogenesis in a microfluidic system

Abstract Cancer cell metastases arise due to a series of biological processes starting from malignant tumor cells invading from primary to distal organs. Bone is one of the preferred sites of metastasis for various tumors including breast cancer. Understanding metastatic invasion to the secondary site requires studying the individual steps in metastasis, particularly those leading up to and immediately following extravasation of a circulating tumor cell from a blood vessel into the remote tissue. Microfluidic systems can provide cells with a controllable and reproducible 3D bone micro-environment to study cancer extravasation to a degree not possible by standard tissue culture methods while also simultaneously providing in situ imaging capabilities for visualization. We investigate the critical steps of cancer extravasation: tumor cells adhering to the endothelium and subsequently transmigrate across it into a hydrogel model for the bone extracellular space. Our model system consists of a functional microvascular network generated through vasculogenesis in a bone-mimicking microenvironment all within a microfluidic system. The microfluidic system contains a central hydrogel region flanked by two lateral media channels. The hydrogel region is filled with cells dispersed inside a fibrin gel. The two channel system enables easy access to the microvascular network for the addition of cancer cells for extravasation. Cells of passage 6 or lower MSCs, harvested from patients undergoing hip arthroplasty, were differentiated to osteo lineage. GFP-human umbilical vein endothelial cells (HUVECs) were suspended at 2·107 cells/ml in EMG2 + thrombin and combined with osteo-differentiated and non-differentiated MSCs mixed at a 9:1 ratio, and inserted into the gel channel to complete the seeding. RFP-expressing BOKL, which is an MDA-MB-231 cell line specific to bone metastasis enabled live-cell imaging via fluorescent microscopy. We created a functional microvascular network in a bone cell-conditioned microenvironment with the addition of osteo-differentiated and non-differentiated MSCs. Immunofluorescent imaging of α-smooth muscle actin in the system confirmed the presence of mural cells differentiated from MSCs wrapped around the generated vascular network. Visual confirmation of 3μm microspheres flowing through the vessels confirmed that the vascular network generated in this system was indeed perfusable. Finally, the osteo-cell conditioned microenvironment was confirmed by staining for osteocalcin. BOKL were then perfused into the microvasculature to model the extravasation process. The percentage of cancer cell extravasation in a device containing a microvascular network seeded with osteo-differentiated MSCs was 3.8 fold higher (56.5±4.8%) than the case with HUVECs alone (14.7±3.7%). Permeability values of the vasculature were analyzed by monitoring the leakage of fluorescent 70kDa dextran from the microvessels over time. Vasculature in the bone cell-conditioned microenvironment exhibited a higher permeability (4.12±0.75)·10-6 cm/s compared to the HUVEC only condition (0.89±0.31)·10-6 cm/s. While the increase in extravasation rates may due to a combination of factors, the higher permeability of the vasculature in osteo-cell conditioned environment may also be one of the contributing factors. Microfluidics offers the capability to create organ-specific microenvironments that can capture extravasation events, leading to tumor metastasis. This system therefore captures certain aspects of cell-cell and cell-matrix interactions for preferential metastasis to bone, offering potential for drug screening. Citation Format: Jessie S. Jeon, Simone Bersini, Gabriele Dubini, Joseph Charest, Matteo Moretti, Roger D. Kamm. Extravasation of breast cancer cells to a bone-cell conditioned microenvironment in functional 3D microvascular networks generated by vasculogenesis in a microfluidic system. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B23. doi:10.1158/1538-7445.CHTME14-B23

Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation

A key aspect of cancer metastases is the tendency for specific cancer cells to home to defined subsets of secondary organs. Despite these known tendencies, the underlying mechanisms remain poorly understood. Here we develop a microfluidic 3D in vitro model to analyze organ-specific human breast cancer cell extravasation into bone- and muscle-mimicking microenvironments through a microvascular network concentrically wrapped with mural cells. Extravasation rates and microvasculature permeabilities were significantly different in the bone-mimicking microenvironment compared with unconditioned or myoblast containing matrices. Blocking breast cancer cell A3 adenosine receptors resulted in higher extravasation rates of cancer cells into the myoblast-containing matrices compared with untreated cells, suggesting a role for adenosine in reducing extravasation. These results demonstrate the efficacy of our model as a drug screening platform and a promising tool to investigate specific molecular pathways involved in cancer biology, with potential applications to personalized medicine.