Mechanisms of triple-negative breast cancer extravasation: Impact of the physical environment and endothelial glycocalyx.

Cancer metastasis is the leading cause of death for those afflicted with cancer. In cancer metastasis, the cancer cells break off from the primary tumor, penetrate nearby blood vessels, and attach and extravasate out of the vessels to form secondary tumors at distant organs. This makes extravasation a critical step of the metastatic cascade. Herein, with a focus on triple-negative breast cancer, the role that the prospective secondary tumor microenvironment's mechanical properties play in circulating tumor cells' extravasation is reviewed. Specifically, the effects of the physically regulated vascular endothelial glycocalyx barrier element, vascular flow factors, and subendothelial extracellular matrix mechanical properties on cancer cell extravasation are examined. The ultimate goal of this review is to clarify the physical mechanisms that drive triple-negative breast cancer extravasation, as these mechanisms may be potential new targets for anti-metastasis therapy.

Interaction of Drug-Sensitive and -Resistant Human Melanoma Cells with HUVEC Cells: A Label-Free Cell-Based Impedance Study.

Cancer cell extravasation is a crucial step in cancer metastasis. However, many of the mechanisms involved in this process are only now being elucidated. Thus, in the present study we analysed the trans-endothelial invasion of melanoma cells by a high throughput label-free cell impedance assay applied to transwell chamber invasion assay. This technique monitors and quantifies in real-time the invasion of endothelial cells by malignant tumour cells, for a long time, avoiding artefacts due to preparation of the end point measurements. Results obtained by impedance analysis were compared with endpoint measurements. In this study, we used human melanoma M14 wild type (WT) cells and their drug resistant counterparts, M14 multidrug resistant (ADR) melanoma cells, selected by prolonged exposure to doxorubicin (DOX). Tumour cells were co-cultured with monolayers of human umbilical vein endothelial cells (HUVEC). Results herein reported demonstrated that: (i) the trans-endothelial migration of resistant melanoma cells was faster than sensitive ones; (ii) the endothelial cells appeared to be strongly affected by the transmigration of melanoma cells which showed the ability to degrade their cytoplasm; (iii) resistant cells preferentially adopted the transcellular invasion vs. the paracellular one; (iv) the endothelial damage mediated by tumour metalloproteinases seemed to be reversible.

Endothelium and Subendothelial Matrix Mechanics Modulate Cancer Cell Transendothelial Migration.

Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push-pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.

Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22.

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Circulating extracellular vesicles and tumor cells: sticky partners in metastasis.

The intravascular behavior of tumor-derived extracellular vesicles (EVs) and circulating tumor cells (CTCs) lies at the heart of the metastatic cascade. Their capacity to disseminate and stop at specific vascular regions precedes and determines the formation of metastatic foci. We discuss in detail the central role of cellular adhesion molecules (CAMs) that are present on EV/CTC surface, as well as their endothelial ligands, in dictating their arrest site and their capacity to exit the vasculature. We focus on the differences and similarities between CAMs on CTCs and EVs, and speculate about their role in the organotropism of different cancer types. Better understanding of the binding mechanisms might pinpoint potential targets for novel therapies.

Lectin Staining of Microvascular Glycocalyx in Microfluidic Cancer Cell Extravasation Assays.

The endothelial glycocalyx forms the inner-most lining of human microvasculature. It ensures the physiological function of blood vessels and plays a crucial role in the occurrence and progression of microvascular diseases. The present communication aims to highlight the usefulness of high-resolution imaging of lectin (Bandeiraea Simplicifolia) stained endothelial glycocalyx in 3-dimensional microfluidic cell cultures. The microfluidic system allowed visualizing cancer cell extravasation, which is a key event in metastasis formation in cancer pathologies. In brief, microvascular networks were created through spontaneous vasculogenesis. This occurred from 3 dimensional (3D) suspensions of human umbilical vein endothelial cells (HUVECs) in hydrogels confined within microfluidic devices. Extravasation of MDA-MB-231 breast cancer cells from perfusable endothelial lumens was observed with confocal imaging of lectin-stained microvascular networks. The present work provides guidance towards optimizing the methodology used to elucidate the role of the endothelial glycocalyx during cancer cell extravasation. In particular, a high-resolution view of the endothelial glycocalyx at the site of extravasation is presented. The occurrence of glycocalyx defects is well aligned with the contemporary notion in the field that glycocalyx shedding precedes cancer cell extravasation.

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.

Visualizing cancer extravasation: from mechanistic studies to drug development.

Metastasis is a multistep process that accounts for the majority of cancer-related death. By the end of metastasize dissemination, circulating tumor cells (CTC) need to extravasate the blood vessels at metastatic sites to form new colonization. Although cancer cell extravasation is a crucial step in cancer metastasis, it has not been successfully targeted by current anti-metastasis strategies due to the lack of a thorough understanding of the molecular mechanisms that regulate this process. This review focuses on recent progress in cancer extravasation visualization techniques, including the development of both in vitro and in vivo cancer extravasation models, that shed light on the underlying mechanisms. Specifically, multiple cancer extravasation stages, such as the adhesion to the endothelium and transendothelial migration, are successfully probed using these technologies. Moreover, the roles of different cell adhesive molecules, chemokines, and growth factors, as well as the mechanical factors in these stages are well illustrated. Deeper understandings of cancer extravasation mechanisms offer us new opportunities to escalate the discovery of anti-extravasation drugs and therapies and improve the prognosis of cancer patients.

The effects of monocytes on tumor cell extravasation in a 3D vascularized microfluidic model.

Metastasis is the leading cause of cancer-related deaths. Recent developments in cancer immunotherapy have shown exciting therapeutic promise for metastatic patients. While most therapies target T cells, other immune cells, such as monocytes, hold great promise for therapeutic intervention. In our study, we provide primary evidence of direct engagement between human monocytes and tumor cells in a 3D vascularized microfluidic model. We first characterize the novel application of our model to investigate and visualize at high resolution the evolution of monocytes as they migrate from the intravascular to the extravascular micro-environment. We also demonstrate their differentiation into macrophages in our all-human model. Our model replicates physiological differences between different monocyte subsets. In particular, we report that inflammatory, but not patrolling, monocytes rely on actomyosin based motility. Finally, we exploit this platform to study the effect of monocytes, at different stages of their life cycle, on cancer cell extravasation. Our data demonstrates that monocytes can directly reduce cancer cell extravasation in a non-contact dependent manner. In contrast, we see little effect of monocytes on cancer cell extravasation once monocytes transmigrate through the vasculature and are macrophage-like. Taken together, our study brings novel insight into the role of monocytes in cancer cell extravasation, which is an important step in the metastatic cascade. These findings establish our microfluidic platform as a powerful tool to investigate the characteristics and function of monocytes and monocyte-derived macrophages in normal and diseased states. We propose that monocyte-cancer cell interactions could be targeted to potentiate the anti-metastatic effect we observe in vitro, possibly expanding the milieu of immunotherapies available to tame metastasis.

A combined microfluidic-transcriptomic approach to characterize the extravasation potential of cancer cells.

The reciprocal interaction between circulating tumor cells (CTCs) and tissue-specific cells is influential for the progression of metastases. In particular, the process of extravasation relies on the complex cross-talk between cancer cells and other cellular players such as the endothelium and the secondary tissue. However, most in vitro studies only focus on one heterotypic cell-cell interaction and often lack of physiological relevance. In this project, we investigated both CTC-endothelium and CTC-secondary site interactions during cancer cell extravasation. We first used a microarray analysis of extravasated MDA-MB-231 breast cancer cells to identify key markers involved in extravasation. Then, we developed a tri-culture microfluidic platform combining cancer cells, endothelium and a bone-mimicking (BMi) microenvironment to assess how organ tropism influences the extravasation potential of cancer cells from different tissues. Through the microarray analyses of extravasated cancer cells we found that extravasation is associated with upregulation of late-metastatic markers along with specific proteases, such as matrix metalloprotease (MMP), a-disintegrin and metalloprotease (ADAM) and a-disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family members, which are all involved in endothelium glycocalyx shedding. Through the microfluidic extravasation assay, we found that the bone-like microenvironment increased invasion and motility of breast, bladder and ovarian cancer cell (MDA-MB-231, T24 and OVCAR-3). Among the three cell types, ovarian cancer cells presented the lowest migration rate and bladder cancer cells the highest, hence recapitulating their different level of bone tropism observed in vivo. Taken together, our results shed light on the importance of intercellular communication between CTCs and other non-tumor cells essential for promoting cancer cell extravasation.

Vimentin intermediate filament and plectin provide a scaffold for invadopodia, facilitating cancer cell invasion and extravasation for metastasis.

To investigate the molecular mechanisms of cancer metastasis, we have isolated a high-metastatic bladder cancer cell subpopulation from a low-metastatic cell line by using an in vivo selection system. Cells in the subpopulation showed a high ability to form invadopodia, the filamentous actin (F-actin)-based membrane protrusions that play an essential role in cancer cell invasion. Analysis of the gene expression profile revealed that the expression of an intermediate filament (IF) protein, vimentin and a cytoskeletal linker protein, plectin was up-regulated in the high-metastatic subpopulation compared with the low metastatic cell line. Here we report a novel role of vimentin IF and plectin in metastasis. In invasive bladder cancer cells, the vimentin IF-plectin-invadopodia F-actin link was formed. Disruption of this link severely impaired invadopodia formation, reducing the capacities of extracellular matrix degradation, transendothelial migration and metastasis. In addition, the vimentin assembly into the filaments was required for invadopodia formation. Our results suggest that plectin anchoring invadopodia to vimentin IF scaffolds and stabilizes invadopodia, which is a critical molecular process for cancer cell invasion and extravasation for metastasis.