Metastasis of Circulating Tumor Cells: Speed Matters

Abstract

The mechanisms involved in tumor cell extravasation during metastasis remain incompletely understood. In this issue of Developmental Cell, Follain and colleagues (2018) demonstrate that blood flow velocity is an important regulator of circulating tumor cell exit from the bloodstream.

To metastasize to distant organ sites, cancer cells take advantage of a natural network of transport: the vascular system. However, entrance to and exit from the circulation is not simple. Seeding of metastasis requires the acquisition of a specific set of skills that allows tumor cells to detach from the primary tumor, intravasate into adjacent blood vessels, survive in the bloodstream, adhere, and finally transmigrate across the vascular wall to gain access to new sites (Chambers et al., 2002; Strilic and Offermanns, 2017). Because prevention of metastasis is critical to limit cancer progression, understanding how this process works has become highly relevant in order to seek opportunities for therapeutic intervention.

The question of why certain cancers preferentially spread to specific organs— the “seed and soil” hypothesis— has been under experimental scrutiny for years. There is concordance that the anatomical routes of the vascular system play a role in metastatic spreading, because preferred sites for a given cancer type often include the first capillary bed downstream of the primary tumor. Examples include metastasis of colon cancer cells to the liver and of breast cancer cells to the lungs, where the initial arrest of tumor cells may be caused by physical restriction of small-diameter capillaries (Chambers et al., 2002; Labelle and Hynes, 2012). However, this does not explain why some breast cancers also metastasize to the brain, for example. More recently, the phenomenon of organ tropism driven by the development of a “pre-metastatic niche” has gained impetus. The pre-metastatic niche infers that even prior to gaining access to the circulation, the primary tumor modifies the microenvironment in distant organs to ensure its survival and expansion through metastasis. The primary tumor prepares the future “soil” by secreting soluble factors and by shedding extracellular vesicles that further modify the microenvironment (Psaila and Lyden, 2009). Vascular leakiness is the earliest event in the preparation of the pre-metastatic niche, followed by the education of local resident cells, such as fibroblasts, and the recruitment of non-resident cells, like bone-marrow derived cells. The outcome is the survival and thriving of circulating tumor cells (CTCs) upon exiting the bloodstream (Peinado et al., 2017). In this issue of Developmental Cell, Follain and colleagues (2018) highlight a new critical factor that can affect the success rate and site of tumor cell extravasation: vascular flow patterns.

By using in vitro, in vivo, and in silico techniques, Follain and colleagues (2018) demonstrated that blood flow rate influences arrest, adhesion, and extravasation of CTCs. The authors comprehensively followed sites of tumor cell extravasation in zebrafish embryos expressing EGFP in the endothelium (Tg(Fli1a:EGFP)). High-resolution imaging and flow manipulations allowed them to conclude that the majority of tumor cells become arrested at arterio-venous junctions, a region of relatively low blood flow velocity. The relevance of flow has been unclear until now, and although high flow appears to be unproductive for extravasation, the authors find that some level of flow is needed. Why is it that flow is required? Do tumor cells bind to the endothelium under a preferential flow rate? And if so, what is the relevance of flow for such interaction? To answer these questions, the authors used a microfluidic device associated with optical tweezers. They established that 80pN was the force needed to detach tumor cells from an endothelial monolayer, proving that proper flow rates allowed stable adhesion between CTCs and endothelial cells. This last point also implies that flow was required for adhesion. Subsequent measurements in vivo revealed that forces higher than 200pN were required to dislodge CTCs from endothelial cells. This increase in force was explained by the presence of fingerlike contacts between endothelial cells and CTCs, as quickly as 15 min post-injection. Surprisingly, pharmacological tuning of shear stress forces impacted the location of CTC arrest in the vasculature. When flow was increased, CTC arrest site was posterior to the arteriovenous junction. In contrast, reduced blood flow by lidocaine promoted CTC arrest in a region anterior to the arterio-venous junction. The experiments revealed that extravasation was flow-specific rather than site-specific (Figure 1).

Figure 1. Permissive Blood Flow Facilitates Arrest and Extravasation of CTCs.

Brain metastases are located in regions presenting lower blood flow rates, compatible with smaller vessel size (A). Low flow rate, found on vessels modulated by lidocaine, allows CTCs to arrest and adhere, but not to extravasate (B, upper vessel). Intermediate flow rates are required to permit endothelial remodeling (pocketing) and CTC extravasation beyond arrest and adhesion (B, lower vessel). High blood flow in larger vessels prevents cell arrest and subsequent extravasation (C).

The relevance of flow and shear forces in relation to leukocyte adhesion and extravasation has been previously documented and extensively studied (Ando and Yamamoto, 2009). Flow appears to augment the initial tethering of circulating leukocytes to a stationary surface, slow the velocity, and consequently increase the number of rolling adherent cells. Mechanisms for flow-enhanced leukocyte adhesion are mediated by a number of adhesion molecules such as selectins, lectins, ICAMs, and integrins expressed on the surface of both endothelial cells and leukocytes (Zhu et al., 2008). CTC adhesion to the endothelium shares some of the same molecular partners, including selectins, integrins, cadherins, and members of the immunoglobulin (Ig) superfamily of receptors (Lambert et al., 2017; Reymond et al., 2013).

To understand how tumor cells extravasate, the molecular crosstalk between the endothelium and CTCs must first be decoded. Follain et al. (2018) demonstrated that endothelial cells respond to flow by remodeling their membrane and extending cell processes that facilitate the extravasation of tumor cells. Live-cell imaging revealed that endothelial cells actively engulf single or clustered CTCs, enabling their transmigration across the vessel wall (Figure 1). The authors further found that reduction of flow forces drastically diminished the number of extravascular cells. Follain et al. (2018) reproduced this finding in vitro, observing that endothelial monolayers exposed to laminar flow of 400 μm/s, but not lower, displayed membrane processes that facilitated CTC transmigration.

Finally, the biological significance of the findings was tested in a mouse model of brain micrometastasis. Importantly, arrested CTCs were present in brain capillaries with reduced flow profiles. Interestingly, endothelial cells from vessels with arrested CTCs displayed membrane extensions that expanded into the lumen and were found wrapping around arrested tumor cells. Importantly, evaluation of a cohort of 100 patients with brain metastasis (n = 580) at the supratentorial region revealed that brain metastases preferentially developed in regions with lower cerebral blood flow. These findings further support the notion that blood flow rates are pivotal to metastatic extravasation.

The emphasized concept of flow forces in the context of cancer cell extravasation and distant hematogenous metastasis provides important insights for the metastasis field. Different flow profiles within and between organs might, at least in part, explain the phenomenon of organ tropism and heterogeneous distribution of distant metastasis for certain types of cancer. It would be of interest to explore whether distinct types of circulating tumor cells require different levels of shear for optimal adhesion and extravasation. The implications of flow and shear forces in organization of the pre-metastatic niche and in the context of distinct types of tumors are also important questions to pursue. Despite these questions, Follain and colleagues have certainly placed flow on the metastasis map.