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Feature Review Dissemination from a Solid Tumor: Examining the Multiple Parallel Pathways Moriah
E. Katt,1,2,4 Andrew D. Wong,1,2,4 and Peter C. Searson1,2,3,* Metastasis can be generalized as a linear sequence of events whereby halting one or more steps in the cascade may reduce tumor cell dissemination and ultimately improve patient outcomes. However, metastasis is a complex process with multiple parallel mechanisms of dissemination. Clinical strategies focus on removing the primary tumor and/or treating distant metastases through chemo- or immunotherapies. Successful strategies for blocking metastasis will need to address the parallel mechanisms of dissemination and identify common bottlenecks.
Here, we review the current understanding of common dissemination pathways for tumors. Understanding the complexities of metastasis will guide the design of new therapies that halt dissemination. Tumor Dissemination Dissemination of tumor cells to distant organs ultimately leads to most cancer-related deaths [1,2]. The steps in the metastatic cascade are often described in terms of a linear sequence of events involving a single tumor cell; however, accumulating evidence suggests that there are multiple parallel pathways for tumor cell dissemination and colonization (Figure 1, Key Figure) that depend on the local microenvironment and highlights the important roles of the endothelium and immune system (Box 1). Tumor cells are able to invade short distances, either individually or collectively, through the local microenvironment (e.g., tissue, lymphatic, or vascular interfaces), but generally gain widespread dissemination by intravasating into local microvessels and detaching when exposed to high shear flow. Tumor cells enter circulation as either single cells or aggregates (herein referred to as ‘microemboli’). The intravasation (see Glossary) of single tumor cells or microemboli is dependent on the tumor phenotype and the local microenvironment, which have key roles in determining the degree of endothelial activation and dysfunction. Intravasation can occur across intact, defective, or nonexistent endothelial barriers, and is dependent on vessel size and location in a tumor. After entering circulation, tumor cells can interact with blood components and other cell types [i.e., platelets, neutrophils, red blood cells (RBCs)] resulting in the formation of complex microemboli. The range of mechanisms for arrest, and the corresponding diversity in microenvironments, results in many possible pathways for extravasation and colonization at a secondary site. A consequence of the linear-sequence-of-events model is the widely held view that intervention at any one step can stop metastasis. By contrast, the multiple parallel pathways viewpoint underscores that intervening in the dissemination of tumor cells is challenging. Here, we evaluate the biological and physiological factors that control these pathways, and highlight the involvement of the vascular and immune systems in intravasation, circulation, and extravasation. We examine tumor cell circulation and extravasation in the context of microthrombi formation and the resulting impact on the growth and/or dormancy of a secondary tumor site. A Highlights The range of mechanisms for the dissemination of cells from a solid tumor, along with the corresponding diversity in microenvironments, results in multiple parallel pathways for colonization of a secondary site. Shedding from a solid tumor can occur as single cells or clusters, and occurs across intact, defective, or nonexistent endothelial barriers. Due to the heterogeneous nature of a tumor and the tumor vasculature, these processes may occur simultaneously at different locations within the tumor. Tumor cells in circulation can form clusters with platelets, neutrophils, and natural killer cells that modulate their survival. Extravasation can occur by occlusion of a circulating tumor cell (CTC) in a capillary bed, occlusion of tumorassociated thrombi in a capillary or microvessel, by capture and adhesion in a larger vessel, or by occlusion in defective vasculature. 1 Institute for Nanobiotechnology, 100 Croft Hall, Johns Hopkins University, Baltimore, MD 21218, USA 2 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA 3 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA 4 These authors contributed equally *Correspondence: searson@jhu.edu (P.C. Searson). 20 Trends in Cancer, January 2018, Vol. 4, No. 1 https://doi.org/10.1016/j.trecan.2017.12.002 © 2017 Elsevier Inc. All rights reserved. Glossary Circulating tumor cells (CTCs): single tumor cells or aggregates of fewer than three cells in circulation. Circulating tumor microemboli (CTM): clusters of three or more CTCs, often including other cell types, such as leukocytes and platelets, and proteins. Enhanced permeation and retention (EPR) effect: the defective or dysfunctional vasculature in a tumor results in increased permeability and preferential accumulation of a molecule or nanomedicine. Extravasation: exit of a tumor cell from circulation. The mechanism may include arrest, dissociation of tumor cells from CTCs or CTMs, transendothelial transport across the endothelium, or direct entry into tissue through defective vasculature. Intravasation: entry of a tumor cell into circulation (or lymphatic vessel). Mechanisms include transendothelial transport of a tumor cell across the endothelium into a blood vessel, and shedding of a tumor cell or cluster from a mosaic vessel. Mosaic vessels: vessels where the lumen is formed from endothelial cells and tumor cells. Such vessels may result from rapid growth of tumor neovasculature without sufficient proliferation to form a complete monolayer, or from the shedding or apoptosis of endothelial cells exposing underlying tumor cells. Neutrophil extracellular traps (NETs): adhesion and arrest of neutrophils in capillaries can result in a cell death pathway known as NETosis, resulting in the release of chromatin and granular material. These neutrophil extracellular traps can cause obstruction or ‘plugging’ of the capillary. Tumor microenvironment for metastasis (TMEM): often defined as a site where both a tumor cell and TAMs are in contact with the endothelium. Vascular mimicry: the formation of channels lined with tumor cells that mimic blood vessels and supply nutrients to a growing tumor. Key Figure Multiple Parallel Pathways for Cancer Cell Dissemination from a Primary Tumor Tumor Vessel disrupƟon ParƟal or no endothelium Occlusion Thrombus? MigraƟon along vasculature Platelets, NFs, NETs CTMs (CTC clusters) AggregaƟon fragmentaƟon Shedding TEM / vessel disrupƟon IntravasaƟon CirculaƟon ExtravasaƟon Invasion into Ɵssue Dormancy Single CTCs Intact vessel TEM TEM Endothelial acƟvaƟon Transient adhesion Rolling adhesion Perivascular entry TEM Invasion/growth Invasion/growth Shedding Intravascular growth? Extravascular growth? 
Figure 1. Key steps in the metastatic cascade (intravasation, circulation, and extravasation) often exhibit parallel routes. Characteristics of the tumor vasculature and the role of the immune system dictate the mechanism of dissemination and the transition of tumor cells from different microenvironments and states. Cancer cells at the primary site may encounter intact or partially lined vasculature, which may affect the mode of intravasation (i.e., single cells or microemboli). Microemboli exhibit shorter half-lives in circulation than single tumor cells and interact to a larger degree with blood constituents. These interactions determine the mode of arrest in downstream vessels (i.e., rolling adhesion or vessel occlusion) and organs, and ultimately impact extravasation and possibly dormancy and/or extravascular growth. Supporting clinical and preclinical evidence for the transition of tumor cells between states is represented by the broken or solid lines, respectively. Abbreviations: CTC, circulating tumor cell; CTM, circulating tumor microemboli; NET, neutrophil extracellular trap; NF, neutrophil; TEM, transendothelial migration. Trends in Cancer, January 2018, Vol. 4, No. 1 21 broader perspective on the multiple steps in the metastatic cascade will provide a framework for developing strategies that decrease metastatic efficiency and extend the time before cancer relapse. Intravasation Intravasation is one of the first steps in the metastatic cascade and involves tumor cell migration from tissue across and into an endothelial or epithelial vessel [1,2] (Box 2). Factors that contribute to the rate of intravasation include the phenotype of the tumor cell, the local state of the endothelium, and the local microenvironment [3]. Tumor vessels exhibit vast heterogeneity in size, permeability, and endothelial coverage [4,5]. Physical parameters, such as vessel diameter and shear forces, may have a role in the frequency or success of tumor cell escape into vascular flow [6]. The existence of an intact endothelium separating tumor tissue from vascular flow necessitates transendothelial migration (TEM) or vessel disruption before intravasation; however, an incomplete endothelial lining may directly expose tumor cells to vessel flow. The magnitude of vascular shear stress and intercellular and/or focal adhesion also regulate the release of individual tumor cells or microemboli. Both single tumor cells and microemboli have been discovered in circulation; however, the physical and biological factors regulating their entry into circulation remain poorly understood [6]. Although the immune system is known to attack the tumor microenvironment, certain immune cells have been shown to directly enhance intravasation through activation of the tumor vasculature [7]. Here, we explore the role of the tumor vasculature in dictating the parallel pathways of tumor cell intravasation. Heterogeneous Tumor Vasculature: Intact, Partial, or Nonexistent Endothelium Tumor vasculature exhibits a spectrum of barrier properties, ranging from functional to leaky and/or to nonexistent
Figura 1. Os principais passos da cascata metastática (intravasamento, circulação e extravasamento) freqüentemente exibem rotas paralelas. As características da vasculatura tumoral e o papel do sistema imune determinam o mecanismo de disseminação e a transição de células tumorais de diferentes microambientes e estados. As células cancerosas no local primário podem encontrar vasculatura intacta ou parcialmente revestida, o que pode afetar o modo de intravasamento (isto é, células únicas ou microembolias). Os microembolios exibem meias-vidas mais curtas em circulação do que as células de tumor único e interagem em maior grau com os constituintes do sangue. Essas interações determinam o modo de parada nos vasos a jusante (ou seja, adesão ou oclusão de vasos rolantes) e órgãos e, por fim, afetam o extravasamento e, possivelmente, a dormência e / ou o crescimento extravascular. A evidência clínica e pré-clínica de suporte para a transição de células tumorais entre estados é representada pelas linhas quebradas ou sólidas, respectivamente. Abreviaturas: CTC, células tumorais circulantes; CTM, microembolia tumoral circulante; NET, armadilha extracelular de neutrófilos; NF, neutrófilo; TEM, migração transendotelial. Tendências em Câncer, janeiro de 2018, vol. Uma perspectiva mais ampla das múltiplas etapas da cascata metastática fornecerá uma estrutura para o desenvolvimento de estratégias que diminuam a eficiência metastática e aumentem o tempo antes da recaída do câncer. Intravascular O intravasamento é um dos primeiros passos na cascata metastática e envolve a migração de células tumorais do tecido através e para dentro de um vaso endotelial ou epitelial [1,2] (Quadro 2). Fatores que contribuem para a taxa de intravasamento incluem o fenótipo da célula tumoral, o estado local do endotélio e o microambiente local [3]. Vasos tumorais exibem grande heterogeneidade em tamanho, permeabilidade, e cobertura endotelial [4,5]. Parâmetros físicos, como diâmetro do vaso e forças de cisalhamento, podem ter um papel na frequência ou sucesso do escape de células tumorais no fluxo vascular [6]. A existência de um endotélio intacto que separa o tecido tumoral do fluxo vascular requer a migração transendotelial (TEM) ou a ruptura do vaso antes do intravasamento; no entanto, um revestimento endotelial incompleto pode expor diretamente as células tumorais ao fluxo do vaso. A magnitude do estresse de cisalhamento vascular e a adesão intercelular e / ou focal também regulam a liberação de células tumorais individuais ou microembolias. Tanto células tumorais individuais como microembolias foram descobertas em circulação; no entanto, os fatores físicos e biológicos que regulam sua entrada em circulação permanecem pouco compreendidos [6]. Embora o sistema imunológico seja conhecido por atacar o microambiente tumoral, Certas células do sistema imunológico demonstraram melhorar diretamente o intravasamento por meio da ativação da vasculatura do tumor [7]. Aqui, nós exploramos o papel da vasculatura tumoral em ditar as vias paralelas da intravasão de células tumorais. Vasculatura heterogênea do tumor: A vasculatura do tumor endotelial intacto, parcial ou inexistente exibe um espectro de propriedades de barreira, variando de funcional a vazado e / ou inexistente
(Figure 2). In solid tumors, vessel functionality varies based on location Box 1. The Metastatic Cascade Models Linear Sequence of Events Model A single tumor cell undergoes a sequence of steps resulting in the formation of a secondary tumor: invasion ! intravasation ! circulation ! extravasation ! colonization. Blocking any step can reduce the probability of metastasis. Multiple Parallel Pathways Model Multiple pathways exist for the intravasation of tumor cells at intact, defective, or nonexistent endothelial barriers (mosaic vessels) depending on the location within a solid tumor. Circulating tumor cells (CTCs) or circulating tumor microemboli (CTMs) modulate cell survival by association with platelets, neutrophils, natural killer cells, and other soluble factors in circulation Multiple mechanisms exist for arrest and extravasation at multiple sites throughout the body Intervention strategies should consider the wide range of dissemination mechanisms and the corresponding diversity in microenvironments Box 2. Intravasation & Extravasation Intravasation Associated with endothelium activation and/or downregulation of cell-cell junctions The tumor vasculature within a solid tumor is spatially heterogeneous with intact, defective, or non-existent endothelial barriers, all of which can support different mechanisms of intravasation Extravasation Arrest in circulation can occur by occlusion of circulating tumor cells (CTCs) in capillary beds, circulating tumor microemboli (CTMs) in capillary beds or small vessels, or by capture and adhesion of CTCs or CTMs in larger vessels. Occlusion can lead to a successful metastatic event, hypoxia, and tissue necrosis, formation of neutrophil extracellular traps (NETs), and patient death from a thromboembolic event. Occlusion by CTMs is difficult to distinguish from venous thromboembolism. The complex microenvironments established around arrested CTCs and CTMs result in multiple pathways for extravasation at intact (e.g., transendothelial migration), defective endothelium, and nonexistent (e.g., vessel cooption) endothelium. 22 Trends in Cancer, January 2018, Vol. 4, No. 1 within the tumor. For example, replicas (i.e., corrosion casts) of tumor vasculature from human colorectal cancer show that the intervessel spacing, tortuosity, and diameter become more aberrant from the periphery to the core of the tumor [5]. Tumor vasculature is typically less hierarchical than normal vasculature and often exhibits loops, dead-ends, shunts, and other defects [5]. Although normal vasculature also exhibits large functional heterogeneity, ranging from gaps and fenestrae in the liver sinuses to tight junctions of the blood–brain barrier [4], these characteristics are tailored to the functional role of distinct organs, whereas tumor vasculature is leaky due to rapidly and irregularly formed architecture that lacks lymphatic drainage, resulting in dysregulated endothelium with reduced barrier function [8]. Tumor core Tumor Periphery Monocyte infiltraƟon TEM Single cell intravasaƟon Escape Cluster intravasaƟon Endothelial disrupƟon Vascular invasion Detachment CTM release Vasculogenic mimicry Mosaic vessels EC barrier downregulaƟon Figure 2. Tumor Vessel Heterogeneity Influences Intravasation. Vascular integrity and functionality varies from the tumor core to its periphery. An intact endothelium, often observed at the tumor periphery, necessitates the disruption of endothelial cell (EC)–cell junctions before intravasation, possibly through activation by tumor-associated macrophages (TAMs) and/or the transendothelial migration (TEM) of tumor cells. Partially lined (i.e., mosaic vessels) or disrupted vasculature directly exposes tumor cells to shear forces and facilitates vascular invasion and the release of tumor microemboli that may enter systemic circulation (circulating tumor microemboli; CTM) or occlude the primary tumor vasculature depending on cluster size and vessel diameter. Functional tumor vasculature that lacks an endothelium but is lined by endothelial-like tumor cells, known as vasculogenic mimicry, may also contribute to tumor progression by further enabling nutrient transport and conduits for intravasation. Trends in Cancer, January 2018, Vol. 4, No. 1 23