Published OnlineFirst September 18, 2017; DOI: 10.1158/0008-5472.CAN-16-3279 Cancer Integrated Systems and Technologies Research Mitosis-Mediated Intravasation in a Tissue- Engineered Tumor–Microvessel Platform Andrew D. Wong1,2 and Peter C. Searson1,2,3 Abstract Intravasation involves the migration of tumor cells across the cell interactions with functional microvessels and provide evi- local endothelium and escape into vessel flow. Although tumor dence for a mitosis-mediated mechanism where tumor cells cell invasiveness has been correlated to increased intravasation, located along the vessel periphery are able to disrupt the vessel the details of transendothelial migration and detachment into endothelium through cell division and detach into circulation. circulation are still unclear. Here, we analyzed the intravasation of This model provides a framework for understanding the physical invasive human breast cancer cells within a tissue-engineered and biological parameters of the tumor microenvironment microvessel model of the tumor microenvironment. Using live- that mediate intravasation of tumor cells across an intact endo- cell fluorescence microscopy, we captured 2,330 hours of tumor thelium. Cancer Res; 77(22); 1–9. Ó2017 AACR. Introduction without the assistance of TAMs (5, 9, 10). These studies suggest that there are multiple pathways for intravasation, but the lack of Metastasis involves a sequence of steps including invasion, sufficient resolution has hampered our understanding of the intravasation, circulation, arrest, and extravasation that ultimately mechanism of tumor cell transendothelial migration (TEM) and result in tumor cell dormancy and/or growth at a secondary site detachment into circulation. (1). Intravasation, the entry of tumor cells into circulation, is a Recent advances in the development of in vitro microvessel critical step that occurs at both primary and secondary tumor sites models provide the tools to recreate the essential components of (2). The discovery of circulating tumor cells years after primary the tumor microenvironment and enable visualization of the treatment reveals that tumor cells continue to spread through the details of the metastatic cascade (11–13). Here we set out to vasculature and is a negative prognostic indication for cancer investigate the mechanism of intravasation of BCCs and to relapse (2, 3). Reducing intravasation may ultimately improve address the question, how do tumor cells cross endothelial junc- patient outcome; however, the details of these events are difficult tions to enter circulation? Using live-cell imaging in a tissue- to resolve, as it is a dynamic process that occurs at the interface engineered microvessel model of the tumor microenvironment, between tumor cells and the local tumor microvasculature. we analyzed over 2,330 hours of tumor cell interactions with Our understanding of the dynamics of intravasation is derived functional microvessels and found that intravasation events were largely from a relatively small number of intravital microscopy rare but predominately associated with mitosis. We quantified the (IVM) studies and few direct observations at the single cell level. deflection of peripheral tumor cells on the vessel endothelium For example, intravasation has been investigated in zebrafish and and provide evidence for a model where mitotic single-cell round- chick embryos (4, 5), but only directly observed in mouse models ing exerts a force on the endothelium that is sufficiently large to (6, 7). From these studies, we have learned that tumor associated transiently open endothelial cell–cell junctions and expose the macrophages (TAM) enhance the invasion of breast cancer cells tumor cells to shear flow, which pulls the daughter cells into (BCC) towards vessels through EGF and CSF1 chemotaxis (6, 8) circulation. To confirm that this is the dominant mechanism of and local expression of VEGF causes downregulation of endo- intravasation, we showed that tumor cells that extended protru- thelial cell–cell junctions, thus increasing the intravasation fre- sions across the interface did not intravasate. Similarly, tumor quency. However, tumor cells oversecreting growth factors are cells dividing in a larger perivascular space were unable to deflect also capable of intravasation across multiple in vivo systems the vessel endothelium and intravasate. These results demonstrate a simple, yet effective mechanism by which single tumor cells may undergo intravasation and provide a framework for understand- 1Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, Mary- land. 2Department of Materials Science and Engineering, Johns Hopkins Uni- ing the physical and biological parameters that enable intravasa- versity, Baltimore, Maryland. 3Department of Oncology, Johns Hopkins Univer- tion through this pathway. sity School of Medicine, Baltimore, Maryland. Note: Supplementary data for this article are available at Cancer Research Materials and Methods Online (http://cancerres.aacrjournals.org/). Device fabrication Corresponding Author: Peter C. Searson, Johns Hopkins University, 100 Croft The tumor-microvessel platform was fabricated as described Hall, 3400 North Charles Street, Baltimore, MD 21218. Phone: 410-516-8774; previously (13). Briefly, high concentration rat tail collagen type I Fax: 410-516-5293; E-mail: [email protected] (Corning Inc.) is diluted to 7 mg/mL and neutralized with the doi: 10.1158/0008-5472.CAN-16-3279 manufacturer's recommended amounts of DI water, 10Â PBS, Ó2017 American Association for Cancer Research. and 1 N sodium hydroxide. After neutralization, tumor cells are www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 18, 2017; DOI: 10.1158/0008-5472.CAN-16-3279 Wong and Searson introduced into the collagen solution to a final concentration of donkey anti-mouse IgG (H þ L) antibody conjugated to Alexa 5 Â 105 cells/mL and injected around a cylindrical template rod Fluor 647 (A31571; Thermo Fisher Scientific) was incubated for (diameter 150 mm) within the polydimethyl siloxane (PDMS) 1 hour. Cells were washed for 10 minutes, three times between housing of the platform (Supplementary Fig. S1). After collagen fixing and incubation steps with PBS. gelation at room temperature, the rod is removed, leaving behind a cylindrical channel within the collagen gel. The channel is Image analysis fi m subsequently coated with bronectin (50 g/mL) to promote Intravasating tumor cells located at the ECM–vessel interface endothelial adhesion and spreading. Endothelial cells in along the middle of the vessel (i.e., vessel equator) were manually suspension are introduced into the channel at a concentration traced in ImageJ (NIH, Bethesda, MD) at each time-lapse frame. Â 6 of 5 10 cells/mL and allowed to settle and actively adhere to the Tumor cell circularity (i.e., shape factor, 4ÁpÁarea/perimeter2) was channel walls. After the endothelial cells have spread for about calculated as well as the maximum cell width perpendicular to the 2 hours, normal growth media (NGM) is perfused through the vessel wall. The latter was obtained by measuring the widths along < 2 vessel at a low applied shear stress ( 1 dyne/cm ) overnight. the entire length of tumor cell perpendicular to the vessel wall and fl Devices were typically con uent after 1 day and were switched to taking the maximum value with a custom written ImageJ plugin 2 higher shear stress ( 4 dyne/cm ) conditions for at least 24 hours (program provided in Supplementary Materials). These values before live-cell imaging. were plotted versus time. Cell motility descriptors (e.g., persis- tence, RMS speed, and directedness) were measured for tumor Cell lines and culture conditions cells located at the interface (n ¼ 38 cells continuously tracked for Human umbilical vein endothelial cells (HUVEC; Promocell), 12–17 hours from 4 independent microvessels) as well as in the human dermal microvascular endothelial cells (HMVEC; Lonza), bulk ECM (n ¼ 35 cells continuously tracked for 12–18 hours and VeraVec HUVEC-TURBOGFP (HVERA-GFP; Catalog. No. from 7 independent microvessels). Cell locations were approxi- HVERA-UMB-202100; Angiocrine Bioscience) were seeded in the mated in ImageJ from time-lapse images by manually tracing the cylindrical channel of the microvessel platform. Endothelial cells nucleus of each tumor cell every hour. Only data from tumor cells were grown in MCDB 131 (Caisson Labs) supplemented with tracked for a minimum of 12 hours were used, which reduced the 10% heat inactivated FBS (Sigma), 25 mg/mL endothelial mito- variance of each cell's average RMS speed, persistence, and direct- gen (BT-203, Biomedical Technologies), 2 U/mL herparin edness. Persistence is the shortest length connecting the start-to- m (Sigma), 1 g/mL hydrocortisone (Sigma), 0.2 mmol/L ascorbic end distance divided by the total path length acquired over the – – acid 2-phosphate (Sigma), and 1% penicillin streptomycin glu- entire duration of cell tracking. An average RMS speed for each tamine (Life Technologies). Dual-labeled MDA-MB-231 BCCs tumor cell was obtained by averaging the displacement over time (AntiCancer Inc.) were embedded within the collagen type I ECM for each one hour segment of a tumor cell's migration; for around the microvessel (14). Cancer cells were grown in RPMI example, a tumor cell tracked for 12 hours will exhibit 12 – (Corning Inc.) supplemented with 10% FBS and 1% penicillin individual