LeikeXie, Zhe Sun, Nicola J. Brown, Olga V. Glinskii, Gerald A. Meininger , Vladislav V. Glinsky
Abstract
Cancer cell adhesion to the endothelium is a crucial process in hematogenous metastasis, but how the integrity of the endothelial barrier and endothelial cell (EC) mechanical properties influence the adhesion between metastatic cancer cells and the endothelium remain unclear. In the present study, we have measured the adhesion between single cancer cells and two types of ECs at various growth states and their mechanical properties (elasticity) using atomic force microscopy single cell force spectroscopy. We demonstrated that the EC stiffness increased and adhesion with cancer cells decreased, as ECs grew from a single cell to a confluent state and developed cell-cell contacts, but this was reversed when confluent cells returned to a single state in a scratch assay. Our results suggest that the integrity of the endothelial barrier is an important factor in reducing the ability of the metastatic tumor cells to adhere to the vascular endothelium, extravasate and lodge in the vasculature of a distant organ where secondary metastatic tumors would develop.
Introduction
Vascular endothelial cells (ECs) that physiologically form a monolayer lining the interior of blood vessels serve as a dynamic barrier controlling cell and molecule movement into and out of the blood stream [1, 2]. Due to the location and function, the endothelial barrier plays an important role in hematogenous cancer metastasis [3]. Hematogenous cancer metastasis is a complex process involving several major steps, when tumor cells directly interact with ECs including blood borne metastatic tumor cell arrest in a distant organ vasculature and tumor cell extravasation out of the blood stream into the surrounding tissue where metastases may develop [4–7].
Materials and methods
Cell culture and preparations
The human bone marrow endothelial cell line HBMEC-60, kindly provided by Dr. C. E. van der Schoot (University of Amsterdam, Amsterdam, The Netherlands), has been described previously [33] and was maintained in Medium 200 (Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA, USA) and low-serum growth supplement (Invitrogen) in collagen-I coated Petri dishes (BD Bioscience) [23, 34]. Primary human pulmonary microvascular endothelial cells (HPMEC) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and cultured in Endothelial Cell Medium (ECM, ScienCell Research Laboratories) containing 5% FBS and endothelial cell growth supplement (ECGS) in fibronectin coated dishes as recommended by the company. HPMEC at passages 3–5 were used for experiments. Metastatic human triple negative breast cancer cell line MDA-MB-231 (MB231) was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and routinely grown in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS and L-glutamine. All cells were maintained as monolayer cultures in a humidified incubator (Heraeus Instruments, Newtown, CT) in 5% CO2 at 37°C.
Results
Endothelial cell elasticity changes with EC confluency (growth state)
The cortical stiffness of ECs was measured and compared for different degrees of confluence (growth state) using the AFM indentation approach (Fig 4). In HBMEC-60 cells, Young’s elastic modulus was 2.03 ± 0.21 kPa (n = 50) in NCF state, whereas in SCF (4.29 ± 0.33 kPa, n = 54) and CF (4.25 ± 0.57 kPa, n = 66) the elastic modulus was significantly increased by more than two-fold compared to the NCF (p <0.01 for both). The SCF and CF cells had similar values of Young’s moduli, with no significant difference between the two. Mgrt cells (1.75 ± 0.25 kPa) in scratch assay showed a slightly lower stiffness than the NCF cells, but there was no statistical difference between the two single cell states in culture.
Discussion
In this study, using atomic force single cell spectroscopy, we collected novel information regarding changes in elasticity of ECs as a function of cell confluence and dynamics of their adhesive interactions with tumor cells. We investigated mechanical properties (stiffness) in two types of ECs at different degrees of confluence. Specifically, we have detected Young’s moduli of 2.03 kPa in human bone marrow HBMEC-60 cells and 2.33 kPa in human pulmonary microvascular ECs HPMEC in NCF state, both of which are consistent with mean ranges of 2.0–6.67 kPa in ECs reported previously by others [24, 27, 29]. Since multiple factors such as cell type, environment, aging and disease state affect cell mechanical properties [30, 31, 38, 39], we also addressed the question of whether or not the state of confluence (growth status) is a factor influencing the cellular elastic properties. We found that EC stiffness significantly increased with confluence and the establishment of cell-cell contacts, but decreased as they transitioned to a single cell state in scratch assay (Fig 4). Cell stiffness is mainly determined by the state of the actin cytoskeleton, it is therefore dependent on activation state, ratio of actin polymerization/depolymerization, and stress fibers spatial organization and distribution [24, 27, 40]. Topographic and fluorescent imaging of cortical cytoskeleton networks revealed that the stress fibers in SCF and CF ECs were denser and thicker than in the NCF state (Figs 5 and 6). This suggests that increased density and size of actin stress fibers is likely related to increased polymerization and intracellular remodeling of the actin cytoskeleton (i.e., F-actin formation by polymerization of G-actin) [41].
Citation: Xie L, Sun Z, Brown NJ, Glinskii OV, Meininger GA, Glinsky VV (2022) Changes in dynamics of tumor/endothelial cell adhesive interactions depending on endothelial cell growth state and elastic properties. PLoS ONE 17(6): e0269552. https://doi.org/10.1371/journal.pone.0269552
Editor: Mária A. Deli, EötvösLoránd Research Network Biological Research Centre, HUNGARY
Received: January 11, 2022; Accepted: May 23, 2022; Published: June 6, 2022
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: This research was supported by National Institutes of Health Grants R01CA160461 (V.V.G.), R01NS110915 (to O.V. Glinskii), and in part by P01HL095486 (G.A. Meininger). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0269552#abstract0
