Tumor metastasis to the brain occurs in approximately 20% of all cancer cases and often occurs due to tumor cells crossing the blood-brain barrier (BBB). The brain microenvironment is comprised of a soft hyaluronic acid (HA)-rich extracellular matrix with an elastic modulus of 0.1–1 kPa, whose crosslinking is often altered in disease states.
To explore the effects of HA crosslinking on breast tumor cell migration, we developed a biomimetic model of the human brain endothelium, consisting of brain microvascular endothelial cell (HBMEC) monolayers on HA and gelatin (HA/gelatin) films with different degrees of crosslinking, as established by varying the concentration of the crosslinker Extralink.
Results and Discussion
Metastatic breast tumor cell migration speed, diffusion coefficient, spreading area, and aspect ratio increased with decreasing HA crosslinking, a mechanosensing trend that correlated with tumor cell actin organization but not CD44 expression. Meanwhile, breast tumor cell incorporation into endothelial monolayers was independent of HA crosslinking density, suggesting that alterations in HA crosslinking density affect tumor cells only after they exit the vasculature. Tumor cells appeared to exploit both the paracellular and transcellular routes of trans-endothelial migration. Quantitative phenotyping of HBMEC junctions via a novel Python software revealed a VEGF-dependent decrease in punctate VE-cadherin junctions and an increase in continuous and perpendicular junctions when HBMECs were treated with tumor cell-secreted factors.
Overall, our quantitative results suggest that a combination of biochemical and physical factors promote tumor cell migration through the BBB.
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Atomic force microscopy
Analysis of variance
Bovine serum albumin
Dulbecco’s modified eagle’s medium
Endothelial cell growth supplement
Fetal bovine serum
Green fluorescent protein
Human brain microvascular endothelial cell
Junction Analyzer Program
Phosphate buffered saline
Roswell Park Memorial Institute
Short tandem repeat
Tumor conditioned media
Vascular endothelial cadherin
Vascular Endothelial Growth Factor
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We thank Dr. Toshiyuki Yoneda for generously providing MDA-MB-231-BR cells. The University of Maryland Computer, Mathematical, and Natural Sciences imaging incubator is acknowledged for providing training and equipment for confocal imaging. Kyle Thomas at Yellow Basket, LLC (email@example.com) is acknowledged for the JAnaP software development support. We also acknowledge Mary Doolin for help with editing custom Matlab code. We thank Dr. William Luscinskas from the Harvard Medical School for generously providing us with the VE-cadherin-GFP adenovirus.
Funding was provided by Burroughs Wellcome Fund (Career Award at the Scientific Interface). Additional funding was provided by the Ann G. Wylie Dissertation Fellowship from the University of Maryland Graduate School (to MAP), the Fischell Fellowship in Biomedical Engineering (to KMG), the Dr. Mabel S. Spencer Award for Excellence in Graduate Achievement (to KMG), the Clark Doctoral Fellowship (to AJD), the Fischell Department of Bioengineering, and the University of Maryland.
KMS, MAP, and KMG designed the research. MAP and GMD performed experiments for Fig. 1. GMD analyzed all data for Fig. 1. AJLD performed all experiments and data analysis for Fig. 2, with guidance from MAP. MAP performed confocal microscopy for Fig. 3. KMG performed experiments and analysis for Figs. 4, 5, 6, S2, S3, and S4, with help in analysis from JWJ. KMG prepared Fig. S1. MAP performed experiments and analysis for Figs. 7a–7e. AJLD performed experiments for Figs. 7f–7h, with guidance from MAP, and MAP analyzed data for Figs. 7f–7h. MAP performed confocal microscopy for Fig. 8. MAP and AJLD performed experiments for Fig. 9. MAP performed experiments and all analysis for Fig. 10. MAP performed statistical analysis for Figs. 1, 2, 7, and 10. MAP formatted Figs. 1, 2, 3, 7, 8, 9, and 10. KMG performed statistical analysis and/or formatting for Figs. 4, 5, 6, S2, S3, and S4. MAP, KMG, and KMS wrote the manuscript. All authors edited the manuscript, and all authors reviewed and approved final version of the manuscript.
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MAP, KMG, GMD, AJLD, JWJ, and KMS declare that they have no conflict of interest.
No human studies were carried out by the authors for this article.
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Kimberly Stroka, in January 2015, joined the Fischell Department of Bioengineering as an Assistant Professor at the University of Maryland, College Park. She received her B.S. in Physics in 2006 from Denison University and her PhD in Bioengineering from the University of Maryland, College Park. She completed her postdoctoral training at Johns Hopkins University in the Department of Chemical and Biomolecular Engineering and Institute for NanoBioTechnology. Dr. Stroka has received the National Science Foundation Graduate Research Fellowship, NIH NRSA F31 predoctoral fellowship, NIH T32 and F32 postdoctoral fellowships, and Burroughs Wellcome Career Award at the Scientific Interface. She also received the Rita Schaffer Young Investigator Award from the Biomedical Engineering Society (2014), Research and Scholarship Award from the UMD Graduate School (2017), “Outstanding Young Scientist Award” from the Maryland Academy of Sciences (2017), and Fischell Department of Bioengineering Faculty Teaching Award (2018). Dr. Stroka’s lab engineers cells and their microenvironment in order to create model systems that allow them to systematically understand fundamental aspects of cellular and tissue mechanobiology, with applications in vascular biomechanics, tumor cell metastasis, and stem cell engineering.
This article is part of the CMBE 2019 Young Innovators special issue.
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Pranda, M.A., Gray, K.M., DeCastro, A.J.L. et al. Tumor Cell Mechanosensing During Incorporation into the Brain Microvascular Endothelium. Cel. Mol. Bioeng. 12, 455–480 (2019). https://doi.org/10.1007/s12195-019-00591-2