Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay
Summary
This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.
Abstract
Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.
Introduction
Angiogenesis, which refers to the formation of new blood vessels from pre-existing ones1, is a crucial process during physiological development and pathologic conditions. It is indispensable for providing energy to highly metabolically active tissues such as the retina2 or the developing central nervous system3 and during the healing of damaged tissue4. Abnormal angiogenesis, on the other hand, is the basis for numerous diseases. Solid tumors, such as colorectal cancer or non-small cell lung cancer, facilitate their growth and the necessary energy supply by promoting angiogenesis5. Apart from cancer, neovascular diseases of the eye like diabetic retinopathy or age-related macular degeneration, which represent leading causes of blindness in the developed world, result from aberrant vessel growth6,7. A detailed understanding of the underlying pathomechanism is crucial to comprehend how physiological angiogenesis can be enhanced, for instance, in wound healing while better controlling pathological conditions such as vasoproliferative eye diseases.
On a cellular level, vascular endothelial cells are activated by various signaling molecules in angiogenesis, initiating cell proliferation and migration8. The cells subsequently organize into a hierarchy, with non-proliferating tip cells forming filopodia at the leading edge of the developing vessel branch8. Alongside, fast-proliferating stalk cells trail the tip cells, contributing to the formation of the emerging vessel. Subsequently, other cell types, such as pericytes or smooth muscle cells, are recruited to further stabilize the nascent branch9.
To explore molecular processes at the vascular endothelial cell level, numerous in vitro protocols have been developed and recently reviewed10. These assays typically fall into two categories: more simplistic but scalable 2D approaches and more elaborate 3D protocols. In a recent project, we conducted a comprehensive comparative analysis between the 2D scratch wound migration assay and the 3D spheroid sprouting assay11 to assess the extent of their differences and their ability to model various aspects of angiogenesis12.
While both offer the advantages of being reliable and easily implementable, on a molecular level, the 3D spheroid sprouting assay was favorable in addressing key aspects of angiogenesis compared to in vivo data, such as metabolic switches or cell-matrix interactions. Since in vitro angiogenesis assays are used to evaluate the angiomodulatory potential of signaling pathways13 and to screen for therapeutic agents, transferability of in vitro results to in vivo settings is essential. Furthermore, the opportunity for omics-based analyses on the RNA and protein levels to characterize the molecular changes in response to targeted modulation of angiogenic processes under controlled conditions remains an important benefit compared to in vivo settings14,15.
In this publication, we present key assays for exploring angiogenesis-related questions through the utilization of a live-cell imaging migration assay and a spheroid sprouting assay, including subsequent molecular analyses like RNA sequencing for transcriptomic analysis and immunohistochemistry on the protein level.
Protocol
Representative Results
Discussion
In this report, we presented a spectrum of techniques with functional and molecular readouts to study angiogenesis in vitro.
The migration assay represents a well-established technique used across all fields of wet laboratory work. We chose the commercially available live-cell imaging approach to take advantage of the 96-well format suitable for screening and dose-response experiments, the standardized and reproducible wound size created by the WoundMaker tool, the opportunity to ob…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Sophie Krüger and Gabriele Prinz for their excellent technical support. We thank Sebastian Maier for developing the ImageJ plugin to quantify spheroid sprouts and the Lighthouse Core Facility, Zentrum für Translationale Zellforschung (ZTZ), Department of Medicine I, University Hospital Freiburg for the use of the IncuCyte system. The graphics were created with biorender.com. This work was supported by the Deutsche Forschungsgemeinschaft [Bu3135/3-1 + Bu3135/3-2 to F.B], the Medizinische Fakultät der Albert-Ludwigs- Universität Freiburg [Berta-Ottenstein-Program for Clinician Scientists and Advanced Clinician Scientists to F.B.], the Else Kröner-Fresenius-Stiftung [2021_EKEA.80 to F.B.] the German Cancer Consortium [CORTEX fellowship for Clinician Scientists to J.R.] and the Volker Homann Stiftung [to J.N.+F.B.] and the "Freunde der Universitäts-Augenklinik Freiburg e.V." [to P.L.]
Materials
| 10x Medium 199 | Sigma-Aldrich | M0650 | |
| 2-(4-(2-Hydroxyethyl)1-piperazinyl)-ethan-sulfonsäure | PAN-Biotec | P05-01100 | HEPES |
| Alexa Fluor 647-conjugated AffiniPure F(ab)‘2-Fragment | Jackson IR | 115-606-072 | |
| Axio Vert. A1 | Zeiss | ||
| CapturePro 2.10.0.1 | JENOTIK Optical Systems | ||
| Collagen Type 1 rat tail | Corning | 354236 | |
| Collagenase D | Roche | 11088858001 | |
| Endothelial Cell Basal Medium | Lonza | CC-3156 | EBM |
| Endothelial Cell Growth Medium | Lonza | CC-3162 | EGM |
| Ethylenediaminetetraacetic acid | Serva | 11290.02 | EDTA |
| Fetal bovine serum | Bio&SELL | S 0615 | FBS |
| Human Umbilical Vein Endothelial Cells, pooled | Lonza | C2519A | HUVEC |
| IncuCyte ImageLock 96-well plates | Sartorius | 4379 | |
| Incucyte S3 Live-Cell Analysis System | Sartorius | ||
| Methocel | Sigma | m-0512 | |
| Microvascular Endothelial Cell Medium with 10% FBS | PB-MH-100-4090-GFP | PELOBiotech | |
| NaOH | Carl Roth | P031.2 | |
| Phalloidin-Fluorescein Isothiocyanate Labeled (0.5 mg/mL Methanol) | Sigma-Aldrich | P5282-.1MG | Phalloidin-FITC |
| Phosphate-buffered saline | Thermo Fisher Scientfic | 14190-094 | PBS |
| Primary Human Retinal Microvascular Endothelial Cells | Cell Systems | ACBRI 181 | |
| ProLong Glass Antifade Mountant with NucBlue | Invitrogen by ThermoFisher Scientific | 2260939 | |
| QIAzol Lysis Reagent | QIAGEN | 79306 | |
| recombinant human Vascular Endothelial Growth Factor | PeproTech | 100-20 | VEGF |
| Squared petri dish | Greiner | 688102 | |
| Trizol | Qiagen | 79306 | |
| Trypsin | PAN-Biotec | P10-024100 | |
| VEGF-R2 (monoclonal) | ThermoFisher Scientific Inc. | B.309.4 | |
| WoundMaker | Sartorius | 4493 |
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