Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 growth^6,7. 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 settings^14,15.

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.

Protokół

1. HUVEC cell culture

NOTE: Perform all following steps under sterile working conditions (sterile working bench).

1. Expanding HUVECs
   1. For both angiogenesis assays, use pooled human umbilical vein endothelial cells (HUVECs) or human microvascular endothelial cells (HMVECs).
   2. Cultivate the cells until they reach 90% confluence in an uncoated T75 flask with endothelial cell growth medium (EGM) before performing a split. Perform medium change 3 times a week.
      NOTE: To expedite growth, the medium can be changed daily. Do not use HUVECs beyond the sixth passage. The study received the most consistent results running all experiments with HUVECs of the same passage.
2. Splitting HUVECs
   1. When HUVECs reach the desired confluence in the T75 flask, rinse them once with 5 mL of phosphate-buffered saline (PBS), followed by incubating the cells with 0.5% trypsin for 2 min in the incubator.
      ​NOTE: Prolonged exposure to trypsin can damage HUVEC function.
   2. Gently detach the cells by tapping on the flask. Confirm successful detachment under an inverted phase contrast microscope.
   3. Add 8 mL of EGM, transfer the solution to a tube, and pellet the cells by centrifugation at 250 x g for 3 min.
   4. If cell counting is necessary, resuspend the cells in 5 mL of EGM and use a Neubauer chamber. Otherwise, add 40 mL of EGM and distribute it onto 4 fresh T75 cell culture flasks.

2. Scratch wound migration assay

NOTE: The scratch wound migration assay requires a duration of 3 days for completion (Figure 1). Perform all the following steps under sterile working conditions (sterile working bench).

1. Plating and starving of cells (day 1)
   1. Using a specialized 96-well plate for live cell imaging, seed 20,000 HUVECs in 100 µL of EGM per well. Allow the cells to settle for 6 h in the incubator.
      NOTE: It is advisable to optimize cell numbers considering the varying speed of vascular endothelial cell growth between suppliers and laboratories. The objective is to achieve a perfect monolayer the following day before initiating the scratch. Overpopulated wells can alter endothelial cell behavior, e.g., by contact inhibition¹⁶, while an incomplete monolayer hampers the analysis drastically.
   2. Starve all wells overnight with 100 µL of EBM per well supplemented with 2% FBS.
      NOTE: Be careful not to damage the cell monolayer, especially when using a multichannel pipette.
2. Preparing stimulation solutions (day 2)
   1. Dilute desired substances and controls in endothelial cell growth basal medium (EBM) supplemented with 2% FBS. For each well, use 100 µL of solution.
      NOTE: EBM supplemented with 2% FBS serves as a negative control, while recombinant human vascular endothelial growth factor (VEGF) at 25 ng/mL can be used as a positive control. Using 96-well plates, it is recommended that the experiments be designed with a minimum of technical replicates (4 wells with identical conditions), although it is suggested that 8 be used for optimal results. Strive to minimize potential edge or corner effects when planning the layout.
3. Creating the scratch (day 2)
   1. Check cells under a microscope to verify a confluent cell monolayer and cell viability. Position the 96-well plate in a 96-well wound maker tool and generate the scratch by pressing down the lever of the device. Carefully lift off the lid of the wound maker tool before releasing the lever to prevent double scratching.
   2. Wash all wells twice using 200 µL of EBM per well supplemented with 2% FBS. Confirm under a microscope that debris was successfully removed.
4. Cell stimulation (day 2)
   1. Add 100 µL of the prepared stimulation or control solutions per well.
5. Imaging (day 2-3)
   1. Acquire images once per hour for a period of up to 24 h using an automated schedule provided by the live-cell imaging microscope.
      NOTE: To ensure good image quality, scan every well immediately after transferring it into the incubator housing the live-cell imaging system. A 30 min acclimation time in the incubator helps to get a better picture quality. The microscope software is programmed to acquire one image per hour for each well at the midline of the defined scratch.
6. In the absence of a wound maker tool and a live cell imager, follow the steps described below.
   1. Use a pipette tip in a 24-well plate to induce a scratch. Take images with a classic cell culture microscope at specific intervals. For this purpose, utilize a motorized microscope equipped with precise x and y tracking capabilities. This enables automatic positioning, ensuring consistent image capture at predefined locations.
      NOTE: When manual imaging is necessary it is a viable option to only image T0 as well as one time point 'x' hours following treatment. In the described protocol using HUVECs the time point T12 (12 h post-stimulation) was an attractive time point with a clear separation between negative and VEGF-stimulated positive control. However, running initial time course experiments with imaging every 2 h or 1 h is recommended to determine the optimal time point for the specific experimental settings in each lab.
7. Analysis (day 3)
   1. The live-cell imaging microscope software may enable direct analysis of the acquired images. Define masks delineating the cell lawn, initial scratch, and relative wound density regions. Determine cell boundaries based on contrast differences.
      NOTE: A segmentation adjustment of 1.6, a hole file of 500 µm², an adjusted size of 1 pixel, an area >500 µm, and an eccentricity >0.6 were used for experiments run with HUVECs. Thorough visual vetting of the cell detection parameters relative to actual images is essential. With optimized configurations, the software calculates relative wound density (RWD) over time automatically and generates corresponding temporal graphs with the standard deviation (SD) of the technical replicates. This data can then be used for statistical analysis.

3. RNA extraction with 2D cultivated cells

NOTE: Perform all the following steps until step 3.3 under sterile working conditions (sterile working bench).

1. Plating cells (day 1)
   1. Detach HUVECs per step 1.2 of the HUVEC cell culture protocol.
   2. Seed 50,000 cells per well in a 12-well plate with 2 mL of EGM per well.
   3. Change the medium after 6 h to EBM containing 2% FBS (starving media) for starving overnight.
2. Treatment of the cells (day 2)
   1. Dilute agents of interest in EBM supplemented with 2% FBS. Replace starving media with the prepared dilution and incubate cells for the desired time in an incubator.
3. RNA extraction (day 2-3)
   1. Lyse the cells with 750 µL of Trizol per well and incubate for 10 min on an orbital shaker.
   2. Resuspend the samples with a 1000 µL pipette a few times and then store them in specific low-binding RNA tubes (volume 1.5 mL). Store at -80 °C.

4. Immunocytochemistry with 2D cultivated cells

NOTE: Perform all following steps until step 4.4 under sterile working conditions (sterile working bench).

1. Preparation of coverslips
   1. Incubate 100 coverslips with a diameter of 12 mm in 5% potassium hydroxide in methanol in a 50 mL tube for 30 min while shaking the applied solution.
      NOTE: This is a very important step in deprotonating the surface of the coverslips and improving the conditions for coating and culture. At this point, ensuring that the coverslips are well distributed within the medium and don’t get dry and glued together is highly recommended.
   2. Wash the coverslips for 30 min 4 times with demineralized water.
   3. After washing, transfer them into 70% isopropyl solution for storage.
2. Coating of coverslips
   1. Lean the coverslips individually against the wall of a square Petri dish to allow the evaporation of isopropyl alcohol.
   2. After 5 min transfer the coverslips into a 24 well plate (one coverslips per well). Apply 1 mL of a solution of 10 mg/mL collagen I in PBS to each well and incubate for 60 min at 37 °C.
   3. Then, wash the coverslips 4-5 times for 5 min with PBS. 
3. Cultivating cells
   1. Detach HUVECs following the procedures outlined in step 1.2 of the HUVEC cell culture protocol.
   2. Seed 50,000 cells per well in a 24-well plate with 2 mL of EGM per well.
   3. After 6 h, change the medium to EBM containing 2% FBS to allow the cells to attach to the well and starve the cells overnight.
   4. On the next day, dilute agents of interest in EBM supplemented with 2% FBS. Replace the starving solution in wells with the prepared dilution and incubate cells for the desired time in an incubator.
4. Fixation and blocking
   1. Cultivate the cells for the desired time, then wash the cells with PBS for 5 min.
   2. Fix the cells with 2% paraformaldehyde (PFA) in PBS for 20 min at room temperature (RT).
   3. Wash with PBS 3-4 times for 5 min.
   4. Incubate samples with blocking buffer containing 5% normal goat serum (NGS), 0.1% Triton-X-100 in PBS for 1 h at RT.
      NOTE: At this step, it is important to wash the samples to guarantee that PFA is removed completely. Choose the species of the serum based on the origin of the secondary antibody.
5. Staining
   1. Incubate the samples overnight at 4 °C in the blocking buffer with the primary antibody.
   2. On the next day, to remove any unbound primary antibody, wash the samples 3-4 times with PBS for 5 min each.
   3. Subsequently, incubate the samples for 1 h at RT with the corresponding secondary antibody and Phalloidin-FITC, diluted together in the blocking buffer.
   4. Wash an additional 3-4 times.
   5. Invert the coverslips onto slides with a small droplet of DAPI-containing mounting medium, allow to dry in the dark, and seal with nail polish. Store samples in the dark at 4 °C.

5. Spheroid sprouting assay

NOTE: The spheroid sprouting assay requires 3 days for completion (Figure 2). Perform all following steps until step 5.7 under sterile working conditions (sterile working bench).

1. Generating spheroids in hanging drops (day 1)
   1. Detach HUVECs following the procedures outlined in step 1.2 of the HUVEC cell culture protocol.
   2. Combine 200,000 cells with 8 mL of EGM and 2 mL of methocel stock solution, ensuring thorough mixing. Please refer to Supplementary File 1 for instructions on preparing the methocel stock solution.
   3. Dispense 25 µL droplets of the solution onto the inverted inner surface of a large, squared Petri dish. Gently rotate the lid by 180° and position it on the bottom of the cell culture vessel so that the droplets are hanging. Place the vessel in a cell culture incubator overnight.
2. Prepare the collagen gel (day 2)
   1. Mix thoroughly 2.3 mL of rat tail collagen type I with 0.280 mL of 10x Medium 199 until the mixture achieves a consistently even yellow hue. Keep this mixture on ice throughout the entire process.
3. Titrate the collagen gel (day 2)
   1. Titrate the mixture to achieve a physiological pH, indicated by an orange color, using NaOH (2 N).
   2. Add 50 µL of HEPES buffer to the final product.
      NOTE: To ensure an optimal dynamic range for the assay, it is imperative to approach as closely as possible to a physiological pH. Various batches of collagen may require different amounts of NaOH. Keep the mixture strictly on ice throughout the process and homogeneously mixed all the time.
4. Harvesting spheroids (day 2)
   1. Rinse hanging drops from cell culture lids with 20 mL of PBS.
   2. Centrifuge the solution for 7 min at 250 x g. Discard the supernatant and resuspend the spheroids in 0.1 mL of FBS and 0.4 mL of EGM by gently tapping the tube.
   3. Add 2 mL of methocel stock solution and mix thoroughly. Use a pipette with a minimum capacity of 5 mL to avoid damaging the spheroids.
5. Adding spheroids to the 3D collagen matrix (day 2)
   1. Add 2 mL of the prepared collagen mixture to the resuspended spheroids and mix thoroughly.
   2. Dispense 0.5 mL of the resulting mixture into the wells of a 24-well plate and incubate in a cell incubator for 30 min.
6. Stimulating spheroids in a 3D collagen matrix (day 2)
   1. Prepare a dilution of desired substances in EBM. For each well, use 100 µL of solution.
      NOTE: EBM serves as a negative control, while vascular endothelial growth factor (VEGF) at 25 ng/mL (final concentration in 500 µL of collagen matrix and 100 µL EBM layer) can be used as a positive control.
   2. Incubate for the desired time.
      NOTE: In a standard assay, 18-24 h is recommended, but timing needs to be carefully optimized in each lab.
7. Imaging spheroids (day 3)
   1. Utilize an inverted microscope and imaging platform to capture images of all spheroids that are not in contact with the well rim, with each other, or show signs of damage. Maintain consistent settings and zoom levels across all conditions.
8. Analysis (day 3)
   1. Utilize the measuring tool in ImageJ Fiji to ascertain the length of all sprouts from each spheroid in every image.
      NOTE: Use the plugin provided in Supplementary File 2, which makes the analysis more efficient and generates an output .txt file.
   2. Import the data into a spreadsheet, R, or any other preferred software, and use the average cumulative length of all sprouts per spheroid as the readout for each condition.
      NOTE: Results from different gels cannot be combined in the form of absolute sprouting lengths. Data from each treatment group need to be normalized to the EBM or VEGF control group (relative sprouting length) before data can be merged.

6. RNA extraction with 3D cultivated cells

1. Spheroid sprouting assay
   1. Perform the spheroid sprouting assay as described in section 5.
2. Lysis of collagen gel
   1. Wash spheroid gels with warm PBS for 5 min.
   2. Cut the spheroid gels into quarters and transfer all 4 quarters into a 50 mL tube. Use a scalpel for the mechanical dissection of the gels.
   3. Treat gel samples with lysis solution (containing 2 mg/mL Collagenase D in PBS) and incubate for 45 min at 37 °C on a shaker.
   4. Wash gels gently with PBS supplemented by 2 mM EDTA to stop the reaction of the lysis buffer.
3. RNA extraction
   1. Resuspend the lysed gels in 750 µL of Trizol and incubate for 10 min to guarantee sufficient RNA extraction as described in step 3.3.
   2. Resuspend the samples with a 1000 µL pipette a few times and transfer samples to specific low-binding RNA tubes (volume 1.5 mL). Store samples at -80 °C.

7. Immunocytochemistry of spheroids in a 3D collagen matrix

1. Spheroid sprouting assay
   1. Perform the spheroid sprouting assay as described in section 5.
2. Fixation and blocking
   1. Fix the gels with 4% PFA in PBS for 1 h.
      ​NOTE: To ensure good fixation of the cells in the gel, gels need to be detached carefully on the sides of the wells with a scalpel and tweezers following rinsing with PBS.
   2. Wash gels gently 3-4 times with PBS and then fully detach them from the surface of the wells and transfer them into new 24-well plates.
   3. Incubate gels for 1 h at RT with the blocking solution (containing 5% NGS and 0.1% Triton-X-100 in PBS) on an orbital shaker.
3. Staining
   1. Incubate the samples overnight at 4 °C on an orbital shaker with the primary antibody diluted in blocking buffer.
      NOTE: Turning the gels during this incubation did not improve staining quality.
   2. The next day, wash the gels gently 4-5 times with PBS for 5 min each.
   3. Incubate the corresponding secondary antibody, diluted with Phalloidin-FITC in blocking buffer overnight at 4 °C on an orbital shaker.
   4. Conduct 4-5 additional washing steps with PBS.
   5. Transfer the gels onto microscope slides and mount them with two drops of DAPI-containing mounting medium on a coverslip placed onto each gel. Allow the samples to dry before sealing with nail polish and store them in the dark at 4 °C.
4. Microscopy
   1. Image all samples on a confocal microscope. Use the 20x objective and focus on the region of interest. Acquire images as Z-stacks, as well as individual focal plane images.
      NOTE: Acquiring Z-stack and focal plane images at multiple sections throughout the 3D sample is essential to capture the complete representation, especially for thick 3D samples.

8. Human retinal microvascular endothelial cells (HRMVECs)

NOTE: All the described steps can also be performed with microvascular endothelial cells, e.g., human retinal microvascular endothelial cells (HRMVECs). In that case, the medium needs to be switched to a specific microvascular endothelial cell medium containing 10% fetal bovine serum (FBS) for cultivation as well as specific steps in each assay. HRMVEC-specific differences to the assay protocols are outlined below:

1. Scratch wound assay
   1. Cultivate cells in 10% FBS microvascular endothelial cell medium instead of EGM.
   2. Seed 20,000 cells/well.
   3. Do not starve the cells overnight.
   4. After performing the scratch, change the medium to basal endothelial cell medium containing 5% FBS instead of EBM with 2% FBS.
2. Immunocytochemistry of 2D cultivated HRMVECs
   1. Cultivate the cells in 10% microvascular endothelial cell medium instead of EGM.
   2. Do not starve the cells overnight.
   3. Change the medium right before treatment to EBM + 5% FBS instead of EBM+2% FBS.
3. Spheroid sprouting assay
   1. Seed the cells as hanging drops with microvascular endothelial cell medium containing 10% FBS instead of EGM.
4. Immunocytochemistry of spheroids in a 3D collagen matrix
   1. Seed the cells as hanging drops with microvascular endothelial cell medium containing 10% FBS instead of EGM.

Wyniki

For the migration assay, it is crucial to thoroughly examine the images captured at the t = 0 h time point to ensure the presence of a fully formed cell monolayer is accurately detected by the system (Figure 1B). Additionally, the clarity and straightness of the scratch border should be confirmed (Figure 1B). The cell-free area ought to be largely free of debris. At the end of the assay, a group stimulated with, for example, 25 ng/mL VEGF as a positive control s...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
10x Medium 199	Sigma-Aldrich	M0650
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