A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

Summary

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

Abstract

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

Introduction

The vascular endothelial growth factor (VEGF) family of secreted protein growth factors and their cognate cell surface receptors is an important and diverse group of soluble ligands and membrane-embedded receptors, respectively that function in transducing signals across cellular membranes. They function mainly in endothelial cells but also in cells of epithelial origin and those of the immune system ^1,2. Signaling pathways engaged by ligand-activated VEGF receptors (VEGFRs) are critical in major pathologies, such as age-related macular degeneration and cancer, and therapeutics targeting them are in frequent clinical use (e.g., the monoclonal antibody bevacizumab that targets VEGF-A) ^3,4.

One of the complexities of the VEGF family is the diversity of soluble ligands present in nature (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF proteins encoded by the parapox virus family orf and snake venom VEGF, plus other inhibitory isoforms of VEGF-A)².

These ligands interact with three members of the receptor tyrosine kinase family, namely VEGFR-1, VEGFR-2 and VEGFR-3. These receptors are variably expressed on different cell types but are often co-expressed on the surface of endothelial cells that line blood and lymphatic vessels of all sizes ⁵. VEGFR-2 can bind the mammalian ligands VEGF-A ⁶, VEGF-C ⁷ and VEGF-D ^8,9 as well as orf virus VEGF¹⁰ and snake venom VEGF¹¹. VEGFR-2 plays a major role in driving angiogenesis (the growth of new blood vessels from pre-existing vessels) in embryonic development, wound healing, cancer and eye diseases. In these contexts, ligands such as VEGF-A, -C and -D bind and activate the receptor on blood vascular endothelial cells^12-15. On lymphatic endothelial cells, VEGFR-2 plays a role in lymphangiogenesis, the formation of new lymphatic vessels ¹⁶. VEGFR-2 can also promote dilation and expansion of major arteries and lymphatics in healthy tissues and disease ¹⁷. A complete understanding of VEGFR-2:ligand interactions is therefore important for the development of inhibitors for use in treating angiogenesis-dependent diseases¹⁸. While most isoforms of VEGF-A bind to VEGFR-2, proteolytic cleavage of VEGF-C and VEGF-D is required to release a fragment consisting of the VEGF-homology domain that exhibits high affinity binding to VEGFR-2 ^19,20.

We have developed a bioassay to monitor ligands of VEGFR-2 that is designed to circumvent the need for primary endothelial cells, which are technically difficult to passage, expensive to purchase and culture (requiring specialized medium) ²¹ and express multiple VEGFRs and associated co-receptors ²². Heterodimerization of VEGFR-2 with other VEGF receptors or co-receptors can cause unwanted complexity when aiming to study binary receptor-ligand interactions, evaluating activity attributable to a specific receptor, or assessing the effect of inhibitory reagents.²³. The bioassay retains mobility of the relevant receptor in the cell membrane and allows evaluation of a ligand's ability to bind and cross-link the VEGFR-2 extracellular region.

The bioassay relies on the creation of a chimeric receptor in which the extracellular region of a VEGF receptor (in this case VEGFR-2) is fused to the transmembrane and intracellular regions of the erythropoietin receptor (EpoR), a member of the cytokine receptor family ^8,24. This fusion protein is then expressed in the factor-dependent pro-B cell line Ba/F3, upon which stimulation with a ligand capable of binding and cross-linking the extracellular domain of the receptor causes activation of the cytoplasmic effector region, which is capable of transducing a survival signal via Janus kinases (JAKs) to promote cell survival and/or proliferation. In contrast, expression of full-length VEGFR-2 in the same cell type, and stimulation with ligand, does not promote cell survival and proliferation, indicating that the proximal signaling effectors of the VEGFR-2 pathway are not available in this cell type.

We have used the assay in a variety of contexts to explore binding of novel VEGFR-2 ligands ^{10,19,20,24-29}. In combination with a VEGFR-3-EpoR-Ba/F3 assay, we have compared the relative activities of the VEGF-C and VEGF-D growth factors for binding and cross-linking VEGFR-2 and VEGFR-3 ³⁰. The assay has been used to characterize the inhibitory activity of neutralizing monoclonal antibodies to VEGFR-2 or VEGF-D, soluble VEGFR-2 trap and peptidomimetics targeting the VEGF family³¹. The assay was also used to show the ability of VEGFs from different orf virus strains to bind and cross-link VEGFR-2 prior to testing in primary endothelial cells ^10,26. The assay is particularly useful for the rapid screening of mutants of VEGFs which can be quickly assessed for activity before they are introduced to the more laborious endothelial cell assays ²⁵ or when assessing protocols for purifying growth factors ²⁷.

The assay we describe is easy to perform, and the semi-quantitative version allows for quick determinations that are sometimes required when monitoring the production or purification of growth factors, antibodies or soluble receptor domains for other experiments. The ease of use of the assay makes it an ideal complement to further and more complete studies performed with primary endothelial cells derived from blood or lymphatic vessels from specific tissues or organ systems.

Protocol

Source of IL-3 and Preparation of WEHI-3D-conditioned Medium

Note: The mouse granulocytic leukemia cell line WEHI-3D is cultured to generate a conditioned medium containing IL-3.

1. Culture WEHI-3D in Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), 1% long-life glutamine supplement, 50 μg/ml gentamicin. Inoculate 5 × 10⁶ cells in the log phase of growth into 50 ml of fresh culture medium in a T175 cm³ tissue culture flask and grow for about 7 days or until the cells have passed their log phase of growth by about 24 - 48 hr (avoid excessive cell death in the culture). Conditioned media is capable of being stored for multiple years at −20 °C, so batches of 200 − 500 ml can be produced at one time.
2. Decant fluid and cells from flasks and spin at 1,000 x g for 15 min to remove cells and cellular debris. Remove the top 90% of supernatant. Filter through a 0.22 μm filter unit.
3. Aliquot WEHI-3D conditioned medium (CM) into 1 ml, 50 ml and 200 ml volumes for storage at −20 °C or −70 °C. Smaller aliquots are useful for assay preparation, intermediate volumes for making culture medium for passage of factor-dependent cell lines, and larger volumes for long-term storage. IL-3 is relatively stable at 4 °C so thawed medium can be stored for a few weeks if used under sterile conditions.
4. Alternatively, use recombinant mouse IL-3 at levels of 50 ng/ml diluted into culture medium, after being filtered through a 0.22 µm filter unit.

2. Culture and Evaluation of VEGFR-2-EpoR-Ba/F3 and Control Ba/F3 Cell Lines

1. Culture control Ba/F3 cells in DMEM, 10% FBS, 50 µg/ml gentamicin or penicillin/streptomycin supplement, 1% stabilized L-glutamine and 10% WEHI-3D-CM. Passage cells at 1:15 dilutions about every three days from cells growing in log phase.
2. Culture VEGFR2-EpoR-Ba/F3 cells in DMEM, 10% FBS, 50 µg/ml gentamycin, 1% stabilized L-glutamine and 10% WEHI-3D-CM and 1 mg/ml G418. Passage cells at 1:15 dilutions from cells growing in log phase. See ²⁴ for more details about the construction and expression of the chimeric receptor.
3. Harvest control Ba/F3 or VEGFR2-EpoR-Ba/F3 cells from mid log-phase cultures. Gently pipette to remove these non-adherent cells from the bottom of the flask. Wash three times in mouse tonicity phosphate-buffered saline, pH 7.3 (PBS), (10 ml, centrifuge at 750 x g for 5 min to recover cell pellet) to remove medium containing IL-3.
4. Wash cells once with DMEM and additives, without the WEHI-3D-CM or recombinant IL-3, and then resuspend in this medium at a concentration of 7.4 × 10⁴ cells/ml (i.e., 1,000 cells per 13.5 µl; 10,000 cells per 135 µl as determined by counting using a hemocytometer) for use in the semi-quantitative or quantitative versions of the assay respectively.
5. Assess cells for viability by Trypan Blue exclusion (CAUTION). Mix Trypan Blue in PBS (0.4%) 1:1 with the cell population and count a minimum of 100 cells on a hemocytometer. Cells that take up the dye are considered dead or dying. Viability of greater than 98% is required to perform the assay.

3. Semi-quantitative Assay

1. Add washed cells (1,000 cells) contained in 13.5 μl of IL-3-deficient medium to the wells of a 72-well microwell plate at RT. Take care to mix the cell suspension during aliquotting to ensure cell settling by gravity does not bias cell concentration. Use a well-calibrated P20 automated pipette, and autoclaved tips.
2. Add test samples and controls to the wells at 10% volume (1.5 μl, making a final volume of 15 µl containing 1,000 cells per well) using a calibrated P20 pipette or, preferably, P2 pipette. Take care to ensure that samples are compatible with the culture conditions for Ba/F3 cells in terms of pH, salt and other potentially cytotoxic/cytostatic substances. Where possible, use a compatible medium or buffer (e.g., DMEM or PBS, respectively) or dilute in such medium or buffer. Trituration with the same tip will ensure mixing.
3. For control samples, include (i) Medium alone-containing no IL-3; (ii) WEHI-3BD-CM added to medium alone at 10% final volume; and/or (iii) VEGF-A diluted to 100 ng/ml in medium alone, containing no IL-3.
4. Fill any unused wells of the microwell plate with sterile water, PBS or medium and place the plate within a humidified container (with water soaked tissue paper) allowing gas exchange. Incubate cells within the containers in a humidified atmosphere of 10% CO₂. This assay can be comfortably set up in 3 hr by one person if test samples and media components are available.
5. Assess plates typically after 16 hr of incubation at which time discrimination between positive and negative samples is evident. See Figure 3 for examples of how cells appear in culture. Analyze plates using a standard inverted phase-contrast microscope at 40 − 100× magnification.
   Note: Wells containing supportive growth factors such as IL-3 or VEGF-A will contain cells which appear round and translucent. Wells without supportive growth factors or with medium alone will have reduced numbers of round cells, as well as dead or dying cells and cellular debris (e.g., Figure 3C). Evaluating the assay at 24−48 hr post incubation is the optimum for sensitivity.

4. Quantitative Bioassay

1. Add washed cells (10,000 cells in 135 μl) in IL-3-deficient medium to the wells of a standard 96-well microtiter plate at RT. Take care to mix the cell suspension during aliquotting to ensure cell settling by gravity does not affect cell concentration.
2. Add test samples and controls to the wells at 10% volume (15 μl) using a calibrated pipette (P20). Take care to ensure that samples are compatible with the culture conditions for Ba/F3 cells in terms of pH, salt or other potentially cytotoxic/cytostatic substances. Where possible, use a compatible medium or buffer (e.g., DMEM or PBS) or dilute in such medium or buffer. Filter sterilize small volumes using a 0.22 µm pore cellulose acetate centrifuge tube filter unit.
3. Incubate the mixture of cells, growth factors and/or inhibitor agents in a humidified atmosphere of 10% CO₂ for 48 hr. Perform the assay within a humidified container that has water-soaked tissue paper and allows gas-exchange. At the completion of the incubation period, evaluate the assay using one of the alternative methods listed below which are surrogate markers of the viable cell number in the well.
4. Alternative#1: ³H-thymidine Incorporation
   Note: This quantification is designed for the 96-well plate format.
   1. After incubation of assay plates for 48 hr at 37 °C (150 µl volume per well), add ³H-thymidine in a volume of 50 µl of assay medium (Ba/F3 culture medium without IL-3/WEHI-3D CM) per well at a concentration of 20 µCi/ml, giving a final dose of 1 µCi per well.
   2. Incubate plates for a further 4 hr at 37 °C before harvesting cells using a cell harvester. Extract individual samples by placing the filters in scintillant and quantify with a Liquid Scintillation Counter.
5. Alternative#2: Bioluminescence Detection of Cellular ATP
   Note: This approach uses an ATP monitoring reagent which when combined with cell lysate can generate a bioluminescence signal which reflects the viable cells in the population.
   1. Lyse cells to extract ATP and use an ATP Monitoring Reagent to generate a luminescent signal according to the manufacturer's instructions. Read luminescence using a luminometer compatible with a 96-well plate format.
6. Alternative#3: Enzymatic Reduction of an Indicator Dye by Viable Cells
   Note: Resazurin based assays show good correlation to cell viability.
   1. Combine cultured cells with the resazurin based dye and quantify assays using a fluorescence plate reader according to the manufacturer's instructions.

Results

In this section, we show the results of an experiment demonstrating the essential features of a VEGFR-2-EpoR-Ba/F3 bioassay (see Figure 1 for principles of the assay). Other published studies demonstrate broader applications of the assay for alternative VEGFR-2 ligands, mutant VEGF molecules and inhibitory monoclonal antibodies ^{8,10,19,24-30}.

The data presented here represent an assay in which the VEG...

Discussion

The assay described here relies on using cells of high viability, which are dependent on growth factors. Cells therefore need to be carefully cultured to ensure they are factor-dependent, and retain expression of the chimeric receptor. Ensuring that the medium is freshly made and not stored for an excessively long period and that WEHI-3D CM is highly active is important. Cells need to be thoroughly washed from IL-3 containing medium into the assay medium to ensure that no residual IL-3 contaminates the assay when exposin...

Materials

Name	Company	Catalog Number	Comments
Trypan Blue	Sigma-Aldrich	T8154	0.4% solution in PBS is used 1:1 with cell suspensions to measure viable cells. Hazard-may cause cancer
G418 Sulphate (Geneticin)	Invivogen	ant-gn-5	Agent for selecting transfected eukaryotic cells. Hazard-may cause allergy or asthma symptoms or breathing difficluties.
3H-Thymidine	PerkinElmer	NET-027	This radioactive nucleoside is incorporated into chromosomal DNA during mitosis. Hazard-radiation
Vialight Plus Kit	Lonza	LT07-221	Bioluminescent detection of cellular ATP to quantify viability, using ATP Monitoring Reagent
Prestoblue Cell Viability Reagent	Invitrogen	A13261	Resazurin-based indicator of cell viability. Turns red in color in the reducing environment of the cell
Nunc Minitray with Nunclon Delta Surface (72 well)	Thermo Scientific	136528	Small microtitre plate
96 well Tissue Culture Plate	Falcon, Corning Inc.	353072
DMEM (1X)	Gibco	11965-92
GlutaMAX (100X)	Gibco	35050-061
Fetal Bovine Serum	Gibco	10099-141
Cell Harvester	Tomtec Life Sciences		Tomtec Harvester, 96 Mach 3M Cell Harvester
Liquid Scintillation Counter	LKB Wallac	1205	LKB Wallac 1205 Betaplate Scintillation Counter
UniFilter-96 GF/B	Perkin Elmer	6005177	White 96-well Barex Microplate with GF/B filterof 1 µm poresize
Gentamicin	Gibco, Life Technologies	15750-060
Penicillin/Streptomycin	Gibco, Life Technologies	15140-122
0.22 um pore cellulose acetate centrifuge tube filter unit	Costar, Corning Inc.	8160	Centrifuge tube filters have a 0.22 µm pore CA membrane-containing filter unit within a 500 µl capacity polypropylene microcentrifuge tube.
Fluorescence Reader	BioTek		BioTek Synergy 4 Hybrid Microplate Reader