Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove

Developmental Biology

A Patient-Derived Xenograft Model for Venous Malformation

doi: 10.3791/61501 Published: June 15, 2020
Sandra Schrenk*1, Jillian Goines*1, Elisa Boscolo1,2
* These authors contributed equally

Abstract

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7‒9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

Introduction

Defects in the development of the vasculature are the underlying cause of many diseases including venous malformation (VM). VM is a congenital disease characterized by abnormal morphogenesis and expansion of veins1. Important studies on VM tissue and endothelial cells (EC) have identified gain-of-function mutations in two genes: TEK, which encodes the tyrosine kinase receptor TIE2, and PIK3CA, which encodes the p110α (catalytic subunit) isoform of PI3-kinase (PI3K)2,3,4,5. These somatic mutations result in ligand-independent hyper-activation of key angiogenic/growth signaling pathways, including PI3K/AKT, thereby resulting in dilated ectatic veins3. Despite these important genetic discoveries, the subsequent cellular and molecular mechanisms triggering abnormal angiogenesis and the formation of enlarged vascular channels are still not fully understood.

During normal and pathological angiogenesis, new vessels sprout from a pre-existing vascular network and EC undergo a sequence of important cellular processes including proliferation, migration, extracellular matrix (ECM) remodeling and lumen formation6. Two- and three- dimensional (2D/3D) in vitro cultures of EC are important tools to investigate each of these cellular properties individually. Nevertheless, there is a clear demand for a mouse model recapitulating pathological vessel enlargement within the host microenvironment while providing an efficient platform for preclinical evaluation of targeted drugs for translational research.

Up to date, a transgenic murine model of VM associated with TIE2 gain-of-function mutations has not been reported. Current transgenic VM mouse models rely on the ubiquitous or tissue-restricted expression of the activating mutation PIK3CA p.H1047R3,5. These transgenic animals provide significant insight into whole-body or tissue-specific effects of this hotspot PIK3CA mutation. The limitation of these models is the formation of a highly pathological vascular network resulting in early lethality. Thus, these mouse models do not fully reflect the sporadic occurrence of mutational events and localized nature of VM pathology.

On the contrary, patient-derived xenograft models are based on the transplantation or injection of pathological tissue or cells derived from patients into immunodeficient mice7. Xenograft models are a powerful tool to broaden knowledge about disease development and discovery of novel therapeutic agents8. In addition, using patient-derived cells allows scientists to recapitulate mutation heterogeneity to study the spectrum of patient phenotypes.

Here, we describe a protocol where patient-derived VM EC which express a mutant constitutively-active form of TIE2 and/or PIK3CA are injected subcutaneously in the back of athymic nude mice. Injected vascular cells are suspended in an ECM framework in order to promote angiogenesis as described in previous vascular xenograft models9,10,11. These VM EC undergo significant morphogenesis and generate enlarged, perfused pathological vessels in the absence of supporting cells. The described xenograft model of VM provides an efficient platform for preclinical evaluation of targeted drugs for their ability to inhibit uncontrolled lumen expansion.

Subscription Required. Please recommend JoVE to your librarian.

Protocol

Patient tissue samples were obtained from participants after informed consent from the Collection and Repository of Tissue Samples and Data from Patients with Tumors and Vascular Anomalies under an approved Institutional Review Board (IRB) per institutional policies at Cincinnati Children’s Hospital Medical Center (CCHMC), Cancer and Blood Disease Institute and with approval of the Committee on Clinical Investigation. All animal procedures described below have been reviewed and approved by the CCHMC Institutional Animal Care and Use Committee.

1. Preparation of materials and stock solutions

  1. Preparation of complete endothelial cell growth medium (EGM)
    1. Supplement endothelial basal medium (EBM) with the following growth factors present in the kit (see Table of Materials): human Fibroblast Growth Factor-Beta (hFGF-β), vascular endothelial growth factor (VEGF), Long Arg3 Insulin-Like Growth Factor- I (R3-IGF-I), ascorbic acid, epidermal growth factor (EGF), gentamycin sulphate and amphotericin-B, and heparin. Add 1% Penicillin/Streptomycin/L-Glutamine (PSG) solution and 20% fetal bovine serum (FBS).
      NOTE: We do not recommend adding hydrocortisone.
    2. Sterile filter the solution under a laminar flow hood through a 0.2 µm bottle top filter into an autoclaved glass bottle and aliquot media into 50 mL conical tubes and store at 4 °C up to a week or at -20 °C for up to one year.
  2. Prepare collagenase A stock solution
    1. Prepare 50 mg/mL collagenase A stock solution in 1x phosphate buffer saline (PBS).
    2. Sterile filter the solution under a laminar flow hood with 0.2 µm filter and syringe, and store 100 µL aliquots at -20 °C.
  3. Prepare tissue collection medium (Buffer A)
    1. Prepare a Ca2+/Mg2+ stock solution by adding 0.927 g of calcium chloride dihydrate (CaCl2.2H2O) and 1 g of magnesium sulfate heptahydrate (MgSO4.7H2O) to 500 mL of distilled water. Sterile filter the solution through a 0.2 µm bottle top filter into an autoclaved glass bottle and store at room temperature (RT).
    2. Supplement Dulbecco's Modified Eagle Medium (DMEM) with 10% Ca2+/Mg2+ solution and 2% FBS.
    3. Sterile filter the solution under a laminar flow hood with 0.2 µm filter and syringe and store aliquots of 5 mL at -20 °C.
  4. Fibronectin coating of tissue culture plates
    1. Prepare coating buffer (Buffer B) by dissolving 5.3 g of sodium carbonate (Na2CO3; 0.1 M) in 500 mL of deionized water. Adjust the pH of the buffer to 9.4 using 1 M hydrochloric acid (HCl). Filter under a laminar flow hood through a 0.2 µm bottle top filter into an autoclaved glass bottle and store at RT.
    2. For coating, pipette 2 mL/5 mL/10 mL of Buffer B per 60 mm/100 mm/145 mm tissue culture plate, respectively. Add 1 µg/cm2 of human plasma fibronectin purified protein solution and gently distribute the liquid onto the plate.
    3. Incubate plate at 37 °C, 5% CO2 for 20 min.
    4. Aspirate Buffer B and wash the plate with PBS prior to culturing cells.

2. Isolation of endothelial cells from VM patient tissue

  1. Isolation of EC from solid VM tissue
    NOTE: VM tissue is resected by debulking surgery12,13 under an IRB approved protocol.
    1. Wash VM tissue (tissue sample weight typically ranges between 0.5 g and 1.5 g) in 5% PSG in PBS.
    2. Transfer tissue to a 100 mm cell culture dish, mince tissue sample into small pieces using sterile surgical dissection tools, and transfer into a 50 mL conical tube.
    3. Add 100 µL of collagenase A stock solution to 5 mL of Buffer A for a final concentration of 1 mg/mL, then add this to the tissue and digest the minced tissue at 37 °C for 30 min while shaking contents every 5 min.
    4. Carefully grind the digested tissue at RT using a 6 mm, smooth-surface pestle grinder within the 50 mL conical tube.
    5. Continue to carefully grind digested tissue using a pestle while adding 5 mL of cold PBS supplemented with 0.5% bovine serum albumin (BSA) and 2% PSG. Repeat this step four times.
    6. Filter the solution through a 100 μm cell strainer into a 50 mL conical tube to remove tissue fragments.
    7. Centrifuge cell suspension for 5 min at 400 x g at RT. Proceed to step 2.3.
  2. Isolation of VM EC from lesional blood obtained from patient sclerotherapy
    1. Obtain human VM lesional blood from sclerotherapy under an IRB approved protocol.
    2. Dilute lesional blood (sample volume typically ranges between 0.5 mL and 5 mL) in PBS to a final volume of 40 mL.
    3. Centrifuge cell suspension for 5 min at 200 x g at RT.
  3. Initial cell plating
    1. Discard supernatant and resuspend the single-cell pellet in 1 mL of EGM.
    2. Add 9 mL of complete EGM onto fibronectin-coated (1 µg/cm²) 100 mm plates and seed 1 mL of cell suspension.
    3. Incubate cells at 37 °C in a humidified 5% CO2 atmosphere.
    4. Every other day remove 2 mL of media and add 2 mL of sterile filtered FBS.
    5. Once cell cultures reach a confluency of 40‒50% change the medium to complete EGM. It typically takes between 2‒3 weeks for the cells to reach this confluency.
    6. Observe daily for the appearance of EC colonies. They can be recognized by their typical “cobblestone-like” morphology (Figure 1A). Between 3‒5 EC-colonies appear in each sample.
    7. Change the medium every other day for another 5‒7 days until individual EC colonies start to touch one another.
  4. Manual isolation of individual EC colonies
    1. To harvest EC colonies, wash the plate with 5 mL of PBS and manually aspirate with a serological pipette.
    2. Take the plate to a microscope and circle the locations of multiple EC colonies using a marking pen both on lid and bottom before returning it to laminar flow hood.
    3. Detach EC colonies by pipetting 50 µL of 0.05% trypsin-EDTA solution on the marked areas.
    4. Using a small cell scraper or a pipette tip, gently scrape cells from plate.
    5. Tilt the plate to the nearest edge, and rinse with 1 mL of EGM per marked area and collect cells.
    6. Count the number of cells using a hemocytometer or automated cell counter.
    7. Plate the collected EC colonies at a density of 1 x 104 cells/cm2 onto fibronectin-coated (1 µg/cm²) cell culture dishes containing fresh EGM. The next day, change medium to complete EGM.
    8. Change the medium to complete EGM every other day for 2‒3 weeks until cells reach 80% confluency.
    9. Trypsinize EC with 2 mL of pre-warmed 0.05% trypsin-EDTA per 100 mm dish at 37 °C for 2 min and neutralize trypsin by adding 4 mL of EGM.
    10. Collect the cell suspension into one 15 mL conical tube and centrifuge at 400 x g at RT for 5 min. Aspirate the supernatant and resuspend cells in 2 mL of EGM.
    11. Count the number of cells using a hemocytometer or automated cell counter. Typical cell numbers obtained are 1 x 106 cells per 60 mm cell culture plate or 2 x 106 cells per 100 mm tissue culture plate.
    12. Pellet the cells by centrifugation at 400 x g at RT for 5 min and aspirate the supernatant. Proceed to step 3.2.1.

3. Endothelial cell selection and expansion

  1. Preparation of anti-CD31-conjugated magnetic beads
    1. Vortex the vial containing the anti-CD31-conjugated magnetic beads for 30 s. The number of beads required is 8 x 106 beads/2 x 106 cells.
    2. Wash the desired amount of anti-CD31-conjugated magnetic beads with 1 mL of wash solution containing 0.1% BSA in PBS in a 1.5 mL microcentrifuge tube.
    3. Place the microcentrifuge tube in a cell isolation magnet for 1 min.
    4. Aspirate the supernatant and repeat the washing step.
  2. Endothelial cell purification and plating
    1. Resuspend cell pellet obtained in 2.4.12 in 500 µL of wash solution containing 0.1% BSA in PBS.
    2. Add cell solution to microcentrifuge tube containing magnetic beads and resuspend thoroughly.
    3. Incubate for 20 min at 4 °C with gentle tilting.
    4. Add 500 µL of 0.1% BSA in PBS and mix well.
    5. Place the tube on a cell isolation magnet for 1 min.
    6. Gently aspirate all of the liquid, which contains the CD31-negative fraction of cells without touching the beads.
    7. Wash the bead pellet containing the CD31-positive cell fraction with 1 mL of 0.1% BSA in PBS and repeat magnetic separation.
    8. Repeat washing and magnetic separation step for a minimum of 3 times to purify CD31-positive cell fraction.
    9. Resuspend purified endothelial cells into a 15 mL conical tube and spin down for 5 min at 400 x g at RT.
    10. Remove supernatant and resuspend cell pellet in 1 mL of EGM.
    11. Add 9 mL of complete EGM onto fibronectin-coated (1 µg/cm²) 100 mm plates and seed 1 mL of cell suspension. Note that some magnetic beads are still attached to the cells in this initial seeding step. Most beads wash away during cell expansion, but small number of beads may persist in early passages.
    12. Incubate cells at 37 °C in a humidified 5% CO2 atmosphere.
    13. Change the medium every other day until cells reach 80 % confluency (Figure 1B).
  3. Endothelial cell expansion
    1. Once cells reach 80 % confluency, detach with 2 mL of 0.05% trypsin-EDTA per 100 mm dish at 37 °C for 2 min.
    2. Neutralize trypsin by adding 4 mL of EGM and collect cells into one 15 mL conical tube.
    3. Centrifuge at 400 x g at RT for 5 min.
    4. Aspirate the supernatant, resuspend cells in 2 mL of EGM, and count the number of cells with a hemocytometer or by automated cell counting.
    5. Seed cells at a density of 1 x 104 cells/cm² into 145 mm fibronectin-coated (1 µg/cm²) tissue culture plates.
    6. Continue to passage cells until the desired cell number has been met. Consider that a confluent 145 mm tissue culture plate contains about 8‒9 x 106 cells and the number of cells needed is 2.5 x 106 cells per injection. Only cells between passage 3 and 8 should be considered for the xenograft.

4. VM patient-derived xenograft protocol

NOTE: In this protocol we use 5‒6 week old, male immunodeficient, athymic nude Foxn1nu mice.

All animal procedures must be approved by the Institutional Animal Care and Use Committee (IACUC).

  1. Preparation of materials on the day before injection
    1. Pre-chill syringes, needles, and pipet tips in -20 °C freezer overnight.
    2. Slowly thaw the basement membrane extracellular matrix (BMEM) overnight on ice bucket placed at 4 °C to avoid increased viscosity of the gel.
  2. Preparation of cell suspension for injection
    1. Trypsinize EC with 5 mL of pre-warmed 0.05% trypsin-EDTA per 145 mm dish at 37 °C for 2 min.
    2. Neutralize trypsin by adding 5 mL of EGM and collect cells into one 15 mL conical tube.
    3. Centrifuge at 400 x g at RT for 5 min.
    4. Aspirate the supernatant, resuspend cells in 3 mL of EGM, and count the number of cells using a hemocytometer or automated cell counter.
    5. Determine the total number of cells that are needed for all planned injections.
      NOTE: The recommended number of cells is 2.5 x 106 cells per injection. A total of 2 injections are typically performed in each mouse for technical duplicates. It is necessary to calculate a 10% excess of cell number to account for loss during transfer into the syringe.
    6. Transfer the volume containing the calculated cell number into a new 50 mL conical tube and pellet cells by centrifugation at 400 x g at RT for 5 min.
    7. Aspirate the supernatant, leaving a small volume (about 50‒70 µL) to loosen the pellet.
  3. Syringe preparation
    1. Calculate an excess volume of 20 µL/injection to account for loss during transfer to the syringe. Resuspend the cell pellet with 220 µL of BMEM per injection on ice. The injected volume of cell suspension will be 200 μL per lesion.
    2. Mix the cell suspension thoroughly on ice to obtain a homogenous cell suspension and avoid creating bubbles.
    3. Using a 1 mL pipet and 1 mL syringe, simultaneously pipet BMEM-cell mixture into the syringe opening by suction force while pulling plunger of syringe.
    4. Luer lock a 26G x 5/8 inch sterile needle to the syringe and keep prepared syringes flat on ice prior to injection.
  4. Subcutaneous injection into mouse
    1. Anesthetize the mice with 5% isoflurane/oxygen mixture at a flow rate of 1 L/min using an isoflurane vaporizer. Ensure proper sedation of animals (e.g., unresponsiveness to toe pinches). Maintain anesthesia via continuous administration of 1.5% isoflurane/oxygen delivered via nose cone.
    2. Place mice on their stomach, exposing the back region where grafting will occur and disinfect the injection region with 70% ethanol.
    3. Gently roll the prepared syringe to resuspend any settled cells. Flick bubbles to the needle end of the syringe and expel a small volume of the cell suspension to ensure the removal of all bubbles.
      NOTE: Two injections can be performed for each mouse – on the left and the right side of mouse back.
    4. Pinch and create a ‘tent-like’ structure using your thumb and index finger and insert the needle subcutaneously right under the skin. Ensure that the needle is only skin deep by releasing pinched skin to prevent injection into muscle tissue.
    5. Holding needle at 45° angle carefully inject 200 µL of the cell-suspension to create a small spherical mass (Figure 1C).
    6. Record mouse weight with a scale, ear tag the mouse, and return to cage.
    7. Monitor mice following sedation to ensure they return to normal activity.
  5. Lesion growth monitoring
    1. Using a caliper, measure the length and width of each plug (Figure 1D).
    2. Document measurements every other day up until lesion collection.

5. Tissue collection and processing

  1. Euthanize mice 9 days post-implantation in a CO2 chamber and check vital signs to confirm death. Perform cervical dislocation on the mice as a secondary method to euthanize the mouse.
  2. Harvest the xenograft lesion/plug from the flank of the mouse by dissection using surgical forceps and scissors.
    NOTE: In order to prevent rupturing the blood-filled vessels within the plug, it is important to avoid touching the lesion plug with dissection tools and leave excessive surrounding tissue such as skin attached to the plug.
  3. Immerse the resected lesion in PBS to wash.
  4. Set up a camera stage with a camera. Align plugs onto a cutting board with a ruler. Take an image of all plugs to record gross vascularity of lesions (Figure 1E).
  5. Fix plugs by submerging in 10% formalin overnight at RT.
  6. Wash plugs in PBS the following day and move them into 70% ethanol.
  7. Process lesion plugs for paraffin embedding (pathology core).

6. Lesion sectioning

  1. Use a microtome to cut 5 μm sections from the collected murine lesions onto positively charged slides.
    NOTE: For the subsequent analysis, sections in the center of the plug (about 50‒70 µm into the tissue) are of importance.
  2. Melt paraffin at 60 °C for 1 h prior to staining.
  3. De-paraffinize and re-hydrate tissue sequentially under a chemical fume flow hood. Therefore, incubate slide in xylene for 10 min, 100% ethanol (EtOH) for 5 min, 90% EtOH for 3 min, and 80% EtOH for 3 min.
  4. Rinse slide in deionized water for 5 min.

7. Hematoxylin and Eosin (H&E)

  1. Incubate sections in Hematoxylin for 2 min.
  2. Place slides in a staining jar and rinse in a sink by a steady stream of tap water until water is clear.
  3. De-hydrate slides by incubating tissue sequentially in 70% EtOH for 1 min, 80% EtOH for 1 min, 90% EtOH for 1 min, 100% EtOH for 1 min, and fresh 100% EtOH for 1 min.
  4. Stain sections in Eosin Y for 30 s.
  5. Rinse in fresh 100% EtOH until solution is clear.
  6. Incubate slide in xylenes for 2 min. Let slides dry for 5‒10 min under the fume hood.
  7. Dispense a drop of permanent, non-aqueous mounting medium over xenografts sections and place coverslip on top.
  8. Allow slides to dry overnight before imaging.

8. Immunohistochemistry

  1. Prepare antigen retrieval buffer (Tris-EDTA) by weighing 0.6 g of Tris-base and 1 mL of 0.5 M EDTA to 500 mL of deionized water. Adjust pH to 9.0 using 1 M HCl. Add 250 µL of Tween-20.
  2. Incubate de-paraffinized tissue slides (as obtained in step 6.2‒6.3) in a beaker with antigen retrieval buffer, stirring on a heating block, for 20 min at 95 °C.
  3. Remove the beaker from heating block, allow solution to cool to 35 °C then wash in PBS for 3 min.
  4. Block tissue sections in 5% normal horse serum in PBS for 30 min at RT.
  5. Prepare a biotinylated Ulex europaeus agglutinin-I (UEA-I) working solution by diluting 20 µg/mL of biotinylated UEA-I in 5% normal horse serum in PBS.
  6. Pipet 50‒100 µL of UEA-I working solution per section and incubate for 1 h at RT in a humidifying chamber.
  7. Wash slides two times in PBS for 3 min.
  8. Quench slide sections in 3% hydrogen peroxide for 5 min at RT.
  9. Wash slides two times in PBS for 3 min.
  10. Prepare 5 µg/mL of Streptavidin horseradish peroxidase-conjugated in 5% normal horse serum in PBS.
  11. Pipet 50‒100 µL on each tissue slide and incubate for 1 h at RT in a humidifying chamber.
  12. Wash slides two times in PBS for 3 min.
  13. Prepare 3,3'Diaminobenzidine (DAB) solution according to manufacturer’s instructions and add 50‒100 µL per section.
  14. Incubate sections for 10‒15 min, checking and monitoring for development of stain every 2‒5 min.
  15. Wash slides three times in PBS for 3 min.
  16. Add a drop of Hematoxylin and incubate for 3 min.
  17. Place slides in a staining jar and rinse in a sink by a steady stream of tap water until water is clear.
  18. Sequentially incubate slide in 80% EtOH for 1 min, 90% EtOH for 1 min, 100% EtOH for 1 min, and xylene for 2 min.
  19. Let slides dry for 5‒10 min under the fume hood.
  20. Dispense a drop of permanent, non-aqueous mounting medium over xenografts sections and place coverslip on top.
  21. Allow slides to dry overnight before imaging.

9. Analysis of human-derived Vascular Channels

NOTE: Vascularity of VM lesions is quantified by measuring vascular area and vascular density. Only UEA-I positive, human-derived vascular channels are considered for quantification.

  1. Take four to five images per lesion section with a bright field microscope at a 20x magnification (high power fields [HPF]). Take HPF images in an x-plane pattern within the lesion section to avoid overlap (Figure 1F-H). Include a scale bar on the images taken.
  2. Open the HPF images in Image J (File > Open). Calibrate the pixels of the scale bar as follows. Use the straight line tool and go over the scale bar. To convert the measured pixels into mm click on Analyze > Set scale.
  3. Click on Analyze > Set Measurements and select Area and Add to overlay.
  4. Measure the total field area in a HPF using Analyze > Measure. Save this measurement for quantification in step 9.8.
  5. Using the freehand selections tool, manually outline UEA-I+ vascular channels.
    NOTE: A vascular channel is defined as any area that is lined with UEA-I+ - EC that may contain blood cells.
  6. Click on Analyze > Measure to quantify the outlined UEA-I+ vascular area (mm2/HPF).
  7. Repeat this measurement for all five HPF taken within one plug.
  8. Average the total vascular area of all five HPF. The obtained vascular area per HPF is subsequently divided by the HPF field area (in mm2, step 9.5) and expressed as a percent (%).
  9. For quantification of vascular density, count the number of UEA-I+ vascular channels of each HPF taken. The vascular density is the average number of UEA-I+ vascular channels counted per HPF area (vessels/mm2).

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

This protocol describes the process of generating a murine xenograft model of VM based on the subcutaneous injection of patient-derived EC into the back of immunodeficient nude mice. Endothelial cell colonies can be harvested within 4 weeks after initial cell isolation from VM tissue or lesional blood (Figure 1A,B). The day after injection, the xenograft lesion plug covers a surface area of approximately 80‒100 mm2. In our hands, lesion plugs with TIE2/PIK3CA-mutant EC are visibly vascularized and perfused within 7‒9 days from injection 14,15 (Figure 1C-E). However, the extent of lesion growth is variable and reflects on patient and sample heterogeneity.

Lesion plugs closely recapitulate the histopathological features of human VM tissue: enlarged vascular channels lined by a thin layer of endothelial cells (Figure 1F‒H). These vascular structures typically contain erythrocytes, confirming functional anastomoses with the host mouse vasculature (Figure 1F‒H). Immunohistochemical staining using the human specific lectin UEA-I can confirm that cells lining vascular lesions are derived from human implanted cells rather than mouse vasculature (Figure 1H). A scheme summarizing the steps from VM-EC isolation to dissection of lesion plug is presented in Figure 2

Figure 1
Figure 1: Representative results.
(A) Representative image of primary mixed cell culture three weeks after isolation from VM tissue before EC selection. Typical endothelial cell colony (EC) and contaminating fibroblast (FB). (B) Image of a purified (CD31 bead-selection) endothelial cell culture from VM patient-derived tissue. Scale bar = 200 µm. (C) The lesion will form a spherical structure. Vascularization is visible due to blueish color through the skin of nude mice. (D) Dashed lines show how lesion size is recoded by measuring the length (L) and the width (W) using a caliper. (E) Photo of visibly vascularized, xenograft lesion explant at day 9. Scale bar = 1 cm. (F) Representative image of a lesion plug section. The x-plane pattern in which five high power field images are taken for quantification are indicated by white dashed boxes. Scale bar = 1000 µm. (G‒H) Representative images of VM lesion plug sections. (G) Hematoxylin and Eosin staining and (H) immunohistochemistry of human specific lectin UEA-I. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Schematic of the workflow to generate a patient-derived xenograft of VM.
(A) Endothelial cells isolated from patient VM lesion solid tissue or lesional blood are plated and, when 80% confluency is reached, are selected by anti CD31-conjugated immunomagnetic beads and expanded. (B) For subcutaneous injection of EC, on day 0, skin on the backside of the mouse is pinched using forefinger and thumb to create a tent-like structure. Lesions are measured at day 1 and then every other day (red arrows) using a caliper through experimental day 9. Lesions are dissected and processed for histological analysis. Please click here to view a larger version of this figure.

Subscription Required. Please recommend JoVE to your librarian.

Discussion

Here, we describe a method to generate a patient-derived xenograft model of VM. This murine model presents an excellent system that allows researchers to gain a deeper understanding of pathological lumen enlargement and will be instrumental in developing more effective and targeted therapies for the treatment of VM. This can be easily adapted to investigate other types of vascular anomalies such as capillary lymphatic venous malformation16. There are several steps that are crucial for the successful generation of reproducible vascular lesions. First, the patient-derived endothelial cells must be pure (without the presence of other cell types) and growing exponentially at the time of injection. Contaminating fibroblast or other mesenchymal non-EC can be easily recognized by elongated morphology. On rare occasion, it is possible that even after purification using anti-CD31 antibody conjugated magnetic beads, a small number of non-EC remain in the culture. These cultures require further purification with endothelial specific cell surface markers. As an alternative approach, single cell clonal expansion of endothelial cells is possible. This would reinsure the homogeneity of mutant-EC as all of the cells within one culture would derive from one single cell. However, this approach is not recommended for VM-derived EC as cells tend to top their proliferation capabilities and convert into a senescent phenotype after 9‒10 passages. It is critical to use cells between passages 3‒8 for xenograft experiments and to not passage cells the day before the injection.

The xenograft model can be modified to investigate other vascular anomalies carrying different activating mutations. Moreover, as patient tissue samples are difficult to access for some laboratories, the xenograft model can be adapted by using EC, such as human umbilical cord blood endothelial cells (HUVEC), genetically engineered to express the mutation/s known to cause dysfunctional vascular growth15,17.

The number of cells recommended for the injection in the xenograft is 2.5 x 106 cells/200 µL of BMEM. However, if the cell number is insufficient it is possible to either reduce the number of injections per animal to one or to reduce the injection volume to a minimum of 100 µL. For the latter, it is however important to maintain the cell density ratio e.g., 1.25 x 106 cells/100 µL BMEM. When working with BMEM, all the steps must be performed on ice to avoid solidification of the cell suspension before injection. During injection, it is important and that the needle is inserted at an angle of 45° directly under the skin and away from the muscle tissue, as injecting into muscle impedes lesion reproducibility and makes the lesion dissection difficult. A total of two injections can be performed on each mouse—one on the right and one on the left side of each animal. The second injection in the same mouse can serve as a technical replicate. More injections on the back are not recommended as lesions grow over time and might interfere with each other. For statistical analysis in pre-clinical studies comparing xenograft plugs of treated versus untreated (vehicle only) mice, we recommend the use of a minimum of 5 animals (10 xenograft plugs) per study group. If available, the second injection could alternatively be used as a ‘internal control’ using non-mutant EC. We have used primary non-mutant EC, such as HUVEC, as a control and have shown that these cells formed a negligible number of small channels14,15. Furthermore, in these HUVEC control lesion plugs, we have noticed infiltration of murine-derived vascular channels into the plug after day 9. If the experimental design requires longer incubation times, these infiltrating channels can be easily excluded from analysis by staining for a human-specific marker such as human-specific CD31 antibody or Ulex europaeus agglutinin I (UEA-I) that does not cross react with mouse.

To ensure that the lesion does not become a burden to animal health and wellbeing it is important to observe lesion size, record mouse weight daily, and pay attention to any side complications such as bleeding and bruising. If the lesion volume exceeds 500 mm3, the experiment has to be terminated.

When vascular lesions are enlarged and perfused, extreme attention must be paid during dissection to avoid rupturing the lesion. It is important to avoid touching the lesion plug with dissection tools and leave excessive surrounding tissue (such as skin) attached to the plug. This prevents collapse of the vascular structures within the xenograft plug which would interfere with accurate analysis.

Finally, to maintain consistency, it is important that the initial histological analysis begins in the center of the plug (about 50‒70 µm into the tissue) rather than the border regions where anastomosing mouse vasculature might be present. It is highly recommended to stain the tissue sections with a human-specific EC marker, such as UEA-I or an alternative human-specific antibody which will not cross-react with mouse, in order to confirm that vascular structures are formed by human-derived EC rather than invading mouse EC.

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

The authors have no conflicts-of-interests to disclose.

Acknowledgments

The authors would like to thank Nora Lakes for proofreading. Research reported in this manuscript was supported by the National Heart, Lung, and Blood Institute, under Award Number R01 HL117952 (E.B.), part of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Materials

Name Company Catalog Number Comments
Athymic nude mice, (Foxn1-nu); 5-6 weeks, males Envigo 069(nu)/070(nu/+) Subcutaneous injection
Biotinylated Ulex europeaus Agglutinin-I (UEA-I) Vector Laboratories B-1065 Histological anlaysis
Bottle top filter (500 ml; 0.2 µM) Thermo Fisher 974106 Cell culture
Bovine Serum Albumin (BSA) BSA A7906-50MG Cell culture; Histological analysis
Calcium cloride dihydrate (CaCl2.2H2O) Sigma C7902-500G Cell culture
Caliper Electron Microscopy Sciences 50996491 Lesion plug measurment
CD31-conjugated magnetic beads (Dynabeads) Life Technologies 11155D EC separation
Cell strainer (100 μM) Greiner 542000 Cell culture
Collagenase A Roche 10103578001 Cell culture
Conical Tube; polypropylene (15 mL) Greiner 07 000 241 Cell culture
Conical Tube; polypropylene (50 mL) Greiner 07 000 239 Cell culture
Coplin staining jar Ted Pella 21029 Histological anlaysis
Coverglass (50 X 22 mm) Fisher Scientific 12545E Histological anlaysis
DAB: 3,3'Diaminobenzidine Reagent (ImmPACT DAB) Vector Laboratories SK-4105 Histological anlaysis
Dulbecco's Modification of Eagle's Medium (DMEM) Corning 10-027-CV Cell culture
DynaMag-2 Life Technologies 12321D EC separation
Ear punch VWR 10806-286 Subcutaneous injection
EDTA (0.5M, pH 8.0) Life Technologies 15575-020 Histological anlaysis
Endothelial Cell Growth Medium-2 (EGM2) Bulletkit (basal medium and supplements) Lonza CC-3162 Cell culture
Eosin Y (alcohol-based) Thermo Scientific 71211 Histological anlaysis
Ethanol Decon Labs 2716 Histological anlaysis
Fetal Bovine Serum (FBS) , HyClone GE Healthcare SH30910.03 Cell culture
Filter tip 1,250 μL MidSci AV1250-H Multiple steps
Filter tip 20 μL VWR 10017-064 Multiple steps
Filter tip 200 μL VWR 10017-068 Multiple steps
Formalin buffered solution (10%) Sigma F04586 Lesion plug dissection
Hemacytometer (INCYTO; Disposable) SKC FILMS DHCN015 Cell culture
Hematoxylin Vector Hematoxylin H-3401 Histological anlaysis
Human plasma fibronectin purified protein (1mg/mL) Sigma FC010-10MG Cell culture
Hydrogen Peroxide solution (30% w/w) Sigma H1009 Histological anlaysis
ImageJ Software Analysis
Isoflurane, USP Akorn Animal Health 59399-106-01 Subcutaneous injection
magnesium sulfate heptahydrate (MgSO4.7H2O) Sigma M1880-500G Cell culture
Basement Membrane Matrix (Phenol Red-Free; LDEV-free) Corning 356237 Subcutaneous injection
Microcentrifuge tube (1.5 mL) VWR 87003-294 EC separation
Microscope Slide Superfrost (75mm X 25mm) Fisher Scientific 1255015-CS Histological anlaysis
Needles, 26G x 5/8 inch Sub-Q sterile needles Becton Dickinson (BD) BD305115 Subcutaneous injection
Normal horse serum Vector Laboratories S-2000 Histological anlaysis
Penicillin-Streptomycin-L-Glutamine (100X) Corning 30-009-CI Cell culture
Permanent mounting medium (VectaMount) Vector Laboratories H-5000 Histological anlaysis
Pestle Size C, Plain Thomas Scientific 3431F55 EC isolation
Phosphate Buffered Saline (PBS) Fisher Scientific BP3994 Cell culture
Scale VWR 65500-202 Subcutaneous injection
Serological pipettes (10 ml) VWR 89130-898 Cell culture
Serological pipettes (5ml) VWR 89130-896 Cell culture
Sodium carbonate (Na2CO3) Sigma 223530 Cell culture
Streptavidin, Horseradish Peroxidase, Concentrate, for IHC Vector Laboratories SA-5004 Cell culture
Syringe (60ml) BD Biosciences 309653 Cel culture
SYRINGE FILTER (0.2 µM) Corning 431219 Cell culture
Syringes (1 mL with Luer Lock) Becton Dickinson (BD) BD-309628 Subcutaneous injection
Tissue culture-treated plate (100 X 20 mm) Greiner 664160 Cell culture
Tissue culture-treated plate (145X20 mm) Greiner 639160 Cell culture
Tissue culture-treated plates (60 X 15) mm Eppendorf 30701119 Cell culture
Tris-base (Trizma base) Sigma T6066 Histological anlaysis
Trypan Blue Solution (0.4 %) Life Technologies 15250061 Cell culture
Trypsin EDTA, 1X (0.05% Trypsin/0.53mM EDTA) Corning 25-052-Cl Cell culture
Tween-20 Biorad 170-6531 Histological anlaysis
Wheaton bottle VWR 16159-798 Cell culture
Xylenes Fisher Scientific X3P-1GAL Histological anlaysis

DOWNLOAD MATERIALS LIST

References

  1. Dompmartin, A., Vikkula, M., Boon, L. M. Venous malformation: update on aetiopathogenesis, diagnosis and management. Phlebology: The Journal of Venous Disease. 25, (5), 224-235 (2010).
  2. Limaye, N., et al. Somatic mutations in angiopoietin receptor gene TEK cause solitary and multiple sporadic venous malformations. Nature Genetics. 41, (1), 118-124 (2009).
  3. Castel, P., et al. Somatic PIK3CA mutations as a driver of sporadic venous malformations. Science Translational Medicine. 8, (332), 42 (2016).
  4. Limaye, N., et al. Somatic Activating PIK3CA Mutations Cause Venous Malformation. The American Journal of Human Genetics. 97, (6), 914-921 (2015).
  5. Castillo, S. D., et al. Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans. Science Translational Medicine. 8, (332), 43 (2016).
  6. Stratman, A. N., et al. Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP-dependent proteolysis in 3-dimensional collagen matrices. Blood. 114, (2), 237-247 (2009).
  7. Okada, S., Vaeteewoottacharn, K., Kariya, R. Application of Highly Immunocompromised Mice for the Establishment of Patient-Derived Xenograft (PDX) Models. Cells. 8, (8), 889 (2019).
  8. Byrne, A. T., et al. Interrogating open issues in cancer precision medicine with patient-derived xenografts. Nature Reviews Cancer. 17, (4), 254-268 (2017).
  9. Allen, P., Melero-Martin, J., Bischoff, J. Type I collagen, fibrin and PuraMatrix matrices provide permissive environments for human endothelial and mesenchymal progenitor cells to form neovascular networks. Journal of Tissue Engineering and Regenerative Medicine. 5, (4), 74 (2011).
  10. Allen, P., Kang, K. T., Bischoff, J. Rapid onset of perfused blood vessels after implantation of ECFCs and MPCs in collagen, PuraMatrix and fibrin provisional matrices. Journal of Tissue Engineering and Regenerative. 9, (5), 632-636 (2015).
  11. Nowak-Sliwinska, P., et al. Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis. 21, (3), 425 (2018).
  12. Roh, Y. N., et al. The results of surgical treatment for patients with venous malformations. Annals of Vascular Surgery. 26, (5), 665-673 (2012).
  13. Marler, J. J., Mulliken, J. B. Current management of hemangiomas and vascular malformations. Clinics in Plastic Surgery. 32, (1), 99-116 (2005).
  14. Goines, J., et al. A xenograft model for venous malformation. Angiogenesis. 21, (4), 725-735 (2018).
  15. Li, X., et al. Ponatinib Combined With Rapamycin Causes Regression of Murine Venous Malformation. Arteriosclerosis, thrombosis, and vascular biology. 39, (3), 496-512 (2019).
  16. Le Cras, T. D., et al. Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation. Angiogenesis. 1-18 (2020).
  17. Boscolo, E., et al. Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects. Journal of Clinical Investigation. 125, (9), 3491-3504 (2015).
This article has been published
Video Coming Soon
PDF DOI

Cite this Article

Schrenk, S., Goines, J., Boscolo, E. A Patient-Derived Xenograft Model for Venous Malformation. J. Vis. Exp. (160), e61501, doi:10.3791/61501 (2020).More

Schrenk, S., Goines, J., Boscolo, E. A Patient-Derived Xenograft Model for Venous Malformation. J. Vis. Exp. (160), e61501, doi:10.3791/61501 (2020).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
simple hit counter