This protocol can be used to create patient-derived xenografts that recapitulate the histopathology of venous malformations. This was further allowed to carry our preclinical therapeutic testing. This technique provides a fast and reliable NVivo system that allows daily monitoring of the growth of patient direct endothelial cells and the response to experimental therapy.
This method could be easily adapted to investigate other types of vascular or lymphatic anomalies. Begin by preparing the cell suspension for injection. Trypsinize the endothelial cells with five milliliters of pre-warmed 0.05%tripsin-EDTA at 37 degrees Celsius for two minutes.
Neutralize the tripsin and by adding five milliliters of EGM and collect the cells into 150 milliliter conical tube. Centrifuge it to 400 times G for five minutes then aspirate the supernatant and resuspend the cells in three milliliters of EGM. Count the cells with a hemocytometer.
Venture the volume with the desired cell number into a new 50 milliliter conical tube and pellet the cells again by centrifugation. Aspirate the supernatant leaving a small volume to loosen the cell pellet. Resuspend the cell pellet with 220 microliters of BMEM per injection.
Mix the cell suspension thoroughly on ice making sure to avoid creating bubbles. Aspirate the BMEM cell culture mix into a one milliliter syringe opening by pulling the plunger. You'll lock a 26 gauge sterile needle to the syringe and keep the prepared syringes flat on ice prior to injection.
After ensuring that the mouse has properly anesthetized place it on its stomach and disinfect the injection region with 70%ethanol. Gently roll the prepared syringe to resuspend any settled cells. Flake bubbles to the end of the syringe and expel a small volume of the cell suspension.
Pinch the skin at the injection site to create a tent like structure. Then insert the needle right under the skin and sure that the needle is only skin deep by releasing pinch skin to prevent injection into muscle tissue. Holding the needle steadily carefully inject 200 microliters of the cell suspension to create a small spherical mass.
Take care to avoid injecting the cell suspension into muscle. Measure the length and width of each plug with a caliper. Align the dissected xenograft plugs on a cutting board next to a ruler and take an image to record the gross vascularity of the lesions.
Then fix the plugs by submerging them in 10%formalin overnight at room temperature. After dissection, the lesion plugs are processed for histological analysis and stained for UEA-1 to detect human derived endothelial cells within the plug. Take five HPF images per lesion section with a bright field microscope at at 20 X magnification in an X plane pattern within the lesion section to avoid overlap.
Make sure to include a scale bar on the images taken. Open the HPF images in image J.To calibrate the pixels of the scale bar, use the straight line tool and go over the scale bar. Then convert the measured pixels into millimeters by clicking on Analyze and Set Scale.
Next click on Analyze Set Measurements and select area and add to overlay. Measure the total field area of the HPF using analyze and measure saving this measurement for quantification. Using the free hand selections tool manually outline the UEA-1 positive vascular channel.
Click on Analyze and Measure to quantify the outlined area. Measure all vascular channels within the HPF. Repeat the process for the five HPF taken within each section and transfer the values to Excel for further analysis.
Then calculate the percentage of vascular area and vascular density according to manuscript directions. Using this protocol the lesion plugs with TIE2 or PIK3CA hyperactive mutant endothelial cells are visibly vascularized and perfused within seven to nine days after injection. The lesion plugs closely resemble the histopathological features of human VM tissue and large vascular channels aligned by a thin layer of endothelial cells are visible.
These vascular structures typically contain erythrocytes confirming functional anastomosis with the host mouse vasculature. Immunohistochemical staining using the human specific lectin UEA-1 confirms that the cells lining vascular regions are derived from human implanted cells rather than mouse vasculature. Following this procedure, additional staining of lesion sections for markers of proliferation or apoptosis can be performed to analyze specific effects of experimental treatments.
This scenographic model has been used for preclinical studies of new therapies for venous malformation, such as the mTOR inhibitor rapamycin, which has shown efficacy in several clinical trials for VM patients.
