The overall goal of this experimental procedure is to develop a preclinical mouse model to study the role of extracellular vesicle-mediated communication between tumor and mesenchymal stem cells. This method can help answer several key questions in the cancer biology field, as to how tumor and mesenchymal stem cells communicate by means of extracellular vesicles, and whether this supports cancer progression. The main advantage of this technique is that it allows to study the role of mesenchymal stem cell adaptation by cancer EVs, on the progression and metastasis formation in vivo.
The implication of this procedure extends to our therapy of cancer, because we can use GSA to interfere with EV communication and androgen signaling to inhibit cancer progression. Besides providing insights into the EV-mediated communication in osteosarcoma, this method can be also applied to many other cancer types. Indeed, bone marrow derived from the mesenchymal stem cells are readily recruited to more sites where they can differentiate into cancer-supporting cells.
Begin by seeding eight 175 square centimeter flasks with 3 million 143B cells each. Culture the cells using EV-depleted medium for 36 to 48 hours. When the cells are 80 to 90%confluent, collect the supernatants from the eight flasks for EV isolation.
Next, use differential centrifugation to remove all of the cells and cell debris from the supernatant. This consists of repeated centrifugations at four degrees Celsius, with maximum acceleration and deceleration. After each centrifugation step, collect the supernatant, and leave behind one milliliter of the supernatant in the centrifugation tube.
Now, load a pre-cleared supernatant into ultracentrifuge tubes at 38.5 milliliters per tube. Then, isolate the EVs using an ultracentrifuge at 70, 000g for one hour, with a slow break and at four degrees Celsius. After the ultracentrifugation, carefully remove the supernatant, leaving behind about one milliliter.
Then, resuspend the EV-containing pellets in the remaining volume. Pool all the suspensions. And wash the pooled suspensions with PBS.
Fill the tube to 38.5 milliliters. Then, repeat the ultracentrifugation cycle, but use no break. The rotor should stop spinning after about an hour.
Next, carefully remove the supernatant without disturbing the pellet, and leave 100 to 200 microliters of solution. Finally, resuspend the EV pellet, and adjust the final volume to 200 microliters with PBS. The EVs can then be characterized by TEM, labeled with dyes for uptake experiments, or used to educate mesenchymal stem cells for in vivo experiments.
It is crucial that the primary MSCs are at the right cell density before exposure to cancer EVs. The cell should be around 70%confluent to achieve optimal cell EV ratio, and to also prevent overgrowth during education periods. This experiment uses acclimated adult Athymic Nude-Foxn1nu mice.
One day before the surgical procedure, provide paracetamol via the drinking water. On the day of the surgery, prepare 143B cells at 200, 000 per microliter in PBS. 20 minutes before the surgery, provide the animal with buprenorfine.
Just before anesthesia, load a 10 microliter syringe with the concentrated 143B cells. After confirming proper anesthesia and applying eye ointment, position the animal on its back with its rear legs towards the operator. Next, secure an anesthesia mask to the animal.
Then, flex the left knee and wipe the skin with three alternating scrubs of 70%ethanol and povidone-iodine, and then apply 2%lidocaine as a local analgesic. Now, make a small incision into the skin just below the knee to expose the tibia. Then, using a 0.8 millimiter microtwist drill, make a pinhole in the tibia, approximately 2 millimeters below the knee.
Avoid drilling through both tibia cortices. Proceed by slowly injecting one microliter of cells into the hole over about five seconds. Then, close the hole with a droplet of tissue glue to prevent backflow of the suspension.
Complete the surgery by closing the skin with sutures and one drop of tissue glue. Allow the animal to recover with heating, and treat it with paracetamol for 24 hours. Two days later, use a 0.5 millimeter insulin syringe to inject the MSCs into the tail vein.
Correct administration during a tail vein injection can be visually confirmed. If a white area appears, or if you encounter resistance, please inject again at an area above the previous injection site. Then, twice per week, follow the tumor growth using bioluminescence.
Inject the mice with D-luciferin via an insulin syringe, and 10 minutes later, measure the photon flux generated from the tumor cells using an imaging system. When an animal's tumor diameter becomes greater than 15 millimeters, or its weight loss exceeds 15%euthanize the animal and collect all the relevant tissues. 10 minutes before euthanization, inject D-luciferin, as done prior to imaging.
After euthanasia, collect the lungs, liver, spleen, and kidneys, using sterilized scissors and tweezers. Wash the blood from the organs using PBS, and arrange them on a Petri dish for imaging. Using a bioluminescence imaging system, document both sides of the organs.
Once images are taken, immediately fix the organs for future histological analysis. To process the photo documents, simply apply manual thresholding, and count the number of nodules in each photo. Be certain to use the same threshold for each photo, and to document the tissues from every direction, especially the lungs.
After decalcifying the mouse tibia, proceed with dehydration and paraffin embedding. Then, use a microtome to prepare six-micron paraffin sections, and collect the sections on glass slides. Once the slides have dried, store them overnight at room temperature.
The next day, perform a heat-mediated antigen retrieval, using citrate buffer. Then, stain the tissues with anti-GFP antibody at one to 900, and follow with a DAPI counterstain. Finally, document the stained tissue sections using fluorescence microscopy.
The purity of isolated EVs was confirmed by electron microscopy. The preparations contained vesicles between 40 and 100 nanometers in diameter. To assess whether cancer EVs interact with MSCs, the EVs were labeled with a green fluorescent lipophilic linker dye, and incubated overnight with MSCs.
Efficient EV uptake by MSCs was thus confirmed. An immunomodulatory property of EVs on MSCs was confirmed using a multiplex B-based cytokine immunoassay. Tumor EVs caused increased IL-8 and IL-6 production in MSCs, compared with human fibroblast control EVs.
Next, in an orthotopic xenograft mouse model of osteosarcoma, mice treated with EV-educated MSCs were shown to have accelerated tumor growth. Four days after systemic injection of GFP-expressing MSCs, GFP-expressing cells were detected in the bone marrow and in the tumor tissue, demonstrating MSC homing to the tumor site. Ex vivo imaging of various organs highlighted lung nodule formation.
Ultimately, mice receiving EV-educated MSCs had more lung metastases. After watching this video, you should have a good understanding of how to educate stromal cells with cancer EVs ex vivo, and study the effects of educated stromal cells in vivo using preclinical mouse models. Once mastered, this procedure can be performed within four weeks, provided that enough cancer EVs are available.
While attempting this procedure, it is important to plan ahead and get a reproducible purification of cancer extracellular vesicles, as well as to use low-passage proliferating mesenchymal stem cells.
