This study explores the use of genetically engineered mesenchymal stem cells (MSCs) expressing TRAIL to induce apoptosis in tumor cells. The methodology involves the implantation of MSC-seeded scaffolds into a tumor-bearing mouse model, allowing for targeted therapy against brain tumors.
Mesenchymal stem cells or MSCs can be genetically engineered to express tumor necrosis factor-related apoptosis-inducing ligand or TRAIL, which binds to specific receptors on tumor cells inducing apoptosis.
To deliver these MSCs into a tumor-bearing mouse model, first, prepare a TRAIL-expressing MSC-seeded biopolymer scaffold. Next, secure an anesthetized mouse bearing fluorescent-labeled brain tumor on a stereotactic frame. The mouse has a pre-established cranial window for accessing the brain.
Maintain the mouse's body temperature with a heating pad. Sterilize the surgical site and make a midline incision on the scalp. Remove the subdermal fat to reveal the cranial window. Peel off the dura mater from the window, exposing the underlying parenchymal tissue and tumor.
Now, under a fluorescence stereomicroscope, visualize the fluorescent tumor. Aspirate the fluorescent tumor tissue from the brain. Wash with saline to restore the physiological pH. Implant the MSC-seeded scaffold into the resection cavity. Apply a suitable hemostatic agent to secure the scaffold in place. Close the incision and allow the mouse to recover.
Following implantation, tumor cells within the residual tumor foci secrete soluble factors, which facilitate the migration of MSCs from the scaffold towards the tumor cells. The TRAIL proteins secreted by MSCs bind to specific receptors on tumor cells, inducing apoptotic cell death.
Begin by placing the stereotaxic frame on the stage of a fluorescence dissecting stereomicroscope. Then, secure the anesthetized mouse on the stereotaxic frame with continuous inhaled anesthesia supply via an isoflurane nose cone adapter. Maintain body temperature with a heating pad. Apply ophthalmic ointment to the eyes to prevent drying of the cornea. Then, sterilize the incision site of the scalp with a series of three alcohol and three betadine wipes.
Perform the toe pinch reflex test on each limb and confirm negative response to ensure proper anesthetization. Using forceps, pinch and gently lift the scalp. After making a midline linear rostral-caudal incision with surgical scissors, irrigate the incision site with PBS and remove subdermal fat with a cotton tip applicator in a circular brushing motion.
Arrange the skin so that the previously established cranial window is fully visible. Use an 18 gauge needle to gently puncture the dura just interior to the borders of the cranial window. Repeat until the incision fully traces the interior of the window. Remove the dura by peeling it away using fine forceps, revealing the underlying parenchyma and tumor.
Next, dim the room lights and turn the stereomicroscope fluorescence mode on. Locate the U87 mCherry and firefly luciferase-expressing tumor. Load a 200-microliter pipette tip into the end of the tubing of a vacuum pump. Then, turn on the vacuum pump. Resect the tumor by gently aspirating the fluorescent tissue until no signal remains. To control bleeding, irrigate with cold PBS and apply steady pressure with a cotton tip applicator.
Use a combination of saline, steady pressure with a cotton tip applicator, and/or a hemostatic agent, such as SURGICEL, to stop bleeding prior to implanting the scaffold. If not properly controlled, a potentially dangerous hematoma may form. After resection, turn off the vacuum pump and discard the pipette tip. Turn the fluorescence off and the room lights back up. Immediately prior to implantation, slowly dip an MSC-seeded PLA scaffold in PBS to remove unwanted media and associated components. Implant the scaffold into the resection cavity.
If needed, add 1 microliter of fibrinogen, followed by 1 microliter of thrombin to secure the scaffold in place. With the dura removed and the bone flap from the cranial window already discarded, seal the wound by closing the skin and applying surgical glue. Remove the animal from inhaled anesthetic and allow it to recover on a heated surface.
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