Glioblastoma multiforme remains the most frequent and aggressive primary brain tumor in adults. Despite aggressive treatments based on surgery followed by radio and chemotherapy, GBM is associated with an extremely poor prognosis and a median survival lower than 15 months. Since few therapeutic advances have been made over the last decade, cellular immunotherapies are currently explored to eliminate highly-invasive and resistant GBM cells likely involved in rapid tumor relapse.
administrations of selected GBM-reactive cytotoxic immuno factors in the vicinity of the tumor could represent a unique opportunity to deliver concentrated cellular immunotherapy, directly into the site of residual malignancy. Our group recently showed that immunodeficient NSG mice carrying orthotopic primary human GBM xenograft recapitulate GBM to more development in patients. These GBM models were used to evaluate the efficacy of cytotoxic immune effectors on intratumoral injections.
This protocol describes our therapeutical process based on first the isolation and amplification of GBM-reactive immune effector cells. Second, the propagation of these effector cells for intercranial injection. And third, their stereotactic injection at the tumor site within the mouse brain.
The behavior of the immune effector after orthotopic injection was also investigated. Three weeks after PHA fetus stimulation, effector cells, which form cell clump in the bottom of the flask should have returned to rest state and are ready to be used. After checking effector cell count, resuspend and collect cells in 115 milliliters tube and centrifuge them.
Carefully and completely remove a supernatant. And resuspend cells in sterile PBS and centrifuge to wash them. Carefully and completely take off the supernatant.
And then resuspend cells in one milliliter of sterile PBS. Transfer cells in a microtube for centrifugation. Again, carefully and completely take off the supernatant by pipetting slowly.
The next step is critical, resuspend cells in half of the final volume of sterile PBS and homogenize softly and carefully. Verify the final volume with micropipette, final volume per mouse has to be between 15 and 20 microliter for 20 million cells. Be sure it will not exceed 20 microliter per mouse.
Keep cells on ice until injection. Ensure that the animal is adequately anesthetized by a toe pinch and then remove fur from the surgical site, between the two ears and up to the nose. Resuspend cells slowly to prevent cell clumping and load cell suspension into syringe with great care to avoid the presence of bubbles.
Then, place the syringe into the adapted syringe pump. Disinfect the surgical site with salt and povidone iodine 5%solution and place a lubricating ophthalmic ointment in the mouse's eyes to prevent drying of the cornea. Then, position the anesthetized mouse on the stereotactic frame on a warm block.
Appropriately position mouse's nose and teeth above the tooth bar. Tighten ear bars firmly in the mouse ears to immobilize the head. Be careful not to damage the eardrums, compromise the respiration, and make sure that head is well immobilized.
Make a midline sagittal skin incision with sterile scissors along the upper part of the cranium to expose the skull. Identify the intersection of the sagittal and coronal structures to serve as landmarks for stereotactic localization, and place the syringe above this point. Move the syringe at predetermined coordinates.
Two millimeter right lateral, and 0.5 millimeter anterior of the Using a microdrill, make a small hole in the skull with sterile drill bit. Be careful to remain superficial in order to avoid brain injury. Insert syringe carefully into the drilled hole.
Move slowly forward the needle three millimeter down in the dura, and then backward 0.5 millimeter to a final depth of 2.5 millimeter cells injection. Run the cell injection at two or three microliter per minute, and monitor the mice all along the injection time. Once injection is complete, withdraw the needle for only one millimeter and keep syringe in place for an additional minute before slowly withdrawing completely the syringe to prevent any leakage from the infusion site.
Remove carefully the animal from the stereotactic frame. Close skin with appropriate surgical suture and apply 2%Lidocaine gel directly on the wound. Transfer anesthetized mouse to its respective cage above heating pad set to 37 degrees Celsius to maintain appropriate mouse body temperature until full recovery from anesthesia.
As the suspension is very concentrated, the cell viability and reactivity were checked. Effector cells were loaded in syringe in order to mimic in vivo injection. Cells were collected immediately or 10 minutes later and were analyzed by flow cytometry for propidium iodide staining.
As you can see, the preparation and the loading did not affect the viability of effector cells at 24 hours. The reactivity against Glioblastoma tumor cells were also checked using either unprepared cells or effectors prepared and stayed three hours on the eyes. Reactivity is monitored by positive staining of the activation marker CD107A.
These results demonstrate that the effector cells'reactivity were not affected by the preparation. Seven days after effector cells'injection, mice brains were collected, sectioned, and stained for hematic coloration to allow the identification of tumor structure. Then anti-human CD3 staining allowed localization of effector cells.
Interestingly, effector cells were found around the tumor and in the tumor core, but also in contralateral hemisphere. Human effector T-cells isolated from mouse brain 48 hours after the injection represent 2%of brain cells. They were still able to proliferate in presence of PHA, feeders and IL2 stimulation, with a 99%purity after amplification.
Thanks to this protocol, resting human effector cells can survive and patrol with a mouse brain parenchyma for several days following the stereotaxic injection. In conclusion, adoptive transfer of human effector cells by stereotactic injections represent promising approaches to efficiently treat infiltrative malignant brain tumors with limited deleterious effects on healthy cells. This route of administration takes advantage that human cells are delivered closely to the tumor site, limiting their dilution within the tissues and the organism.
Here we described the procedure following the delivering of a large amount of immune cells within the brain tumor without harmful effects on effector cells and, importantly, without any visible deleterious effect on mice. Moreover, locally-injected immune cells in mouse brain are not only able to survive and keep their ability to be activated and amplified ex vivo, but also to and eliminate disseminated tumor cells in the surrounding brain tissue. Collectively, these features are essential to allow the elimination of strong infiltrative tumor cells that characterize GBM tumors.
This protocol opens new opportunity for the establishment of adoptive transfer procedure in mice model of brain tumor. Essential step before considering clinical CD.
