Funding for this work was provided by the Matthew Larson Foundation, Grayson Saves Foundation, Hyundai Hope on Wheels Young Investigator Award, Kortney Rose Foundation, National Institutes of Health NCI K12 CA076931-19 and 1K08CA263179-01, and the Department of Defense W81XWH-21-1-0221.

All protocol procedures were approved by the Institutional Animal Care and Use Committee of the Children&#39;s Hospital of Philadelphia (IAC 19-000907), which is accredited by the AAALAC. This study used 6-12-week-old NOD scid gamma (NSG) mice with orthotopic xenograft tumors; however, the protocol can be utilized on any mouse strain.&#160;NSG mice were housed in sterile barrier conditions and surgery performed under sterile biosafety cabinets. When human material such as tumor cells or T cells are being used, procedures and handling must be completed in ABSL-2 biosafety cabinets.
1. Preparing the mouse for surgery
<ol>
	<li>Anesthetize the mouse in an induction chamber with isoflurane (2-4%) at an oxygen flow rate of 1 L/min&#160;until an adequate plane of anesthesia is reached (approximately 5 min).</li>
	<li>Weigh the mouse using a scale to the nearest 0.1 g and administer subcutaneous slow-release (SR) buprenorphine (1 mg/kg) or other analgesic. 
	NOTE: SR buprenorphine provides analgesia for 72 h.</li>
	<li>Shave the fur on the top of the mouse&#39;s head using electric clippers or depilatory agents.</li>
	<li>Use a spatula to gently open the bottom of the stereotactic arm and insert the guide cannula using forceps. Tighten the screw on the arm to secure the cannula so that approximately 1/2 to 2/3 of the white plastic portion of the cannula is protruding from the bottom of the opening, along with the entire 5 mm metal length of the cannula.</li>
	<li>Insert and secure the mouse&#39;s top teeth in the bite bar of the stereotaxic apparatus. Pull the nose cone forward and tighten it, ensuring the mouse is inhaling isoflurane.</li>
	<li>Mount the mouse on the warmed stereotaxic apparatus using ear cuffs or ear bars, avoiding excessive pressure. 
	NOTE:&#160;Warmed tray should have a rectal thermometer inserted, and the warming tray should adjust to maintain a normal body temperature of the mouse during the procedure.</li>
	<li>Apply sterile ophthalmic ointment to both eyes using a cotton-tipped applicator.</li>
	<li>Wipe the surgical site with povidone-iodine on a pad or applicator, followed by an alcohol pad. Perform this step three times in total.</li>
	<li>Before beginning the procedure, perform a toe pinch with forceps to assess for adequate sedation.</li>
</ol>
2. Surgical procedure
NOTE: All aspects of the surgical procedure utilize sterilized instruments and aseptic techniques. The mice continue under anesthesia with isoflurane (2-4%) throughout the duration of the procedure, approximately 10-20 min.
<ol>
	<li>Gently pick up the scalp between the ears with forceps. Using sterile&#160;scissors, cut the lifted scalp parallel to the skull and remove an oval flap of skin (0.75-1 cm in length) to expose the skull. 
	NOTE: Scissors are preferred over a scalpel to provide a clean, oval-shaped opening and to prevent unnecessary damage to surrounding skin and tissue.&#160;</li>
	<li>Push away fascia using a scalpel or cotton-tipped swabs and a hemostatic cotton pellet to help slow excessive bleeding as needed. 
	NOTE: Using the wooden side of a sterile cotton tip can also push away fascia and help avoid excessive bleeding.</li>
	<li>Identify the landmarks bregma and lambda, the respective anterior and posterior marks on the skull where the cranial plates meet13. 
	NOTE: Identification can be augmented by wiping the top of the exposed skull with hydrogen peroxide.</li>
	<li>Gently score the skull using a scalpel to create a surface for the acrylic to attach. Scoring should include multiple linear lines approximately 0.5-1 cm in length at 90&#176; angles to each other.</li>
	<li>Using the stereotaxic arm, localize the cannula to the landmark of interest (bregma or lambda). Once localized, raise the cannula tip 1-2 mm above the skull surface and move to the desired coordinates. For intratumoral injections, this uses the same A/P and M/L coordinates as the tumor placement.</li>
	<li>On the exposed skull, away from the area where the cannula will enter, make two screw holes with an 18 G needle or a surgical drill. Ensure that the holes are spaced out to include enough room for the cannula. Using a drill bit, twist through the screw holes until they catch on the skull. Insert and fasten two screws into the holes using a flat-tipped screwdriver. Then, gently pull the screws up to ensure they are secured. 
	NOTE: Do not insert the screws until they are flush with the skull, or they may damage the mouse brain underneath. Leave at least a 1-2 mm gap between the screw and the skull.</li>
	<li>Using an 18 G needle or surgical drill, drill through the skull at the identified coordinates to create a hole for the cannula to be inserted.</li>
	<li>Using the stereotactic arm, lower the cannula to the desired D/V coordinate. 
	NOTE: The D/V coordinate of cannula implantation needs to account for the projection length of the dummy and treatment cannulas, and may be more superficial than the orthotopic injection of tumor cells (Figure 1).</li>
	<li>In a porcelain 12-well plate, fill one well with acrylic resin powder (approximately 0.3 g) and 10-15 drops (approximately 0.5-0.75 mL) of acrylic resin liquid. This produces a viscous white-colored material. Draw up the mixture into a 1 mL syringe and use it to coat and cover the skull, filling in the spaces around the cannula and screws. 
	NOTE: The viscous material hardens into cement over time, so this step should be completed promptly after mixing.</li>
	<li>While the cement is still pliable, loosen the screw on the stereotactic arm and use a spatula in the opening at the bottom to gently release the cannula from the holder and slowly retract the stereotactic arm upward away from the mouse.</li>
	<li>Once the cement is completely dry, insert the dummy cannula into the guide cannula and tightly secure it by turning clockwise.</li>
	<li>Once the procedure is complete, place the mouse back in its warmed home cage to carefully monitor, ensuring adequate recovery and recording any post-procedure observations, including that the mouse has fully regained consciousness, before returning to the colony. 
	NOTE: It is generally recommended to place only half of the cage on a heating pad to allow for the animal to move to the cooler side to avoid&#160;overheating.</li>
	<li>Administer additional analgesics as needed if the mice display behaviors indicative of pain post-operatively, such as meloxicam 5 mg/kg subcutaneously delivered once daily for up to 3 days.</li>
</ol>
3. Preparing CAR T cells
<ol>
	<li>Measure the pre-transfected CAR T cell concentration using a cell counter.</li>
	<li>Centrifuge pre-transfected T cells at 200 x g for 5 min at room temperature (RT).</li>
	<li>Aspirate the supernatant using a sterile Pasteur pipet on a vacuum aspiration system and resuspend the pellet in phosphatae buffered saline (PBS) to a desired concentration. Typical delivery volumes are 2-5 &#181;L. Typical cell doses are 0.5-5 x 106 cells.</li>
</ol>
4. CAR T cell infusions
<ol>
	<li>Prepare the treatment cannula by feeding the top through a small piece of PKG tubing.</li>
	<li>Fill the treatment syringe with the CAR T cell suspension and insert it through the other end of the PKG tubing, enough to cover the top of the treatment cannula.</li>
	<li>Anesthetize the mouse with isoflurane (2-4%) at an oxygen flow rate of 1 L/min.</li>
	<li>Stabilize the guide cannula using forceps at the base, and then carefully unscrew and remove the dummy cannula, allowing for access into the guide cannula. 
	NOTE: A stereotaxic setup is not necessary, but can be used to stabilize the head for treatment.</li>
	<li>Infuse the CAR T cells for 1 min and hold the treatment cannula in place for an additional 1 min following the cessation of infusion.</li>
	<li>Remove the treatment cannula and screw the dummy cannula tightly back in place.</li>
	<li>Administer subcutaneous meloxicam (5 mg/kg) for optional pain control.</li>
</ol>

<ol>
	<li>Curtin, S. C., Minino, A. M., Anderson, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Declines+in+cancer+death+rates+among+children+and+adolescents+in+the+United+States,+1999-2014.">Declines in cancer death rates among children and adolescents in the United States, 1999-2014.</a> NCHS Data Brief. 257, 1-8 (2016).</li><li>Rohaan, M. W., Wilgenhof, S., Haanen, J. B. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cellular+therapies:+the+current+landscape.">Adoptive cellular therapies: the current landscape.</a> Virchows Archiv. 474 (4), 449-461 (2019).</li><li>Sadelain, M., Brentjens, R., Riviere, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basic+principles+of+chimeric+antigen+receptor+design.">The basic principles of chimeric antigen receptor design.</a> Cancer Discovery. 3 (4), 388-398 (2013).</li><li>Maude, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tisagenlecleucel+in+children+and+young+adults+with+B-cell+lymphoblastic+leukemia.">Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia.</a> The New England Journal of Medicine. 378 (5), 439-448 (2018).</li><li>Wagner, J., Wickman, E., DeRenzo, C., Gottschalk, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR+T+cell+therapy+for+solid+tumors:+Bright+future+or+dark+reality.">CAR T cell therapy for solid tumors: Bright future or dark reality.</a> Molecular Therapy. 28 (11), 2320-2339 (2020).</li><li>Vitanza, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locoregional+infusion+of+HER2-specific+CAR+T+cells+in+children+and+young+adults+with+recurrent+or+refractory+CNS+tumors:+an+interim+analysis.">Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis.</a> Nature Medicine. 27 (9), 1544-1552 (2021).</li><li>Majzner, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GD2-CAR+T+cell+therapy+for+H3K27M-mutated+diffuse+midline+gliomas.">GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas.</a> Nature. 603 (7903), 934-941 (2022).</li><li>Vitanza, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraventricular+B7-H3+CAR+T+cells+for+diffuse+intrinsic+pontine+glioma:+preliminary+first-in-human+bioactivity+and+safety.">Intraventricular B7-H3 CAR T cells for diffuse intrinsic pontine glioma: preliminary first-in-human bioactivity and safety.</a> Cancer Discovery. 13 (1), 114-131 (2023).</li><li>Theruvath, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locoregionally+administered+B7-H3-targeted+CAR+T+cells+for+treatment+of+atypical+teratoid/rhabdoid+tumors.">Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors.</a> Nature Medicine. 26 (5), 712-719 (2020).</li><li>Donovan, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locoregional+delivery+of+CAR+T+cells+to+the+cerebrospinal+fluid+for+treatment+of+metastatic+medulloblastoma+and+ependymoma.">Locoregional delivery of CAR T cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma.</a> Nature Medicine. 26 (5), 720-731 (2020).</li><li>Brown, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+glioblastoma+after+chimeric+antigen+receptor+T-Cell+therapy.">Regression of glioblastoma after chimeric antigen receptor T-Cell therapy.</a> The New England Journal of Medicine. 375 (26), 2561-2569 (2016).</li><li>Bourne, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+microdialysis:+30+years+as+a+tool+for+the+neuroscientist.">Intracerebral microdialysis: 30 years as a tool for the neuroscientist.</a> Clinical and Experimental Pharmacology and Physiology. 30 (1-2), 16-24 (2003).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatically+detecting+bregma+and+lambda+points+in+rodent+skull+anatomy+images.">Automatically detecting bregma and lambda points in rodent skull anatomy images.</a> PLoS One. 15 (12), 0244378 (2020).</li><li>Foster, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+GPC2-directed+chimeric+antigen+receptors+using+mRNA+for+pediatric+brain+tumors.">Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors.</a> Journal for Immunotherapy of Cancer. 10 (9), 004450 (2022).</li><li>Akhavan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+for+brain+tumors:+Lessons+learned+and+road+ahead.">T cells for brain tumors: Lessons learned and road ahead.</a> Immunological Reviews. 290 (1), 60-84 (2019).</li></ol>

JBF holds a patent related to glypican 2 (GPC2)-directed immunotherapies. All other authors have nothing to disclose.

CAR T cell therapy has revolutionized the treatment of hematological cancers and shows promising value in treating solid brain tumors6,7,8. This protocol was designed to enable the preclinical evaluation of locoregional CAR T cell delivery for the treatment of pediatric CNS tumors. The cannula system replicates an Ommaya or Rickham reservoir, an intraventricular catheter system currently being used in ongoing clinical trials of CAR T cell therapy in pediatric CNS tumors6,7,8, underscoring the relevance and translational potential of these methods. This system allows for the repeated delivery of CAR T cells that bypass the blood-brain barrier, again similar to methods being employed in ongoing clinical trials. Locoregional delivery can provide maximal efficacy in the CNS9 and may also reduce the risk of systemic toxicities associated with trafficking from circulation15. While stereotactic delivery can provide a single dose into the CNS, the advantage of this system is the opportunity to provide multiple repeated doses into a specified location in the CNS without the need for multiple surgeries. The limitations of this procedure include a fixed delivery site without the ability to change location or make adjustments once the cannula is in place, and the potential for dislodgement of the cannula.
A critical step in this protocol is the implantation of the fixed guide cannula at a D/V coordinate that takes into consideration the projection of the treatment cannulas. The treatment cannula will protrude beyond the tip of the guide cannula, and so care must be taken to ensure the placement will result in the delivery of CAR T cells to the region of interest. Projection lengths of the treatment cannula can be customized, and in the author&#39;s experience, 0.5 mm is a useful projection length. This length ensures that the therapeutic does not remain in the guide cannula upon dispensing, but also does not require significant adjustment of the D/V coordinates for the guide cannula to the region of interest. An additional important step in this protocol is the time the treatment cannula is left in place following CAR T cell infusion. The treatment cannula should be held in place for at least 1 min following the end of infusion, to prevent leaks and the loss of locoregional delivery of CAR T cell therapy.
Troubleshooting this method is straightforward, with most complications involving difficulty removing the dummy cannula or inserting the treatment cannula into the fixed guide cannula, likely due to dried blood on the interior of the guide cannula. This can be easily resolved by gently passing the dummy cannula through the guide cannula until there is less resistance and the debris has been cleared. The acrylic resin can occasionally dislodge from the skull, resulting in the loss of the cannula system. In our experience, this is generally limited by scoring the skull with a scalpel and the placement of two screws. In addition, any items from the cage that may accidentally apply force to the cannula while the mouse is moving around are removed, such as particular mouse enrichment huts with small openings.
In conclusion, described here is a protocol for the insertion of a cannula system in murine models of CNS tumors for the repeated delivery of CAR T cells. Cannula placement can be adjusted to multiple locoregional delivery locations, testing the efficacy of different delivery sites. In addition, this system can be used for additional therapeutics beyond CAR T cells to evaluate efficacy when bypassing the blood-brain barrier, and may also be useful for the evaluation of therapeutics in murine models of non-oncologic disorders.

Despite improvements in chemotherapy, radiotherapy, and surgery, central nervous system (CNS) tumors are the deadliest malignancy in pediatrics1, underscoring an important need for new approaches with more successful outcomes. With significant advances in the field of immunotherapy, adoptive cellular therapy (ACT) approaches have shown promising results in various cancers, especially hematological malignancies2. Chimeric antigen receptor (CAR) T cell therapy, a specific type of ACT, takes advantage of the immune system&#39;s natural ability to recognize and kill harmful cells by redirecting the specificity of T cells to generate tumor-targeting T cells3. CAR T cell therapy has demonstrated substantial success in the treatment of leukemias and lymphomas4, making it a promising immunotherapeutic approach and encouraging its investigation in solid tumors. However, thus far, CAR T cell therapy in solid tumors has achieved little clinical success and faces many challenges, such as inefficient tumor penetration, limited targetable antigens, and the suppressive tumor microenvironment5.
Recent clinical trials have begun evaluating CAR T cell therapy for pediatric CNS tumors, providing proof of concept and early evidence of T cell activity in preliminary reports6,7,8. While most initial preclinical data focused on intravenous delivery of the CAR T cells, recent preclinical evidence has suggested the superiority of locoregional delivery in the CNS9,10, which has also been utilized successfully in several clinical trials6,7,8,11. Preclinical studies to date that have incorporated the locoregional delivery of CAR T cells in the CNS have relied on a single intracranial dose of CAR T cells delivered stereotactically9,10. However, clinical trials in humans have required repeated infusions of CAR T cells in the CNS6,7,8,11, underscoring a need to evaluate multiple repeated infusions in preclinical development. The goal of this procedure is to successfully test serial CAR T cell infusions using a catheter in orthotopic murine models of pediatric brain tumors. The advantage of this technique is the avoidance of multiple surgical procedures to provide repeated intra-CNS treatments. Cannulas have primarily been used for microdialysis sampling of neurotransmitters and the delivery of neuroactive substances in neuroscience and behavioral research in rodents12, with limited reports of their use for the delivery of anti-cancer therapeutics. Building on the prior reports, this protocol uses a stereotactically placed indwelling cannula system to deliver CAR T cells in xenograft murine models of CNS tumors. The protocol can be utilized to test additional therapeutics in murine models of neurological or neuro-oncologic disorders, and may be helpful to test novel therapeutics where bypassing the blood-brain barrier is critical for efficacy.

<table><tbody><tr><td>18 G needles</td><td>BD</td><td>511097</td><td>1 1/2 inch metal hub</td></tr><tr><td>Acrylic resin liquid</td><td>Lang Dental&amp;nbsp;</td><td>B1323&amp;nbsp;</td><td></td></tr><tr><td>Acrylic resin powder</td><td>Lang Dental&amp;nbsp;</td><td>B1323&amp;nbsp;</td><td></td></tr><tr><td>Alcohol wipes</td><td>BD</td><td>326895</td><td></td></tr><tr><td>Centrifuge 5240</td><td>Eppendorf</td><td>5420000040</td><td>Centrifuge&amp;nbsp;</td></tr><tr><td>Cotton tipped swabs</td><td>Puritan</td><td>826-WC</td><td>Handle Width = 2.11 mm (0.083), Head Width = 1.27 mm (0.050), Handle Length = 147.62 mm (5.812), Overall Length = 152.4 mm (6), Head Length = 12.7 mm (0.500)</td></tr><tr><td>Drill bit holder&amp;nbsp;</td><td>P1 Technologies&amp;nbsp;</td><td>DH-1&amp;nbsp;</td><td>Drill bit holder for D56-D70&amp;nbsp;</td></tr><tr><td>Drill bit&amp;nbsp;</td><td>P1 Technologies&amp;nbsp;</td><td>D58&amp;nbsp;</td><td>1.07 mm&amp;nbsp;</td></tr><tr><td>Dummy cannula&amp;nbsp;</td><td>P1 Technologies&amp;nbsp;</td><td>C315DCS-5/SPC&amp;nbsp;</td><td>Configuration: Small cap; &amp;nbsp;Length: Cut 5.00 mm below pedestal; Projection: 0.50 mm&amp;nbsp;</td></tr><tr><td>Flat tip screwdriver&amp;nbsp;</td><td>P1 Technologies&amp;nbsp;</td><td>SD-80&amp;nbsp;</td><td>Screwdriver&amp;nbsp;</td></tr><tr><td>Graefe forceps</td><td>Fine Science Tools</td><td>11051-10</td><td>Forceps</td></tr><tr><td>Guide cannula&amp;nbsp;</td><td>P1 Technologies&amp;nbsp;</td><td>C315GS-5/SPC&amp;nbsp;</td><td>Configuration: 5.00 mm pedestal height; Length: Cut 5.00 mm below pedestal&amp;nbsp;</td></tr><tr><td>Hemostatic cotton pellets with racemic epinephrine&amp;nbsp;</td><td>Pascal&amp;nbsp;</td><td>1151602&amp;nbsp;</td><td></td></tr><tr><td>MOXI Z Mini automated cell counter Kit&amp;nbsp;</td><td>Moxi</td><td>MXZ001</td><td>Cell counter</td></tr><tr><td>NOD&amp;nbsp;scid&amp;nbsp;gamma (NSG) mice</td><td>Jackson Laboratory</td><td>5557</td><td>6 to 12-week-old males and females</td></tr><tr><td>Pasteur pipet</td><td>VWR</td><td>14673-043</td><td></td></tr><tr><td>PKG tubing&amp;nbsp;</td><td>P1 Technologies&amp;nbsp;</td><td>C313CT&amp;nbsp;</td><td>Diameter: 0.58 mm x 1.27 mm&amp;nbsp;</td></tr><tr><td>Porcelain 12 well plate</td><td>Flinn Scientific</td><td>AP6064&amp;nbsp;</td><td></td></tr><tr><td>Povidone iodine</td><td>Medline</td><td>MDS093943</td><td></td></tr><tr><td>Scalpel</td><td>World Precision Instrument</td><td>50-822-457</td><td>Disposable Scalpel, no.10, sterile, 10/box, Plastic Handle with 6&quot; Ruler</td></tr><tr><td>Screws&amp;nbsp;</td><td>P1 Technologies&amp;nbsp;</td><td>0-80 X 3/32&amp;nbsp;</td><td>2.4 mm&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Stereotaxic Frame&amp;nbsp;</td><td>David Kopf Instruments&amp;nbsp;</td><td>940&amp;nbsp;</td><td>Model 940 Small Animal Stereotaxic Instrument with Digital Display Console&amp;nbsp;</td></tr><tr><td>Student fine scissors</td><td>Fine Science Tools</td><td>91460-12</td><td>Scissors</td></tr><tr><td>Treatment cannula&amp;nbsp;</td><td>P1 Technologies&amp;nbsp;</td><td>C315IS-5/SPC&amp;nbsp;</td><td>33GA;&amp;nbsp;Configuration: Standard internal; Length: Cut 5.00 mm below pedestal; Projection: 0.50 mm&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Treatment syringes&amp;nbsp;</td><td>Hamilton</td><td>87908</td><td>5 &amp;micro;L, Model 75 Cemented Needle Special (SN) Syringe, 75SN/22/0.5&quot;/PT3&amp;nbsp;</td></tr><tr><td>Vactrap XL&amp;nbsp;</td><td>Foxx Life Sciences</td><td>305-4401-FLS</td><td>Vacuum System</td></tr></tbody></table>

intracranial cannula implantation for serial locoregional chimeric antigen receptor (car) t cell infusions in mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Successful cannula implantation into the mouse CNS 
Mice with successful cannulation have a secure guide cannula in place, that does not interfere with activities of daily living (Figure 2A) and that can easily pass a treatment cannula and deliver fluid without resistance (Figure 2B). In our experience, the majority of cannulas remain in place for over 4 weeks, although 0-25% can be dislodged over time. Verification of correct placement can be confirmed with Evans blue dye injected into the cannula. For example, a cannula inserted into the lateral ventricle shows Evans blue dye throughout the ventricular system as it travels through the cerebral spinal fluid, confirming correct placement (Figure 2C). Cannulas inserted into the tumor show Evans blue dye at the site of tumor implantation.
Effective delivery of CAR T cells into the CNS for treatment of xenograft tumors. 
The efficacy of the intracranial cannula system and the therapeutic efficacy of CAR T cells in murine models can be measured through a variety of mechanisms, including bioluminescent imaging and overall survival. GPC2-directed CAR T cells were tested against murine medulloblastoma and diffuse midline glioma models, 7316-4509 and 7316-3058, respectively, using repeated CAR T cell dosing via the stated intracranial cannula system14. Orthotopic tumor placement and engraftment were confirmed by bioluminescent imaging, and cannulas were placed into the tumor bed using the same coordinates as the orthotopic tumor placement. The treatments consisted of GPC2 CAR T cell infusions once to twice a week for 2-4 weeks, totaling four to six doses. Following treatment, GPC2-directed CAR T cell therapy induced significant tumor regression in the medulloblastoma model 7316-4509 (p &#60; 0.01) (Figure 3A) and significantly prolonged survival in thalamic diffuse midline glioma 7316-3058 (p &#60; 0.05) (Figure 3B)14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64886/64886fig01.jpg" /> 
Figure 1: Guide cannula with projection dummy and treatment cannulas. (A) Guide cannula with 0.5 mm projection and 2 mm projection dummy cannulas in place. (B) Guide cannula with 0.5 mm projection and 2 mm projection treatment cannulas in place. <a href="https://www.jove.com/files/ftp_upload/64886/64886fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64886/64886fig02.jpg" /> 
Figure 2: Treatment cannula delivery system for repeated doses of CAR T cells. (A) Cannula implanted in a mouse skull with a cap in place when not in use. (B) Infusion of CAR T cells into a pontine tumor via the treatment cannula while the mouse is under anesthesia. (C) Verification of cannula placement in the lateral ventricle using Evans blue dye. Dye was inserted through the cannula, followed by euthanasia and brain excision, with dye present throughout the ventricles. <a href="https://www.jove.com/files/ftp_upload/64886/64886fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64886/64886fig03.jpg" /> 
Figure 3: GPC2-directed CAR T cells mediate antitumor responses and prolong survival in pediatric brain tumors in vivo. (A) Quantification of bioluminescence of the orthotopic group 4 medulloblastoma xenograft 7316-4509 treated with either GPC2- or CD19-directed mRNA CAR T cells. Doses are indicated by arrows on the graph. Data displayed as mean with SD, n = 9-11 mice per arm. (B) Overall survival of mice implanted with thalamic DMG xenograft 7316-3058 treated with six repeated doses of 2 x 106 CAR T cells. Doses are indicated by arrows on the graph. n = 7 mice per arm. **p &#60; 0.01; *p &#60; 0.05; ns = not significant. This figure has been reproduced with permission from Foster et al.14. <a href="https://www.jove.com/files/ftp_upload/64886/64886fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice at JoVE.com

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

intracranial-cannula-implantation-for-serial-locoregional-chimeric

Research

JoVE Journal

Cancer Research

2.9K Views.  Children’s Hospital of Philadelphia. Dit protocol repliceert een intraventrikelkathetersysteem dat wordt gebruikt in lopende klinische onderzoeken om lokale regionale toediening van CAR T-celtherapie gericht tegen tumoren van het centrale zenuwstelsel te testen. Het belangrijkste voordeel van deze techniek is de mogelijkheid om meerdere herhaalde doses van lokale regionale CAR T-celtherapie te bieden zonder meerdere invasieve chirurgische procedures uit te voeren. Dit systeem kan worden gebruikt om verschillende therapieën op v...

Video: Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Neuroscience

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Medicine

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Immunology and Infection

Common Carotid Artery Repair After Injection in a Mouse Model 

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

Author Spotlight: Advancing Glioblastoma Treatment Through Intraarterial Delivery Strategies for Oncolytic Viruses and MicroRNAs

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Surgical Cisterna Magna Injection: A Method for Administering Tumor Cells Directly into Central Nervous System of Murine Model

Source: Law, V. et al. <a href="https://www.jove.com/v/62033/a-murine-ommaya-xenograft-model-to-study-direct-targeted-therapy">A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease.</a> J. Vis. Exp. (2021)
In this video, we describe the procedure to deliver human-derived tumor cells into the cisterna magna of a mouse&#8217;s brain. The orthotopic mouse model can be used to study metastatic spread of cancer to the cranial meninges.

Source: Law, V. et al. A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease. J. Vis. Exp. (2021)
In this video, we ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vivo studies

Intracranial Orthotopic Model: A Procedure to Implant Cancer Cells in Mouse Brain

Source: Geisler, J. A. et al. <a href="https://www.jove.com/v/61272/modeling-brain-metastases-through-intracranial-injection-magnetic">Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging.</a> J. Vis. Exp. (2020)
This video demonstrates implantation of tumor cells in the mouse brain by performing intracranial injection.

Source: Geisler, J. A. et al. Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging. J. Vis. Exp. (2020)
This video ...

Modeling Brain Tumor Metastasis via Internal Carotid Artery Injection of Cancer Cells

<a href='https://app.jove.com/v/23284/modeling-brain-tumor-metastasis-via-internal-carotid-artery-injection'>Source: Lim, M., et al. Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells. J. Vis. Exp. (2022)</a>In this video, an anesthetized mouse undergoes a surgical procedure in which cancer cells are injected into the common carotid artery (CCA) to facilitate targeted delivery to the brain. The procedure involves precise suturing and manipulation of the carotid arteries to direct the injected cells toward the internal carotid artery (ICA), promoting tumor formation in the brain over time.

Source: Lim, M., et al. Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells. J. Vis. Exp. (2022)In this video, an anesthetized ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

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