Name: stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts
Uploaded: 2018-09-01T13:00:00.000+00:00
Duration: 11 min 15 s
Description: Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

The authors thank the staff of the University Hospital animal facility (UTE) of Nantes for animal husbandry and care, the cellular and tissular imaging core facility of Nantes University (MicroPICell) for imaging, and the Cytometry facility (Cytocell) from Nantes for their expert technical assistance. This work was funded by INSERM, CNRS, Universit&#233; de Nantes, Institut National du Cancer (INCa#PLBio2014-155), Ligue Nationale contre le Cancer (AO InterRegional 2017), and the European consortium ERA-Net Transcan2 (Immunoglio). The team is funded by the Fondation pour la Recherche Medicale (DEQ20170839118). This work was realized in the context of the LabEX IGO and the IHU-Cesti programs, supported by the National Research Agency Investissements d'Avenir via the programs ANR-11-LABX-0016-01 and ANR-10-IBHU-005, respectively. The IHU-Cesti project is also supported by Nantes Metropole and the Pays de la Loire Region. The authors thank Chirine Rafia for providing help in correcting the manuscript.

The following procedure involving animal subjects was performed according to institutional guidelines (Agreement #00186.02; regional ethics committee of the Pays de la Loire [France]). Human PBMCs were isolated from the blood collected from informed healthy donors (Etablissement Fran&#231;ais du Sang Nantes, France). All steps are performed under sterile conditions.
1. Non-specific Expansion of Cytotoxic Effector T Lymphocytes
<ol>
	<li>Prepare and irradiate feeder cells at 35 Gy. For the stimulation of 2 x 105 - 4 x 105 effector cells, prepare 10 x 106 PBMCs and 1 x 106 BLCLs from three distinct, healthy donors.</li>
	<li>Resuspend both feeder cells and effector cells in 15 mL of RPMI supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, penicillin (100 IU/mL), and streptomycin (0.1 &#181;g/mL) and 300 IU/mL recombinant IL-2.</li>
	<li>Add PHA-L at a final concentration of 1 &#181;g/mL, carefully mix, and distribute 150 &#181;L of the cell suspension per well in 96-well U-bottomed plates.</li>
	<li>Incubate at 37 &#176;C and with 5% CO2 in a humidified atmosphere.</li>
	<li>Daily check the plate until large cell clumps form in the culture wells (~day 7).</li>
	<li>Transfer the cells into a culture flask at 1 x 106 cells/mL in fresh culture medium.</li>
	<li>Determine the total cell number by counting (2x a week) and maintain them at 1 x 106 cells/mL in fresh culture medium. 
	NOTE: Effector immune cells should be ready for the therapeutic administration at a resting state (usually 3 weeks after initial amplification stimulus). The purity and the reactivity of these effector cells should be checked prior to in vivo injections (e.g., with in vitro assays).</li>
</ol>
2. Pre-operative Effector Cells Preparation
<ol>
	<li>After checking the effector cell count, collect the effector cells in a 50-mL tube by centrifugation (300 x g for 5 min). 
	NOTE: To compensate for any loss, prepare an excess of cells (e.g., 50 x 106).</li>
	<li>Carefully remove the supernatant and resuspend the cells in 15 mL of sterile PBS and centrifuge for 5 min at 300 x g to perform the wash.</li>
	<li>Carefully and completely remove the supernatant and then resuspend the cell pellet in 1 mL of sterile PBS.</li>
	<li>Transfer the resuspended cells in a 1.5-mL microtube for centrifugation at 300 x g for 5 min.</li>
	<li>Carefully and completely remove the supernatant by pipetting slowly.</li>
	<li>Resuspend the cell pellet in 8 &#181;L of sterile PBS per mouse. 
	NOTE: This is a critical step.</li>
	<li>Measure the volume of the cell suspension by using a micropipette. If necessary, add sterile PBS (20 x 106 cells in 15 &#181;L of PBS per dose) and mix carefully.</li>
	<li>Check, by using a micropipette, that the final volume per mouse is between 15 and 20 &#956;L (imperatively &#60; 20 &#181;L).</li>
	<li>Keep the cells on ice until stereotactic injection. 
	NOTE: More than 3-h timepoints were not tested.</li>
</ol>
3. Stereotactic Injection
<ol>
	<li>Equipment set-up
	<ol>
		<li>Assemble and calibrate a small animal stereotactic frame according to the manufacturer&#39;s instructions to ensure the accuracy of intracranial injections (e.g., syringe size, desired volume, and rate of injection). 
		NOTE: A slow infusion rate is recommended (i.e., 2 - 3 &#181;L/min).</li>
		<li>Install the material under a microbial safety cabinet (MSC) to maintain the sterility of the instruments and to protect the mice from infections. 
		NOTE: Place&#34; isothermal blocs&#34; in a water bath at 37 &#176;C. This system limits the hypothermia of mice during the surgery. Heating pads, which are necessary for post-procedural care, must be used during the continuous temperature monitoring.</li>
	</ol>
	</li>
	<li>Pre-operative animal preparation
	<ol>
		<li>Anesthetize a mouse with an intraperitoneal injection of ketamine (10 mg/mL) and xylazine (0.1 mg/mL) at 10 &#181;L/g of body weight of the mouse.</li>
		<li>Perform a toe pinch test to ensure the complete anesthesia and analgesia of the animal. 
		NOTE: Any movement is an indication of non-deep analgesia and, if that occurs, a few more minutes are required before repeating the operation.</li>
		<li>Once the mouse is properly anesthetized, use scissors to remove the fur from the surgical site (between the two ears, up to the nose).</li>
	</ol>
	</li>
	<li>Pre-operative cell preparation
	<ol>
		<li>Carefully resuspend the cells with a pipette (several times) prior to each injection to prevent any cell clumping.</li>
		<li>Carefully draw the required cell suspension volume (15 - 20 &#181;L) into the 22-G microsyringe to avoid the aspiration of bubbles. 
		NOTE: This cell-loading step into the microsyringe is important to minimize variances in injected volumes. Reload cells for each individual injection between procedures to prevent any cell clumping and to ensure an even number of effector cells administration in the cohort.</li>
		<li>Then, place the syringe into the adapted syringe pump.</li>
	</ol>
	</li>
	<li>Procedural care
	<ol>
		<li>Disinfect the surgical site with swabs soaked in povidone-iodine 5% solution at least 3x.</li>
		<li>Place a lubricating ophthalmic ointment in the mouse&#39;s eyes to prevent any drying of the corneas.</li>
		<li>Position the anesthetized mouse on the stereotactic frame, on a warm isothermal block covered with a sterile plastic wrap to maintain the mouse&#39;s temperature during surgery and limit the mortality. 
		NOTE: The mouse&#39;s nose and teeth should be appropriately positioned above the tooth bar, to ensure adequate respiratory flow during the procedure.</li>
		<li>Once the mouse is positioned above the tooth bar, tighten the ear bars firmly under the mouse&#39;s ears to immobilize the head. 
		NOTE: Be careful to not damage the eardrums or to compromise the respiration.</li>
		<li>Make a 1 - 2 cm midline sagittal skin incision with sterile scissors along the upper part of the cranium from anterior to posterior to expose the skull.</li>
		<li>Identify the intersection of the sagittal and coronal sutures (Bregma) to serve as landmarks for stereotactic localization prior to the injection (shown in Figure 1).</li>
		<li>Place the microsyringe above this point.</li>
		<li>Move the microsyringe 2 mm right lateral and 0.5 mm anterior of the Bregma.</li>
		<li>Using a microdrill, make a small hole in the skull with a sterile drill bit at predetermined coordinates. Be careful to remain superficial in order to avoid any traumatic injury of the brain. 
		NOTE: In this protocol, immune cells were injected within an established tumor (one week after tumor cell injection). The skin should be reopened (scar) and the injection is performed at the same coordinates used for the tumor cell implantation (the hole is generally still present up to 2 weeks after the injection). Coordinates were selected for injecting tumor and effector cells in the brain parenchyma13,14.</li>
	</ol>
	</li>
	<li>Injection of immune effector cells
	<ol>
		<li>Carefully insert the microsyringe into the drilled hole and, moving slowly, forward the needle 3 mm down in the dura and then backward 0.5 mm to a final depth of 2.5 mm prior to injecting the effector cells.</li>
		<li>Run the effector cell injection at 2 - 3 &#181;L/min and monitor the mice all along the injection time.</li>
		<li>Once the injection is complete, withdraw the needle for only 1 mm and keep the microsyringe in place for one additional minute before slowly withdrawing completely the microsyringe, to prevent any leakage from the infusion site. 
		NOTE: Following the removal of the animal from the stereotactic device, immediately clean the injection equipment for upcoming experiments.</li>
	</ol>
	</li>
	<li>Post-operative care and follow-up of mouse
	<ol>
		<li>Remove the animal from the stereotactic frame and immediately apply povidone-iodine 5% solution on the incision site and close the skin with an appropriate surgical suture.</li>
		<li>Apply 2% lidocaine gel directly on the wound and administer 0.15 &#181;g/g of buprenorphine by a subcutaneous injection for post-procedural analgesia.</li>
		<li>Place back the anesthetized mouse to its cage above a heating pad set to 37 &#176;C to maintain an appropriate mouse body temperature and to avoid any hypothermia. 
		NOTE: Separate housing is not necessary.</li>
		<li>Monitor the mouse until it is fully recovered from anesthesia and transfer it to a housing room. 
		NOTE: To date, this protocol is well supported as few unforeseen complications have occurred (&#60; 5% of injected mice).</li>
		<li>Daily monitor the mice and euthanize them when any declining health signs are observed (e.g., hunched posture, reduced mobility, prostration, or significant body weight loss [&#8805; 15%]).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

An adoptive transfer of selected native or engineered immune effector cells represents a promising approach to efficiently treat tumors, such as infiltrative brain cancers, taking care of limiting reactivities against non-transformed cells15,16,17,18. However, the central nervous system, which comprises the brain, has a particular immune status, notably due to the existence of the blood-brain b...

GBM (WHO grade IV astrocytoma), is the most frequent and aggressive primary brain cancer in adults. In spite of aggressive treatments that combine surgery and radio-chemotherapy, GBM remains associated with an extremely poor prognosis (median survival of 14.6 months and a 2-year-mortality &#62; 73%)1. This evidences that few efficient therapeutic advances have been validated over the last decade2. Among candidates for the design of more effective therapeutic strategies3,4,5, immunotherapies6 are currently explored to track and eliminate highly invasive and radio/chemo-resistant tumor cells, suspected for their key contribution to rapid and fatal tumor relapse7. Various potential immunological targets were identified and proposed for immunotherapies, involving natural or modified &#945;&#946; or &#978;&#948; T lymphocytes such as GBM-specific tumor antigens or stress-induced molecules8,9,10. The possibility to administrate selected GBM-reactive immune cell effectors represents a unique opportunity to deliver elevated amounts of effector lymphocytes directly into the site of residual malignancy. To support this strategy, we have recently shown that models based on immunodeficient mice carrying orthotopic primary human GBM xenografts faithfully recapitulate the development of brain tumors in GBM patients9,11. Moreover, these models were used to demonstrate the strong antitumor efficiency of adoptively transferred allogeneic human V&#978;9V&#948;2T lymphocytes.
This protocol describes the critical experimental steps for achieving stereotactic immunotherapies of brain tumors, such as GBM, based on the adoptive transfer of allogeneic T lymphocytes. The article shows: (i) the amplification of therapeutic allogeneic immune effector T lymphocytes, such as human V&#978;9V&#948;2T lymphocytes; (ii) the preparation of these effector T lymphocytes for injection; (iii) the procedure for stereotactic administration within the brain, near the tumor. This article also provides insight into the behavior of these cellular effectors after stereotactic injection.
The therapeutic approach presented here is based on the injection of 20 x 106 effector cells per dose for each brain tumor-bearing immunodeficient mouse. An initial in vitro expansion step is required to produce large quantities of immune cells. Therefore, non-specific cell expansions are performed using phytohemagglutinin (PHA-L) and irradiated allogeneic feeder cells: peripheral-blood mononuclear cells (PBMCs) from healthy donors and Epstein Barr Virus (EBV)-transformed B-lymphoblastoid cell lines (BLCLs), derived from PBMCs by in vitro infection with EBV-containing culture supernatant from the Marmoset B95-8 cell line, in the presence of 1 &#181;g/mL cyclosporin-A.
GBM-reactive effector immune cells are compared and selected from in vitro assays9. These effector cells are activated and amplified using standard protocols, according to their nature (e.g., human V&#947;9V&#948;2 T lymphocytes9 or human anti-herpes virus &#945;&#946; T lymphocytes12) with a minimum purity of &#62; 80%, as routinely checked by cytometric analysis. The cell expansion procedure detailed below applies to various human lymphocyte subsets.

<table><tbody><tr><td>PBMCs</td><td></td><td></td><td>from 3 different healthy donors</td></tr><tr><td>BLCLs</td><td></td><td></td><td>from 3 different donors</td></tr><tr><td>Roswell Park Memorial Institute medium (RPMI)</td><td>Gibco</td><td>31870-025</td><td></td></tr><tr><td>FCS</td><td>Dutscher</td><td>S1810-500</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>IL-2</td><td>Novartis</td><td>proleukin</td><td></td></tr><tr><td>PHA-L</td><td>Sigma</td><td>L4144</td><td></td></tr><tr><td>Stereotaxic frame</td><td>Stoelting Co.</td><td>51600</td><td></td></tr><tr><td>Mouse adaptator for stereotaxic frame&amp;nbsp;&amp;nbsp;</td><td>Stoelting Co.</td><td>51624</td><td></td></tr><tr><td>microsyringe pump injector&amp;nbsp;</td><td>WPI</td><td>UMP3-4</td><td></td></tr><tr><td>NanoFil Syringe</td><td>WPI</td><td>NF34BV-2</td><td></td></tr><tr><td>NSG mice</td><td>Charles River</td><td>NSGSSFE07S</td><td></td></tr><tr><td>Ketamine</td><td>Merial</td><td>Imalg&amp;egrave;ne 1000</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td>Rompur 2%</td><td></td></tr><tr><td>Scissors</td><td>WPI</td><td>201758</td><td></td></tr><tr><td>Forceps</td><td>WPI</td><td>501215</td><td></td></tr><tr><td>OmniDrill 115/230V</td><td>WPI</td><td>503598</td><td></td></tr><tr><td>Vicryl 4-0</td><td>Ethicon</td><td>VCP397H</td><td></td></tr><tr><td>Xylocaine</td><td>Astrazeneca</td><td>3634461</td><td></td></tr></tbody></table>

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.

This study describes the strategy of adoptive transfers of cellular immune effector cells within the brain of tumor-carrying mice, based on stereotactic injections performed directly within the tumor bed.
To minimize any risk of brain injury associated with a large injection volume, the effector cell suspension needs to be concentrated (20 x 106 cells in 15 - 20 &#181;L of PBS). To check whether this cell concentratio...

Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

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

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 ...

stereotactic-adoptive-transfer-cytotoxic-immune-cells-murine-models

Research

JoVE Journal

Immunology and Infection

7.8K Views.  CRCINA. 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.

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

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

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

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 ...

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

Cancer Research

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of neuro-oncology. One of the most widely used techniques is the orthotopic intracranial injection, a method that allows investigating of tumor growth, invasion, and dissemination within a controlled setting. This technique involves transplanting tumor cells from a specific patient region into the corresponding anatomical site in an animal. By doing so, these orthotopic brain tumor models offer a unique advantage, as they more accurately replicate cancer's biological behavior and its interactions with the brain environment seen in human patients. This makes them especially valuable for preclinical therapeutic testing, where a close resemblance to the clinical scenario is essential for evaluating potential treatments. This protocol shares experiences in developing patient-derived xenograft (PDX) models for pediatric brain tumors, including diffuse midline glioma (DMG), glioblastoma (GBM), medulloblastoma, and ependymoma. This method delineates the procedure for conducting intracranial stereotaxic injections in mice, ensuring the correct targeting of the injection site within the brain. Additionally, we describe the post-procedural monitoring system employed to detect signs of successful tumor engraftment. Following tumor injection, a rigorous monitoring system is implemented to observe the animals for any signs of neurological impairment, behavioral changes, and/or weight loss, which are common indicators of tumor progression. This system allows for timely intervention and provides critical data regarding the tumor's growth dynamics. By refining these models and protocols, we aim to enhance the reliability and translational potential of preclinical studies, contributing to the development of more effective treatments for pediatric CNS tumors.

The development of orthotopic pediatric brain tumor models requires meticulous precision, using a stereotaxic device to implant cancer cells precisely. The methodology presented here outlines the steps involved in preparing brain tumor cells, performing intracranial injections, and implementing a post-operative monitoring system to assess brain tumor engraftment.

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

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

Summary

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Chapters in this video

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

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

Translational Orthotopic Models of Glioblastoma Multiforme

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds