Method Article
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 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.
Pediatric central nervous system (CNS) tumors are the most common solid tumors in children, comprising about 20%-25% of primary pediatric tumors, with distinct characteristics from adult tumors in terms of incidence and response to treatment1,2. High-grade gliomas (HGGs), such as glioblastoma (GBM) and diffuse intrinsic pontine glioma/diffuse midline glioma (DIPG/DMG), are particularly aggressive, with dismal prognoses, and despite advancements in treatment modalities, survival rates remain low3,4,5. Medulloblastomas, accounting for nearly 20% of pediatric CNS tumors, have achieved a high 5-year survival rate with standard therapy, yet certain subtypes pose challenges, especially in relapse scenarios6,7. Ependymomas, constituting about 10% of pediatric CNS tumors, exhibit varied prognoses depending on molecular characteristics and tumor location, with recurrence posing significant clinical challenges8,9. Understanding the molecular landscape and developing orthotopic brain tumor (BT) models offer promise for developing effective therapies to improve outcomes for pediatric patients.
Developing clinically relevant BT models is vital for advancing neuro-oncology research and therapeutic discovery. Orthotopic injections using a stereotaxic device are pivotal in this approach, as they allow for the precise placement of tumor cells within the brain, closely mimicking the natural environment of brain tumors10. This method enhances the reliability and reproducibility of tumor models, facilitating more accurate studies of tumor biology and key characteristics such as invasion and angiogenesis, as well as the assessment of therapeutic interventions11. The stereotaxic device's capability to control the exact location and depth of cell injections ensures that the tumor cells are consistently placed in the intended brain regions, thereby improving the validity of experimental outcomes10. Subcutaneous models provide a more accessible and cost-effective option for the initial screening of therapeutic agents. In these models, tumor cells are implanted under the skin, enabling straightforward monitoring of tumor growth and treatment responses12. Although they lack the ability to replicate the complex microenvironment of the brain, subcutaneous models remain highly valuable for high-throughput drug screening and preliminary efficacy evaluations12,13.
Multiple studies have demonstrated the successful development of orthotopic patient-derived xenograft models (PDXs) from brain tumor samples, including direct tissue implantation from autopsy or biopsy samples and primary brain tumor cultures11,14,15. However, it's crucial to acknowledge that the intracranial injection of tumor-dissociated tissue or tumor cells does not always guarantee engraftment, with reasons for failure not fully understood15. Interestingly, HGG biopsy samples tend to exhibit a higher success rate compared to other brain tumor subtypes. For instance, He et al. demonstrated a 56% success rate for HGG orthotopic models, whereas the study by Brabetz et al. reported lower engraftment rates, such as 43% and 30%, respectively, for other pediatric brain tumor samples like ependymoma and medulloblastoma11,14,16. Notably, brain tumor PDX models display a wide range of latency periods, varying from 1 month to 11 months, with later engraftments showing diminished pre-clinical utility. Despite their ability to recapitulate disease at the molecular level, some genomic disparities between biopsies and established models have been observed14,16.
PDX models serve as indispensable tools in the quest for novel therapeutic strategies against pediatric BTs. They are considered the gold standard for performing drug efficacy studies as the models faithfully recapitulate the human disease in terms of tumor growth, symptoms, and drug pharmacokinetics due to the presence of the blood-brain barrier (BBB). Research has demonstrated in the field of pediatric brainstem glioma that the maintained BBB integrity and lack of drug penetration is a feature that leads to diminished therapeutic efficacy for these tumors17,18,19. However, efficacy can be observed in selected treatments that demonstrate adequate drug penetration, such as epigenetic and metabolic inhibitors17,20.
This study presents a method for performing intracranial injections of brain tumor cells in immunocompromised animals, aimed at evaluating engraftment either for PDX development and/or assessing therapeutic drug efficacy. Comprehensive tools for monitoring tumor progression until the ethical experimental endpoint is reached are also provided.
The protocol outlined below follows the ethical guidelines for animal care and use as outlined by the University of New South Wales, and all procedures have been approved by the Institutional Animal Care and Use Committee. Animal welfare was prioritized, and all efforts were made to minimize the suffering of animals used during the intracranial injection procedure.
1. Culturing for expansion of neurosphere-forming brain tumor cultures
NOTE: Specific requirements may vary based on the brain cultures being used. The following steps outline a general culturing protocol for primary brain tumor cells as neurospheres. Brain tumor cultures may be obtained from commercial vendors or established as primary cultures from patient brain tumor biopsies or autopsies. The procedure for establishing primary cultures from patient biopsy or autopsy samples is outlined in published literature15,21.
2. Preparation of brain tumor cells for intracranial injection
NOTE: While the following protocol outlines the preparation of cultured cells from established or short-term cultures for intracranial injection, it can also be adapted for other cell types, including freshly dissociated tissue from human brain tumors22,23.
3. Set up of stereotaxic and anesthetic equipment
NOTE: The procedure listed below might vary slightly depending on the manufacturer of the stereotaxic equipment and the type of anesthesia preferred.
4. Intracranial injection
NOTE: Intracranial injections can be performed in both male and female mice, though female mice are generally preferred, as they are less prone to wound complications caused by aggression among males. The procedure is typically carried out in mice aged 7-10 weeks, allowing sufficient time for tumor development. Typically, animals are randomized in groups of 4-6 per cage. Immunocompromised strains such as BALB/c Nude, NOD-SCID, and NSG are commonly used for patient-derived xenograft (PDX) development, as their immune-deficient status supports tumor engraftment and growth24. While this protocol specifies the use of isoflurane for anesthesia induction, alternative anesthetics such as ketamine and xylazine may also be used25. If isoflurane is not utilized, steps involving the anesthetic chamber and related equipment can be omitted from the procedure.
5. Post-surgical monitoring for engraftment
6. Harvesting of brains
Orthotopic injections using a stereotaxic device have become essential for developing brain tumor models. This technique allows for the precise and reproducible placement of tumor cells, which is crucial for generating consistent and reliable data in neuro-oncology research. The successful implementation of orthotopic injections relies heavily on the accurate setup of stereotaxic equipment, which typically includes a stereotaxic frame, a digital micromanipulator for precise movements, an injection syringe, and an anesthesia system to immobilize the animal (Figure 1A,B). The use of a glass microsyringe enables the intracranial injection of small volumes, typically between 2-5 µL. Proper maintenance and frequent washing are essential to prevent the buildup of biological material, which can block the injection (Figure 1C).
The procedure for intracranial injection of brain tumor cells using stereotaxic equipment in mice involves several key steps. Firstly, the mouse is anesthetized to remain still and comfortable throughout the procedure. Typical cues indicating that the animal is anesthetized include the absence of movement and reflexes upon toe pinching, as well as the presence of a regular breathing pattern. Then, the mouse is placed in a stereotaxic frame, securing its head in a fixed position (Figure 2A). Using landmarks on the skull, such as bregma and lambda, precise coordinates for injection are determined. A small incision is made in the scalp, and a burr hole is drilled through the skull at the chosen coordinates (Figure 2B). A microinjection needle attached to the stereotaxic arm is inserted through the burr hole to deliver the tumor cells into the desired brain region (cortex, cerebellum, or brainstem; Figure 2C). Careful monitoring and adjustment of the needle position ensure accurate targeting. After injection, the needle is slowly withdrawn, and the incision is closed (Figure 2D). The sealed wound is expected to heal within two weeks following intracranial injection.
Following intracranial injection, animals require careful monitoring for overall well-being and the development of neurological symptoms, which often indicate tumor progression (Supplementary Table 1). Typically, animals may experience a slight weight loss during the 1st-week post-injection (up to 10% of their highest weight), which typically stabilizes once the injection sites are fully sealed by the end of the 2nd week. Generally, animals exhibit a steady increase in weight and maintain good overall health until tumor progression occurs. The manifestation of neurological symptoms varies depending on factors such as the type of brain tumor, injection site, and animal strain. For instance, cortical gliomas or ependymomas may present with progressive weight loss and potential forebrain enlargement, while brainstem gliomas and medulloblastomas might exhibit symptoms such as ataxia and head tilting, particularly in immunocompromised animals (Figure 3A). Additionally, circling behavior may be observed in animals with brainstem tumors, especially in NSG or NOD/SCID strains. The progression of neurological symptoms often coincides with gradual weight loss. Proper post-care measures, such as providing water bottles with long nozzles or offering soft food (mushy food pellets) or seeds, are essential for supporting the animals' well-being. Upon reaching the experimental endpoint and the subsequent humane euthanasia of the animals, potential alterations in the brain may become apparent, including enlargement at the injection site and/or the presence of hemorrhagic areas (Figure 3B).
Kaplan-Meier graphs are commonly employed to depict the survival outcomes of brain tumor animal models. They offer a visual depiction of the probability of survival over time following intracranial injection or anticancer treatments. These graphs serve as crucial tools for analyzing and presenting survival data in pre-clinical research settings15,27. We performed intracranial injections of medulloblastoma cell culture D425 at varying cell densities in the cerebellum. All animals intracranially injected developed tumors. Our results indicated that higher cell densities, specifically 50,000 and 100,000 cells per mouse, engrafted more rapidly than 10,000 cells per mouse (Figure 4). Furthermore, we intracranially injected patient-derived high-grade glioma (HGG) cells into the cortex and brainstem to compare overall survival and the pattern of tumor growth, as well as immunohistochemical features, in these different locations. Irrespective of the location, all animals were injected with intracranially developed tumors. The culturing of HGG cells was carried out following the protocol detailed in steps 1.1 to 1.10. Preparation of the cells for intracranial injection was performed as described in steps 2.1 to 2.6, while the intracranial injection procedure adhered to the method outlined in steps 4.1 to 4.1627. When injected into the cortex, HGG cells displayed a median survival of approximately 25 days post-intracranial injection (Figure 5A). Animals were monitored as outlined in steps 5.1 to 5.4. These animals did not exhibit any neurological symptoms but did show progressive weight loss. Animals were humanely euthanized upon reaching the humane endpoint outlined in step 5.5. Brains were harvested following the protocol detailed in steps 6.1 to 6.5 and sent for further analysis27. Immunohistochemical analysis revealed a large, highly nucleated tumor mass with evidence of increased vascularization (Figure 5B) and many proliferative Ki67 cells (Figure 5C). When injected into the brainstem, HGG cells displayed a median survival of approximately 26 days post-intracranial injection (Figure 5D). Immunohistochemical analysis indicated a large tumor mass in the 4th ventricle/upper pons region, as well as leptomeningeal infiltration into the lateral ventricle (Figure 5E). Further immunohistochemical analysis indicated many proliferative cells in the brainstem and other infiltrated areas (Figure 5F). This experiment highlights that injecting the same tumor cells into different anatomical locations does not affect the tumor growth rate, although some differences in the growth pattern are observed.
Figure 1: Representative images of the stereotaxic setup for intracranial injection of brain tumor cells. (A) The anesthetic apparatus is typically positioned next to the BSC hood, necessitating dual administration of anesthesia to both the induction chamber and the stereotaxic apparatus. (B) Glass microsyringe wash station and stereotaxic holder; the syringe requires flushing with saline, ethanol, and subsequently water after each intracranial injection. (C) Stereotaxic setup inside the BSC hood, illustrating the anesthetic chamber, drilling machine, stereotaxic device, stereotaxic coordinate console, cotton tips, scalpel, tweezers, pipette, and tips. Please click here to view a larger version of this figure.
Figure 2: Representative images of intracranial injection being performed on an immunocompromised mouse. (A) Anesthetized animal positioned in an anesthetic cone. (B) Skin incision followed by securing skin to each side with ear bars. (C) Glass microsyringe positioned atop the drilled hole for injection. (D) Skin forms a Mohawk style once glued. Please click here to view a larger version of this figure.
Figure 3: Potential neurological symptoms observed following engraftment of brain tumor cells. (A) Animal exhibiting a head tilt following intracranial injection of DMG cells in the brainstem. (B) Enlargement of the right cortical hemisphere with the injection side revealing tumor formation. The animal was injected with ependymoma cells. Please click here to view a larger version of this figure.
Figure 4: Engraftment of the medulloblastoma culture D425 at different cell densities. Medulloblastoma cells were intracranially injected using stereotaxic equipment at densities of 10,000 cells in 2 µL of ECM hydrogel, 50,000 cells, and 100,000 cells. Animals were monitored for progressive weight loss and were humanely euthanized once they reached 20% below their highest recorded weight. The median survival for animals injected with 10,000 cells was 29 days, while the median survival for those injected with higher cell densities of 50,000 and 100,000 cells was 17.5 days and 15 days, respectively. All animals injected in this study successfully developed tumors. Please click here to view a larger version of this figure.
Figure 5: Representative survival curves and histological analysis of NSG animals intracranially injected with HGG cells in the cortex and brainstem. Approximately 100,000 HGG cells (passage 3) were injected into 2 µL of ECM hydrogel using stereotaxic equipment. Animals were monitored for progressive weight loss and humanely euthanized once they reached 20% below their highest recorded weight. No neurological symptoms were observed for this brain tumor subtype. (A) The median survival of animals with HGG cells injected into the cortex is approximately 25 days post-injection (N=2). (B) H&E staining of the cortex shows a highly nucleated region with increased vascularization, indicated by the presence of red blood cells. (C) The tumor-engrafted area in the cortex displays a high level of proliferative cells, as shown by KI67 staining. (D) The median survival of animals with HGG cells injected into the brainstem is approximately 26 days post-injection (N=2). All animals injected in this study successfully developed tumors. (E) H&E staining of the brainstem shows a highly nucleated region with tumor infiltration observed in the leptomeninges in the lateral ventricle. (F) The tumor-engrafted area in the brainstem displays a high level of proliferative cells, as shown by KI67 staining. The black scale bar in the main images indicates 2 mm, whereas the magnified inserted images indicate 20 µm. Please click here to view a larger version of this figure.
Supplementary Table 1: Monitoring form used for the inspection of animals' post-intracranial injections and potential treatment with anticancer agents. Please click here to download this table.
While the intracranial injection technique detailed in this study provides a robust method for establishing orthotopic patient-derived xenograft (PDX) models for pediatric brain tumors, several areas need improvement. One approach to improving the technique involves optimizing the tumor cell preparation process. This includes refining the materials used for the implantation of the tumor cells. While ECM hydrogel is considered the gold standard, utilizing hydrogels composed of components that more closely resemble those of the brain or tumor ECM could enhance BT cell survival and engraftment. This approach particularly benefits tumor types with longer engraftment periods, such as medulloblastomas and ependymomas. For instance, collagen has been shown to play a critical role in the growth of Group 3 medulloblastoma, whereas laminin supports the SHH type of medulloblastoma28. Additionally, the stiffness of the ECM may also influence tumor growth. High-grade glioblastomas, for example, exhibit higher stiffness in their core compared to normal brain tissue, which may support tumor growth and survival29. By tailoring the ECM composition and stiffness to match the specific requirements of different tumor types, the engraftment and growth of PDX models may be significantly improved.
A few recent studies have provided valuable insights into patient tumor sample handling and the development of PDX models. Tsoli et al. highlighted that direct implantation of DMG tissue typically results in higher engraftment rates than primary cultures15. Smith et al. established 37 PDX models through intracranial injection of brain tumor biopsies or autopsy samples, reporting an overall success rate of 43%, with HGGs demonstrating the highest engraftment rate (100%), followed by medulloblastomas (45%) and ependymomas (25%). Tumor latency varied widely, from 1 to 11 months, with more aggressive tumors engrafting more quickly11. In contrast, Brabetz et al. reported a lower overall success rate of 30%, with HGGs exhibiting a 31% engraftment rate and lower rates for medulloblastomas (37%) and ependymomas (17%)16. Despite these differences, the last two studies conducted by Smith et al. and Brabetz et al. confirmed that the PDX models generally recapitulated the patient tumors at immunohistochemical and molecular levels, although minor genomic discrepancies were observed between the original tumor samples and the established models. These findings underscore the challenges in developing representative PDX models and highlight the variability in engraftment success across tumor types11,16.
One significant limitation of the orthotopic approach is the inability to accurately measure tumor size unless an imaging system is used, such as Xenogen Imaging, if the tumor cells are tagged with luciferase or Magnetic Resonance Imaging (MRI). Tagging tumor cells with luciferase could provide a solution by allowing for bioluminescent imaging to confirm engraftment and monitor tumor progression non-invasively. This method would enhance the ability to track the response to treatment dynamically, thereby offering a more precise evaluation of therapeutic efficacy17,30. An alternative method involves utilizing MRI technology; however, the detectability of tumors is contingent upon their size. Larger tumors are more readily identified, which is often at the experimental endpoint, and detection may also vary depending on the subtype of the tumor, with GBM being more easily detected than diffuse gliomas27,31. Particularly for diffuse gliomas, the presence of an intact blood-brain barrier (BBB) leads to a lack of contrast enhancement. In GBM, however, leaky vasculature and the presence of edema can amplify the contrast between the tumor tissue and the surrounding healthy brain tissue, potentially aiding in the detection process. Generally, contrasting agents are needed to improve detection30,31,32. Alternative imaging techniques such as diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) are sometimes used to improve the detection of non-enhancing tumor regions. However, these methods also have their limitations and may not always provide a precise delineation of tumor boundaries33. Amide Proton Transfer-weighted (APTw) imaging is a non-invasive MRI technique that detects diffuse gliomas by tracking protein-water proton exchange, aiding in tumor grading. While promising, it faces challenges like susceptibility to artifacts and has not yet been adapted for small animal imaging, limiting its broader application34.
Another crucial aspect to consider when utilizing orthotopic BT PDX models for drug efficacy studies is the BBB integrity in the PDX models used. The BBB's permeability significantly affects drug delivery and efficacy in brain tumor treatment. For example, the maintained BBB integrity in brainstem gliomas often results in poor drug penetration, limiting the effectiveness of systemic therapies17. On the other hand, in Group 3 medulloblastomas, BBB integrity has been observed to be heterogeneous on MRI imaging, and it was similarly found to be leaky in the patient-derived xenograft (PDX) models35. Therefore, characterizing the BBB status-whether it is intact or leaky-in each PDX model is essential for designing appropriate therapeutic strategies and accurately interpreting the results of drug efficacy studies.
Overall, the intracranial injection procedure outlined offers a robust approach for establishing BT PDX models and conducting efficacy studies for therapeutic agents. Continued refinements, coupled with a comprehensive understanding of these models, will augment their utility and facilitate the development of therapeutic interventions for pediatric BT patients.
The authors have nothing to disclose.
The authors thank the patients, clinicians, and researchers of the Sydney Children's Hospital Network, the Sydney Children's Tumour Bank Network, and the Zero Childhood Cancer Program for generously providing the essential samples for establishing this methodology. Children's Cancer Institute Australia is affiliated with the University of New South Wales Sydney and the Sydney Children's Hospital Network. This protocol stems from research efforts supported by grants from the National Health and Medical Research Council (Synergy Grant #2019056, Leadership Grant APP2017898 to DZ) and Cancer Institute New South Wales Program Grant (TPG2037 to DZ and MT), as well as support from the Levi’s Project, The Kids Cancer Project, The Cure Starts Now, DIPG Collaborative, Cure Brain Cancer Foundation, The Robert Connor Dawes Foundation and The Benny Wills Foundation.
Name | Company | Catalog Number | Comments |
Anaesthetic agent (e.g. Isoflurane) | Merck | 792632 | |
100% Ethanol | Merck | 459844 | |
70% ethanol | NA | NA | Make 70% ethanol from 100% ethanol diluting it with sterile water |
80% ethanol | NA | NA | Make 80% ethanol from 100% ethanol diluting it with sterile water |
Adhesive microscope slides | Merck | Z681156 | |
Analgesic (Buprenorphine 0.3mg/mL) | Jurox | NA | |
Carbon filter canister | Vet Equip | 931401 | |
Conical tubes 15 mL (Falcon) | Fisher Scientific | 14-959-53A | |
Conical tubes 50 mL (Falcon) | Fisher Scientific | 14-432-22 | |
DMSO | Merck | 472301 | |
Ear bars | Kopf | 1921 | |
ECM hydrogel (Matrigel, Corning) | Merck | E1270 | |
Eye Ointment (Poly Visc) | ChemistDirect | 719986 | |
Fetal Calf Serum | Merck | F0926 | |
glass microsyringe | Hamilton | 7002 | |
Isopropanol wipes | Medisa | SUL_LWMS-1 | |
KI67 antibody | Abcam | ab209897 | |
Microcentrifuge tubes | Eppendorf | 211-2130 | |
Microdrill bit 0.028” | Kopf | 8170 | |
Microinjection unit | Kopf | 5004 | |
PBS | Merck | P4474 | |
Pipette 10mL | Corning | CLS4488 | |
Pipette 1mL | Eppendorf | 3123000063 | |
Pipette 200uL | Eppendorf | 3123000055 | |
Pipette 20uL | Eppendorf | 3123000098 | |
Pipette 25mL | Corning | CLS4489 | |
Pipette tip 1mL | Corning | CLS4868 | |
Pipette tip 200uL | Corning | CLS4860 | |
Polysorbate 20 detergent (F10SC) | Health and Hygiene | G3070 | |
Pulse oximeter (MouseSTAT) | Kent Scientific | SPO2-MSE | |
Riodine (Povidone-Iodine) | Merck | Y0000466 | |
Scalpel (disposable) | Medisa | SUL_SCALPEL | |
Skin Glue Vetbond | Medisa | 3M_1469SB | |
Small Animal stereotactic instrument with digital display console | Kopf | 940 | |
Stereotactic Drill | Kopf | 1474 | |
Temperature monitoring system | Harvard Apparatus | 55-7020 | |
Trypan Blue | Merck | 93595 |
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