Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Summary

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

Abstract

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.

Introduction

Spinal cord gliomas, including those of the cauda equina, are commonly malignant neoplasms of the spinal cord, with 20%-40% classified as astrocytomas and the remainder as ependymomas¹. Based on histological features, spinal cord gliomas are categorized into four grades (I-IV). Grade I and II tumors are considered low-grade gliomas, while grade III and IV tumors are classified as high-grade gliomas. Although spinal cord gliomas can occur at any segment of the spinal cord, they are most frequently found in the cervical region (33% of cases) and are relatively rare in other regions, with 26% of cases in the thoracic region and 24% in the lumbar region².

Surgery, radiotherapy, and alkylating agents are the primary treatment options for spinal cord gliomas, largely extrapolated from clinical trials on brain gliomas³. However, previous research has demonstrated that, although the histological profiles of spinal cord gliomas resemble those of brain gliomas, the presence of distinct molecular signatures differentiates them from their cerebral counterparts⁴. In our cohort, spinal cord glioma patients derived no significant benefit from either adjuvant chemotherapy or radiotherapy, underscoring the limited effectiveness of current treatments and the need for new therapeutic strategies⁵. Therefore, reliable and informative animal models are essential for advancing basic research and preclinical studies.

Currently, several well-established spinal cord glioma models exist, including the method described by Minru et al.⁶. These models primarily utilize thoracic vertebra removal techniques to expose the spinal cord^6,7,8. Although rat models have been employed in the past, they are associated with higher costs, smaller sample sizes, and greater management challenges compared to mouse models. Additionally, more genetically modified experimental mouse models are available than rat models. An immune-competent mouse model is particularly valuable for studying the immune response within the spinal tumor microenvironment and for developing immunotherapeutic strategies for spinal cord gliomas. Furthermore, this method is well-suited for generating patient-derived xenograft models for spinal cord gliomas.

This protocol proposes a safe, technically simple, and rapidly reproducible procedure for creating a spinal cord glioma transplantation model in mice. The model is expected to advance research into the largely unexplored mechanisms underlying glioma progression and facilitate the development of therapeutic drugs for spinal cord gliomas.

Protocol

This protocol was conducted in compliance with the guidelines approved by the Institutional Committee for the Ethics of Animal Care and Treatment in Biomedical Research at Capital Medical University (AEEI-2021-187). Female C57BL/6 mice, aged 8 weeks and weighing 19-21 g, were used in this study. The reagents and equipment utilized are detailed in the Table of Materials.

1. Pre-surgical preparation

1. Clean and sterilize all surgical instruments thoroughly.
2. Spray the surgical table with alcohol and wipe it clean using sterile paper towels.

2. Preparation of GL261-luc and B16-F10-luc cells for transplant

NOTE: The GL261-luc GBM cell line was obtained commercially, while the B16-F10-luc melanoma cell line was a gift from Professor Wang Xi. Both cell lines were confirmed to be free of mycoplasma infection through pre-experimental testing.

1. Prepare complete DMEM (Dulbecco's Modified Eagle Medium) by supplementing it with 10% fetal bovine serum (FBS) and 1% penicillin (100 U/mL)-streptomycin (100 µg/mL).
2. Culture GL261-luc or B16-F10-luc cells in the complete DMEM medium and collect cells during the logarithmic growth phase for implantation.
3. Wash the cells twice with sterile PBS, then incubate them with 0.05% trypsin-EDTA solution for 3 min.
4. Transfer the resulting cell suspension into a tube and centrifuge at 500 × g for 5 min at room temperature.
5. After centrifugation, discard the supernatant using a pipette, resuspend the cells in sterile PBS, and centrifuge once more.
6. Stain the cells with trypan blue and count viable cells using a cell counter.
7. Prepare the cell suspension at a concentration of 5 × 10⁶ cells/mL for GL261-luc cells or 5 × 10⁵ cells/mL for B16-F10-luc cells, making it ready for use.

3. Animal preparation

1. Weigh and anesthetize the mice by intraperitoneal injection of a 2.5% tribromoethanol solution (10 µL/g). Confirm anesthesia by checking for the loss of the pedal reflex. The entire procedure, from preparation to suturing, should take approximately 5-10 min.
   NOTE: Position the animal on a heating pad to maintain body temperature throughout the procedure.
2. Expose the skin and prepare a clean surgical window (Figure 1A). Shave the hair from the dorsal neck region and a 2 cm area extending bilaterally from the midline using hair clippers.
   1. To remove any remaining hair, apply a thin layer of depilatory cream to the shaved areas using a cotton swab and leave it for 1-2 min. Afterward, wipe off the depilatory cream with soap-dampened gauze.
3. Disinfect the skin using iodine solution, applied in a circular motion for 30 s, followed by wiping with 75% alcohol for deiodination.

4. Exposure of cervical spine and determination of insertion point

1. Position the mice with their dorsal side facing upward and secure their limbs to the surgical table using medical tape. Place a 1-2 cm thick gauze pad under the neck area for support, providing better access to the spinal cord.
2. Make a longitudinal incision of approximately 1.5 cm along the neck skin using a surgical scalpel and blade (Figure 1B). Gently separate the neck muscles by blunt dissection, taking care to avoid injuring any blood vessels.
3. Carefully dissect the muscles adjacent to the cervical vertebrae to expose the seventh cervical vertebral spinous process, a distinct bony landmark in mice (Figure 1C and Figure 1G-I).
4. Clear any blood from the surgical area using sterile cotton swabs before proceeding with the injection.
5. Set the puncture point at 0.5-0.9 mm from the midline of the spine, adjusting the injection depth to 0.6-0.9 mm based on the body weight of the mice (16-24 g).
   NOTE: The spinal cord injection depth is 0.9 mm for mice weighing 22-24 g.

5. Injection of tumor cells

1. Rinse a 10 µL flat-needle syringe thoroughly with sterile PBS solution 2-3 times.
2. Draw 2 µL of the cell suspension into the syringe, ensuring no air bubbles are present.
3. Stabilize the cervical vertebral spinous process by gently grasping and lifting it with forceps. Use a beveled needle (1.87 mm in length and 0.48 mm in diameter) to puncture the dura mater (Figure 1D). Then, switch to a flat needle syringe (0.48 mm in diameter) to inject the tumor cells (Figure 1E).
   NOTE: The puncture site is preserved during the needle switch, with accurate placement confirmed by lower limb twitching from nerve stimulation.
4. Inject the cell suspension slowly to avoid disruption.
5. Keep the syringe in place for 30 s post-injection to ensure successful tumor implantation.

6. Post-surgical care

1. Close the skin incision by suturing with a 3-0 nylon suture at the end of the operation (Figure 1F).
2. Position the mouse on its side and place it on a heated mat to maintain warmth and ensure stable breathing during recovery from anesthesia in the cage.
3. Administer Buprenorphine (0.1 mg/kg) subcutaneously twice daily for 3 days to alleviate pain.
4. Monitor the mouse to ensure it regains pre-operative activity without signs of bleeding or wound tearing.
   NOTE: Temporary spinal cord dysfunction, including hind limb weakness, is common after surgery and typically resolves within 3 h. Approximately 5% of mice may develop paralysis but usually recover within 3 days. For these mice, provide a nutritionally complete diet and gel water directly on the cage floor to ensure adequate accessibility. A small percentage (about 5%) of mice that experience paraplegia may require euthanasia.
5. Ensure the mice have continuous access to water and food.
   NOTE: If the animals exhibit signs of weight loss or paralysis, they should be housed individually.

7. In vivo bioluminescence imaging

1. Administer an intraperitoneal injection of 150 mg/kg D-luciferin dissolved in D-PBS to the mice.
2. Place the mice in an anesthesia chamber containing isoflurane for induction.
3. Transfer the mice to the integral anesthetic manifold to maintain anesthesia during the procedure.
4. Perform in vivo bioluminescent imaging as described in the previous report⁹.
   NOTE: The optimal response time for D-luciferin in live imaging of small animals is 10 min post-injection. Ensure imaging is conducted precisely 10 min after the injection.

Results

To establish a stable and reliable animal model of spinal glioma, the intervertebral space between the sixth and seventh cervical vertebrae in C57BL/6 mice was identified as the ideal site for inoculation based on literature review and experimental findings¹⁰. The seventh cervical vertebra provides a distinct bony landmark, the spinous process (Figure 1G-I), which aids in accurately locating the injection site and stabilizing the inje...

Discussion

Spinal cord glioma is the most common type of primary malignant tumor in the spinal cord, accounting for over 80% of intramedullary tumors. Pathologically, spinal cord gliomas are primarily classified as ependymomas or astrocytomas, with a particular focus on astrocytomas¹¹. Among astrocytomas, some harbor H3K27M mutations, also known as diffuse midline gliomas (DMGs), which are associated with poor prognoses. A defining feature of spinal cord gliomas is their infiltrative growth pattern, which ma...

Materials

Name	Company	Catalog Number	Comments
A nutritionally complete food and water gelled diet (Nutra-Gel)	Bio-Serv	N/A
Adhesion microscope slides	CITOTEST	188105
AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)	Jacksonimmuno	115-007-003
B16-F10-luc	Professor Wang Xi's laboratory	N/A
Buprenorphine Related Compound A	Sigma-Aldrich	457071-73-7
CD163 (ABT-CD163) mouse mAb	Immunoway	YM6146
CD86 rabbit pAb	Immunoway	YT7823
Cell counter	Bio-rad	1450102
Cell Counting Slides	Biorad	1450011
DAPI/Sealant Dual Solution (Anti-Quenching)	Immunoway	YS0014
Dilator	Jinzhong	D22178
D-Luciferin	PerkinElmer	122799
DMEM	Gibco	C11995500BT
D-PBS	Solarbio	D1040
Fetal Bovine Serum, qualified	Gibco	10270-106
GL261-luc	Shanghai Zishi Biotechnology	N/A
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Invitrogen	A11029
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647	Life	A21244
Goat Serum	Beyotime	C0265
Hamilton microinjector 10 µL fixed 701N	Hamilton	80383
In vivo bioluminescent imaging (IVIS Spectrum)	PerkinElmer	N/A
Methanol	Fuyu Chemical	67-56-1
Micro Scissors	Jinzhong	WAA320
Microliter Syringes (10 µL, pointed tip)	Shanghai Gaoge	N/A
Microscope cover glass	CITOTEST	10212440C
needle holder 12.5 cm	Jinzhong	JCZ200
Ophthalmic Forceps 10 cm	Jinzhong	JD1060
Ophthalmic Scissors 10 cm	Jinzhong	Y00030
PBS, 10×	Solarbio	P1022
Penicillin-Streptomycin Liquid	Solarbio	P1400
Scalpel Blades	Jinzhong	J0B050
super pap pen	ZSGB-Bio	ZLI-9303
Surgical Knife Handle	Jinzhong	J11010
Surgical scissors 12.5cm straight tip	Jinzhong	J21010
Nylon Surgical Sutures with thread, size 3-0	UNIFY	N/A
Tissue-Tek O.C.T. Compound	SAKURA	4583
Tribromoethanol	Sigma-Aldrich	T48402
Triton X-100	Servicebio	GC204003
Trypan Blue Stain Solution, 0.4%	Solarbio	C0040
Trypsin Digestion solutions, 0.25% (without phenol red)	Solarbio	T1350
Tween-20	Solarbio	T8220