U87-MG-Luc | Establishment of an Orthotopic Xenograft Model Using Human Glioma Cells in Nude Mice

The intracranial orthotopic xenograft model using human U87-MG glioma cells is a classic animal model for studying the pathogenesis and therapeutic strategies of glioma. Compared to subcutaneous transplantation, the orthotopic model preserves the brain tumor microenvironment and more closely mimics clinical features such as angiogenesis, infiltrative growth, and drug permeability. This article describes the complete procedure for implanting luciferase-labeled U87-MG cells into the brains of nude mice using stereotaxic techniques.

I. Experimental Materials

· Cells: U87-MG-Luc (luciferase-labeled)
· Animals: Nude mice, male, 6-8 weeks old
· Equipment: Brain stereotaxic instrument, microinjection pump, cranial drill, micro-syringe, in vivo imaging system (IVIS)

Figure 1. Photograph of a brain stereotaxic instrument.

II. Procedure

2.1 Cell Preparation

1. Culture U87-MG-Luc cells to logarithmic growth phase (approximately 80% confluence).
2. Detach cells using 0.25% trypsin and neutralize with serum-containing medium.
3. Wash cells twice with PBS and resuspend in serum-free DMEM.
4. Adjust the cell suspension to the desired concentration and keep on ice.
5. Mix thoroughly just before implantation to avoid cell aggregation.

2.2 Stereotaxic Implantation

1. Anesthetize the nude mouse.
2. Place the mouse in the prone position on the stereotaxic instrument and adjust the incisor bar to level the skull.
3. Disinfect the scalp with 75% ethanol.
4. Make a 1 cm longitudinal incision along the midline of the scalp. Retract the skin and periosteum to expose the bregma.
5. Locate the right caudate nucleus according to a mouse brain atlas and mark the injection site on the skull.
6. Carefully drill a burr hole through the skull using a dental drill.
7. Load a micro-syringe with 5 μL of the cell suspension. Insert the needle slowly through the burr hole to the target depth.
8. Inject the cell suspension at a rate of 0.5 μL/min. Leave the needle in place for 2 minutes post-injection.
9. Withdraw the needle slowly. Seal the burr hole with bone wax and rinse the surgical field with sterile saline.
10. Suture the scalp incision and disinfect with iodine.
11. Place the mouse on a 37°C warming pad until it recovers from anesthesia, then return it to its cage for routine housing.

III. Post-Operative Monitoring and Tissue Collection

3.1 General Observation

Monitor the mice daily for signs of lethargy, changes in food/water intake, activity level, and body weight. Mice typically resume normal feeding and drinking from day 2 post-surgery. Neurological symptoms such as weight loss, decreased activity, hunched posture, irritability, or hemiparesis usually appear 2-3 weeks post-implantation.

3.2 In Vivo Imaging Monitoring

1. Administer D-luciferin potassium salt (150 mg/kg) via intraperitoneal injection. Image the mice 10-15 minutes post-injection.
2. Anesthetize the mouse with isoflurane and place it prone in the IVIS dark box.
3. Acquire bioluminescent signals. Quantify tumor burden as total photon flux (photons/sec) from the region of interest (ROI).
4. Image mice 1-2 times per week to dynamically monitor tumor growth.

Figure 2. Representative in vivo bioluminescence imaging results from nude mice.

3.3 Tissue Collection and Pathological Examination

1. Deeply anesthetize the mouse, then open the chest to expose the heart.
2. Perfuse the mouse transcardially with heparinized saline until the effluent is clear.
3. Perfuse with 4% paraformaldehyde (PFA) for fixation.
4. Carefully remove the whole brain and post-fix in 4% PFA overnight.
5. Embed the brain tissue in paraffin.
6. Perform Hematoxylin and Eosin (H&E) staining and immunohistochemistry (IHC) to confirm glioma characteristics.

IV. Criteria for Tumor Take Evaluation

· Positive Tumor Take: Stable bioluminescent signal detectable by in vivo imaging + histopathological confirmation (H&E) of glioma cells.
· Quantitative Indicators: Dynamic curves of ROI photon flux.
· Supplementary Indicators: Tumor take rate, survival time, body weight change curves, and pathological morphological characteristics.