Modelling Brain Metastasis: Standardized Analysis of Metastatic Colonization and Histological Growth Patterns by Stereotactic Intracortical Injection

Our group investigates how tumor cells colonize the brain and how metastatic growth patterns influence clinical outcomes and treatment responses. To begin, weigh each mouse using a precision scale to calculate the required volume of anesthetic based on its body weight. After administering the anesthetic, place the mouse on a prewarm heating pad, maintained at 37 to 38 degrees Celsius to support thermoregulation, then apply eye ointment to protect the cornea from drying.

Prepare the mouse using aseptic technique approved by the local animal ethics committee. Thoroughly disinfect the scalp using an iodine-based solution. Confirm the depth of anesthesia with a toe pinch, then stretch the skin taut and make a midline incision of approximately three to four millimeters along the skull using a sterile scalpel.

Using the same scalpel, gently scrape away the periosteum to expose the skull surface. Secure the mouse's head in a stereotactic frame using ear bars. Using the stereotactic arm, locate bregma, then move the arm two millimeters anterior and one millimeter lateral to the right, and mark the target site in the frontal cortex with a surgical marker.

Carefully drill through the skull at the marked site using a precision dental drill, ensuring the dura mater is not penetrated. Confirm the opening by gently probing the site with sterile forceps. Next, resuspend the tumor cell suspension by pipetting up and down to ensure even distribution.

Load three microliters of the suspension into a 10-microliter Hamilton syringe and place it into the stereotactic frame using the appropriate holder. Carefully position the syringe needle over the bur hole, ensuring that the bevel is facing to the left for injection consistency. Insert the needle vertically into the brain tissue to a depth of 3.5 millimeters.

Once the required depth is reached, slowly withdraw the needle by 0.5 millimeters to help prevent reflux of the cell suspension and inject three microliters of the tumor cell suspension slowly over one minute. After injection, keep the needle in place for an additional two minutes to allow the cells and extracellular matrix to settle and minimize reflux. Then carefully withdraw the syringe and remove the mouse from the stereotactic frame.

Gently rinse the skull with sterile 0.9%sodium chloride solution, and seal the bur hole using bone wax. Close the scalp incision using three to four sutures tying each suture with three tight knots placed close to the wound edges for optimal healing. Mark the mouse's ears using an appropriate ear punch pattern corresponding to the designated identification number.

Then reapply eye ointment if necessary to prevent corneal drying during recovery and return the mouse to a prewarm heating pad maintained at 37 to 38 degrees Celsius to aid recovery from anesthesia. Thoroughly clean and disinfect the Hamilton syringe by rinsing the needle and barrel three times sequentially, first with distilled water, then with 70%ethanol, and finally with sterile 0.9%sodium chloride solution. After rinsing store the Hamilton syringe on ice until its next use.

Finally administer analgesic subcutaneously while the mouse is still under anesthesia to ensure immediate postoperative pain relief. House mice individually during recovery for a brief period, not exceeding one hour to prevent injuries from interactions between awake and recovering animals. If signs of distress or abnormal behavior are observed in the monitoring period, evaluate neurological function using the hanging wire test to assess grip strength and coordination.

Suspend a mouse on a horizontal wire and record whether it reaches an escape platform within 60 seconds. An non-healthy mouse fails to reach the platform and falls off the wire. After euthanizing and collecting the animal tissues, evaluate the histological growth pattern of the metastasis and associated features on digital tissue slides.

Macroscopic images confirmed the absence of tumor growth in ECM injected control valve C mice while visible metastatic lesions were present in mice injected with tumor cell ECM suspensions. Mice injected with either 4T1 or 410.4 breast cancer cells showed a significantly higher metastatic burden compared to ECM injected controls with no significant difference between the two tumor cell lines. Kaplan Meyer survival analysis demonstrated that mice injected with 410.4 tumor cells had a significantly longer overall survival compared to those injected with 4T1 cells.

Histological analysis showed that 4T1 and 410.4 brain metastases exhibited distinct growth patterns with 4T1 showing cohort-like and 410.4 showing strand-like epithelial infiltrative growth. Improper stereotactic injection led to tumor growth in the skull and meninges due to cell suspension leakage, while proper intracortical injection resulted in parenchymal metastasis with subsequent meningeal dissemination. We have demonstrated that brain metastasis growth patterns influence prognosis and neurological death supporting the relevance for guiding clinical decision-making.

This protocol is particularly useful for studying late state metastatic colonization, addressing clinically relevant aspects like growth dynamics, and secondary dissemination. Unlike other methods, the stereotactic model ensurers brain specific colonization and preserves full system complexity, enabling long-term studies of tumor progression and therapy response. By providing a reproducible clinically relevant platform, our model contributes to understand the mechanisms of brain colonization and explore new therapeutic strategies.