Engineering High-fidelity Preclinical Mouse Model for Brain Metastasis using Interlock Pulsatile Injection Technique and Microsurgical Vascular Repair

摘要

This study establishes high-fidelity mouse models of brain metastasis via transient intracarotid injection of tumor cells using Interlock pulsatile injection and microsurgical repair. It preserves cerebral blood flow, prevents occlusion of the common carotid artery, and provides superior preclinical models with improved tumor metastasis rates and reduced mortality.

摘要

Brain metastasis remains a devastating clinical problem. A major challenge in brain metastasis research is the lack of high-quality models that accurately recapitulate the metastatic process, thereby enabling mechanistic insights into how cancer cells colonize in the brain. Traditional intracarotid artery injection models of brain metastasis often require permanent ligation of the common carotid artery (CCA), which alters cerebral hemodynamics and compromises the integrity of the blood-brain barrier (BBB). The protocol presents a refined method for establishing a high-fidelity mouse model of brain metastasis. The core innovation involves the Interlock Pulsatile Injection (IPI) technique for tumor cell delivery, followed by microsurgical arterial reconstruction at the puncture site to restore physiological blood flow in the CCA. Compared with the conventional CCA ligation model, the IPI–microsurgical repair approach significantly reduced perioperative mortality (2.86% vs. 25.71%) and increased the rate of brain metastasis establishment (94.12% vs. 65.38%). The IPI technique utilizes a tandem syringe configuration to minimize cell regurgitation during intracarotid injection. After tumor cell infusion, the CCA is meticulously repaired using microsurgical sutures under a stereomicroscope, thereby avoiding permanent occlusion. This preserves the native cerebral hemodynamics and the integrity of the BBB at the time of tumor cell entry, while significantly improving surgical success rates and reducing mortality. The metastatic intracranial lesions are validated by serial bioluminescence imaging and histopathology. The method provides a superior platform for studying the pathophysiology of brain metastasis and for preclinical therapeutic evaluation, thereby recapitulating the metastatic process.

引言

Brain metastases represent the most common type of malignant brain tumor in adults, occurring approximately ten times more frequently than primary brain tumors¹. It is estimated that 10%–20% of patients with systemic cancer will develop brain metastases during the advanced stages of their disease, which is a leading cause of mortality^1,2. Despite significant advances in the treatment of primary cancers, the prognosis for patients with brain metastases remains poor. The one-year survival rate of patients with brain metastasis is less than 20%³. The mechanism of why and how some cancers metastasize to the brain remains unknown. The therapeutic challenge is compounded by the unique biology of brain metastases, including the presence of the BBB, the distinct brain immune microenvironment, and complex tumor cell interactions with neuroglial cells^4,5.

Both pharmacodynamic and pathogenesis research on brain metastasis heavily rely on precise preclinical animal models that accurately recapitulate the human disease process. Current animal models of brain metastasis, though widely used, are mainly based on intracranial orthotopic implantation, intravenous or tail vein injection, intracardiac injection, or intracarotid artery injection^6,7,8,9,10. However, these models imperfectly mirror human disease progression, often failing to capture key steps such as tumor cell extravasation across an intact BBB under physiological flow conditions¹¹. Intravenous and intracardiac injections result in low brain-specific tumor formation efficiency and often lead to widespread extracranial metastases, causing premature animal death before substantial brain metastases development¹². The traditional intracarotid artery injection method increases intracranial tumor formation rates but requires permanent ligation of the CCA. By altering cerebral hemodynamics, this ligation may induce ischemia in ipsilateral brain regions and impair physiological blood flow patterns necessary for tumor cell delivery, thus affecting their spatial distribution and colonization niche within the brain^13,14.

High-quality animal models that can more faithfully mimic the pathophysiology of brain metastasis are crucial¹⁵. An ideal brain metastasis model should recapitulate the entire metastatic cascade, including tumor cell homing to the brain vasculature, extravasation across the blood–brain barrier (BBB), and dynamic interactions with the brain microenvironment¹⁶. High model fidelity is critical for both mechanistic studies and therapeutic evaluation⁶. However, existing intracarotid injection models do not fully reproduce key physiological conditions, particularly the preservation of normal cerebral blood flow and the natural distribution of circulating tumor cells.

To overcome the limitations of existing preclinical models of brain metastasis, a high-fidelity animal model that more accurately recapitulates the metastatic cascade was developed based on the IPI technique and microsurgical arterial repair. This method involves transient arterial occlusion after tumor cell injection, followed by meticulous repair of the arterial puncture site to restore physiological blood flow to the ipsilateral CCA. This approach not only enhances model consistency and success rates but also, crucially, preserves the BBB by maintaining native cerebral hemodynamics. By minimizing disruption of cerebral blood flow, the modified intracarotid injection model provides a high-fidelity platform that more accurately recapitulates the natural process of brain metastatic colonization, thereby offering an improved tool for investigating mechanisms of brain metastasis and evaluating potential therapeutics.

方案

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Biophysics, Chinese Academy of Sciences (approval number: SYXK2021124). Animals reaching predefined endpoints were euthanized in accordance with the Guidelines for the Euthanasia of Animals of the Institutional Animal Care and Use Committee (IACUC) of the Institute of Biophysics, Chinese Academy of Sciences, and the AVMA Guidelines for the Euthanasia of Animals (2020)¹⁷.

1. Preparation of the single cell suspension

1. Culture B16F10 and 4T1 cells in complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
2. Maintain cells at 37 °C in a humidified incubator with 5% CO₂.
3. Confirm luciferase expression and verify absence of mycoplasma contamination one day prior to surgical procedures.
4. On the day of surgical operation, harvest cells when they reach 80% confluence by first washing with phosphate-buffered saline (PBS) before trypsinization (0.25%) for less than 3 min at 37 °C.
5. Add 10% FBS-containing RPMI-1640 media into the cells to quench the 0.25% trypsin.
6. Centrifuge the cells at 300 × g for 3 min. Wash cells twice in PBS to remove residual serum, then count them with a hemocytometer.
7. Use the Trypan blue exclusion assay to confirm that cell viability remains at 90%–95% during injection.
8. Resuspend cells in PBS at 2 × 10⁶ cells/mL. Keep cells on ice until injection.
   NOTE: Luciferase-labeled B16F10 and 4T1 cells can be used. B16F10 is presented as the representative example.

2. Mice preparation and perioperative preparation

1. Use 7–8-week-old male C57BL/6 mice.
2. Place a heating pad under the surgical plate to prevent hypothermia. Turn on the heating pad 30 min before surgery.
3. Sterilize all the surgical instruments, surgical plate, and housing cage with an autoclave. Disinfect the stereomicroscope and surgical table with 75% ethanol.
4. Other items that cannot be sterilized by autoclaving should be disinfected using UV irradiation and 75% ethanol (Figure 1A).
5. Weigh the mouse before anesthetization and record the preoperative weight as baseline.
6. Add 10 g of tribromoethanol to a centrifuge tube, then add 10 mL of tertiary amyl alcohol. Shake the tube until the tribromoethanol is completely dissolved.
   NOTE: Tribromoethanol may cause adverse effects, including mortality; monitor anesthetic depth carefully.
7. Filter the solution through a 0.22 µm filter to prepare a 100% stock solution.
8. Dilute the 100% stock solution with 0.9% NaCl saline or diluent to a 2.5% working solution (1:40 dilution). The efficacy of each freshly prepared working solution must be tested for anesthesia in mice.
9. Anesthetize mice by intraperitoneal (i.p.) injection of 2.5% tribromoethanol at 0.2 mL/10 g body weight. When required, prolong anesthesia by administering a top-up dose at one-third of the original anesthetic volume (Figure 1B).
   NOTE: Adjust anesthetic dosing based on body weight and continuously monitor respiration.
10. Ensure the mouse is fully anesthetized by applying a mild stimulus (e.g., toe pinch) and observing no motor response.
11. Shave the fur in the neck region and make a toe clipping mark after anesthesia.
12. Apply eye ointment to protect eyes from dryness.
13. Place the mouse on a surgical plate and secure the 4 limbs and upper incisors with adhesive tape (Figure 1C).
14. Monitor anesthesia depth by assessing respiratory rate and the pinch reflex to ensure that the mice remain pain-free and immobile during the procedure.
15. Animals are randomly assigned to experimental groups, and imaging analysis was performed by investigators blinded to group allocation.

3. Standard surgical procedures of the IPI technique and microsurgical repair

1. Disinfect the neck region by povidone-iodine solution and allow the skin to dry completely before proceeding (Figure 1D).
2. Drape the mouse aseptically using a sterile surgical drape, exposing only the neck region (Figure 1E).
3. Make a midline cervical incision approximately 0.8–1 cm in length using sterile surgical scissors, with fine forceps for tissue retraction (Figure 2A).
4. Perform blunt dissection of the superficial fascia and underlying muscles using fine angled forceps to expose the right carotid sheath (Figure 2B1, Figure 2C1).
5. Isolate the right External Carotid Artery (ECA) from the carotid sheath and ligate the right ECA to prevent cell leakage into the extracranial branches (Figure 2B2, Figure 2C2).
6. Carefully isolate the right CCA within the carotid sheath, preserving the vagus nerve located laterally and beneath the vessel (Figure 2C1).
7. Place a small piece of a rubber glove and a saline-moistened cotton ball beneath the isolated right CCA as a pillow to elevate and stabilize the vessel.
   NOTE: Elevating the CCA stabilizes the vessel and facilitates accurate puncture and injection. (Figure 2B4, Figure 2C4).
8. Place two untightened knots with open loops on the CCA distal and proximal to the cotton ball, and then tie the proximal knot (Figure 2B3, Figure 2C3).
9. Apply a microvascular clip on the CCA near the distal untightened knot to temporarily block the CCA blood flow (Figure 2B4, Figure 2C4).
10. Cancer cell implantation
    1. Interlock puncture
       1. Puncture the elevated segment of the right CCA using a 32G needle with the bevel facing upward.
       2. Withdraw the initial syringe while maintaining the puncture site.
       3. Vortex-mix the cancer cell suspension and draw 100 µL into the microliter syringe, ensuring that no air bubbles are aspirated.
       4. Insert the needle tip of the microliter syringe into the puncture site created in Step 3.10.1.1 and hold it steadily without initiating injection to ensure stable positioning prior to cell delivery (Figure 2B5, Figure 2C5, Supplementary Figure 1).
          NOTE: Use a 32G syringe for puncture and a microliter syringe for injection to ensure stable positioning and controlled delivery. Supplementary Figure 1 shows the parameters of the two syringes. The outer diameter of the 32G syringe is 0.22 mm, while the microliter syringe needle is 0.26 mm, facilitating secure insertion and controlled injection.
    2. Pulsatile injection (Figure 2C5)
       1. Remove the microvascular clip applied in Step 3.9.
       2. Begin injection of the tumor cell suspension into the ICA through the microliter syringe.
       3. Deliver the suspension using a pulsatile injection motion, gently advancing the syringe plunger in small pulses rather than continuous pressure.
       4. Administer the suspension using either of the following criteria: the cardiac cycle criterion, injecting ~1 µL of cell suspension every 8 cardiac cycles guided by pulsatile retrograde blood flow at the distal internal carotid artery; or the injection rate criterion, injecting ~1 µL/s while ensuring the rate does not exceed 60 µL/min.
       5. Monitor the vessel during injection. Successful delivery is indicated when the red vessel becomes temporarily transparent as the suspension passes through the lumen, with no visible extravasation around the puncture site.
       6. After completing the injection, withdraw the microliter syringe carefully.
       7. Immediately tighten the distal knot in a controlled manner to seal the vessel and minimize regurgitation (Figure 2B6, Figure 2C6).
          NOTE: Pulsatile injection reduces overperfusion and minimizes the risk of intracranial pressure elevation.
11. Microsurgical repair (Figure 2B6, Figure 2C6)
    1. Use a 10-0 nylon microsurgical suture to close the puncture site in a watertight manner, positioning the needle entry and exit points immediately adjacent to the defect margin to minimize vascular leakage and preserve luminal patency.
    2. Pass the microsurgical suture needle through the puncture hole in the vessel wall under high magnification and leave the suture untied temporarily.
    3. Flush the surgical field with heparinized saline.
    4. Secure the suture with two to three square knots.
    5. Trim the free ends of the suture to approximately 1–2 mm to prevent loosening while minimizing foreign body burden.
       ​NOTE: Standard surgical procedures of the IPI technique and microsurgical repair are described in Supplementary Video 1.

4. Restoration of the blood flow of the CCA

1. Gradually loosen the proximal knot.
2. Flush the lumen to remove any potential microscopic clot. Place a dry cotton ball in the surgical field to monitor for excessive bleeding.‌
3. Once no active effusion from the puncture site is confirmed, cautiously loosen the distal knot to restore carotid blood flow (Figure 2B7, Figure 2C7).

5. Confirm successful repair

1. Restoration of pulsation
   1. Observe the CCA for robust pulsations, indicating successful reperfusion.
2. Verify patency and vessel integrity​
   1. Inspect the repair site to ensure it is free of stenosis and that the lumen remains patent and continuous with the proximal vessel segment.

6. Wound irrigation and closure

1. Irrigate the surgical field thoroughly with non-heparinized normal saline to remove debris and blood clots.
2. Close the skin incision using a continuous suture.
   NOTE: Maintain a moist surgical field and perform all manipulations gently to minimize tissue injury.

7. Postoperative care

1. Fluid support: Administer 0.5–1 mL warm sterile saline subcutaneously immediately after surgery to compensate for perioperative fluid loss.
2. Infection prophylaxis: If the operative duration exceeds 40 min, administer enrofloxacin (5 mg/kg, once daily) for 3 consecutive days. Antibiotics are not required if the procedure lasts less than 40 min.
3. Nutrition and hydration: Provide hydrogel or softened food for at least 1 week postoperatively to ensure adequate hydration and nutrition.
4. Daily monitoring: Perform daily health assessments, observing for circling behavior, neurological deficits, wound infection, and weight loss.
5. Expected postoperative course: During the first 48 h, mice may show reduced activity, mild piloerection, and weight loss up to 15% of the preoperative baseline, followed by gradual recovery.
6. Analgesia: Administer meloxicam (5 mg/kg, subcutaneously) once daily for 2–3 days to relieve pain and promote recovery.

8. Evaluation criteria for successful animal model establishment

1. Body weight monitoring
   1. Weigh the mice daily and record body weight.
2. Clinical assessment
   1. Assess clinical signs daily, including posture and circling behavior.
3. Bioluminescence imaging
   1. Turn on the power switches of the imaging instrument and the computer.
   2. Launch the bioluminescence imaging analysis software (Version 4.7.2) and click “Initialize” to initialize the bioluminescence imaging system.
   3. During initialization, the temperature status light on the imaging system control panel will appear red. Perform imaging only after the temperature status light turns green.
   4. Withhold food and water from mice for 6 h prior to imaging to prevent aspiration during anesthesia.
   5. Prepare the imaging substrate at a concentration of 10 mg/mL, protected from light throughout the procedure. Calculate the injection volume at 10 µL/g body weight.
   6. Restrain the mouse for imaging. Clean the injection site in the lower left abdomen with 75% medical alcohol.
   7. Use a cotton swab to gently stimulate the abdominal wall at the injection site to minimize the risk of the needle entering the intestine.
   8. After puncturing the abdominal wall, aspirate gently to confirm the absence of blood, then slowly inject the in vivo imaging substrate.
   9. At 4 min 30 s after substrate injection, transfer the mouse to an anesthesia induction chamber.
   10. Induce anesthesia using 3% isoflurane. Once the righting and pinch reflexes are lost, reduce the isoflurane concentration to 2% for maintenance.
   11. Place the anesthetized mouse in the imaging chamber, ensuring that the nose cone fully covers the mouth and nose. Close the imaging chamber door securely.
   12. In the imaging system control panel, select “Luminescent” as the imaging mode. Set the exposure time to 60 s manually. Select “Photography,” “Overlay,” and “Alignment Grid”.
   13. Set the field of view to D for imaging three mice. Adjust the imaging height to 1.5 cm. Select “Block” for the Excitation Filter and “Open” for the Emission Filter.
   14. At 10 min after substrate injection, click “Acquire” to capture the image.
   15. After image acquisition, navigate to the Tool Palette and select “ROI Tools”. Use the “Circle” tool to delineate the region of interest (ROI), then click “Measure ROIs” to obtain quantitative data.
4. Histological analysis
   1. Harvest brain tissues at the experimental endpoint and subject them to histological analysis.
   2. Identify and confirm metastatic lesions by hematoxylin and eosin (H&E) staining.

9. Humane endpoints

1. To ensure animal welfare and compliance with ethical standards, predefined humane endpoints have been established as criteria for immediate euthanasia.
2. Mice are euthanized by carbon dioxide (CO₂) inhalation, followed by cervical dislocation as a secondary physical method to confirm death.
3. Immediate euthanasia should be performed if any of the following conditions occur.
   1. Neurological deficits
      1. Motor dysfunction: ataxia (unsteady gait, swaying), circling, paralysis, hemiparesis, or tremors.
      2. Behavioral or consciousness alterations: lethargy, stupor, or coma.
      3. Signs of increased intracranial pressure: abnormal posturing (e.g., hunched back indicating distress) or head tilt.
      4. Seizures: generalized or focal convulsions.
   2. Changes in body weight and physiological function
      1. Body weight loss exceeding 20% or inability to eat or drink independently due to tumor burden or severe weakness.
      2. Dyspnea (gasping or panting) or urinary/fecal incontinence.
   3. Tumor status
      1. Terminate the experiment immediately if tumor-associated ulceration, infection, or necrosis is detected.
   4. Maximum tolerated tumor burden
      1. Mice undergo bioluminescence imaging twice weekly.
      2. Once the intracranial tumor signal enters an exponential growth phase (e.g., doubling between two consecutive imaging sessions), neurological monitoring is increased to twice daily.
      3. Euthanasia performed immediately upon the onset of ataxia or seizures, regardless of the bioluminescence signal value.

结果

Bioluminescent imaging and quantitative analysis
Following the standardized surgical protocol, the optimized mouse model of brain metastasis demonstrated stable intracranial bioluminescent signals on serial in vivo imaging postoperatively. Continuous in vivo bioluminescent imaging (Figure 3A) and quantitative analysis (Figure 3B) over 4 weeks after intracarotid inoculation showed that metastatic B16F10 cells proliferated wi...

讨论

Robust preclinical models that accurately recapitulate brain metastasis are essential for elucidating pathophysiological mechanisms and evaluating therapeutic efficacy. The traditional intracarotid injection model requires ligation of the common carotid artery (CCA), which alters cerebral blood flow in a non-physiological manner, potentially inducing ipsilateral ischemic injury and disrupting the blood–brain barrier (BBB)^9,11,15...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
10-0 显微外科缝合针	灵桥	2650798	用于修复颈动脉穿刺部位的显微外科缝合
100 µL 微升注射器	博力格工具	100UL	用于锁定脉动注射时递送肿瘤细胞的微升注射器
100 µL 移液器	艾普多夫	3123-000250	用于手术区盐水冲洗的移液器
32G 1 mL 胰岛素注射器	慈爱	YY0497	用于创建初始动脉穿刺部位的胰岛素注射器
4-0 丝线缝合	艾迪康	W501	用于皮肤切口闭合的缝合材料
4T1 乳腺癌细胞系	获得自中国科学院生物物理研究所盛电王实验室	CRL-2539	用于颈动脉脑转移模型的肿瘤细胞系
75% 乙醇	安杰特	Q/371402AAJ008	用于清洁注射部位的消毒剂
7-8 周龄雄性 C57BL/6J 小鼠	查尔斯河实验室	1.10011E+17	用于 B16F10 脑转移模型的实验动物
8 周龄雌性 BALB/c 小鼠	查尔斯河实验室	1.10011E+17	用于 4T1 脑转移模型的实验动物
尖端钳	科沃工具	—	用于组织解剖和血管操作的显微外科钳
B16F10 黑色素瘤细胞系	获得自中国科学院生物物理研究所盛电王实验室	CRL-6475	用于颈动脉脑转移模型的黑色素瘤细胞系
生物发光成像分析软件	Living Image（版本 4.7.2）	—	用于分析生物发光成像数据的软件
DMEM	西工生物科技	M1805	用于肿瘤细胞维持的细胞培养基
恩诺沙星（抗生素）	MCE	HY-B0502	用于术后感染预防的抗生素
伊红 Y 溶液	贝奥泰姆	E301879	用于伊红染色的组织学染色试剂
眼药膏	百云山	H44023089	用于麻醉期间保护眼睛的眼科软膏
胎牛血清（FBS）	Pan Sera Tech	ST30-3302	细胞培养基的血清补充剂
GraphPad Prism	版本 4.7.2	—	用于生存曲线和定量数据分析的统计分析软件
苏木精溶液	贝奥泰姆	C0105S	用于苏木精染色的组织学染色试剂
异氟烷	H.F.Q. 生物技术	101357015	用于成像过程中的吸入麻醉剂
IVIS Spectrum 体内成像系统	Lumina Series III	—	用于体内生物发光成像的成像系统
美洛昔卡（止痛药）	齐鲁动物保健	150252467	用于术后镇痛的非甾体抗炎药
显微外科剪刀	科沃工具	—	用于组织解剖的显微外科剪刀
小型血管夹	哲勇工具	200521	用于临时闭塞颈动脉的微血管夹
针持钳	江欣工具	1420 20Cr13	用于处理显微外科缝合的手术器械
甲醛（4%）	中升万达科技	20240411	用于组织学分析前脑组织保存的固定剂
青霉素	Gibco	1758-9324	用于细胞培养的抗生素
碘伏溶液	四环牌	Q/PGSHW0009	用于手术皮肤消毒的防腐溶液
RPMI-1640 培养基	西工生物科技	12-115F	用于肿瘤细胞生长的细胞培养基
Scilogex 搅拌器	Scilogex	MX-S	用于均质肿瘤细胞悬液的旋转混合器
蒸汽指示胶带	3M-Comply	1322-24MM	用于高压灭菌手术器械的灭菌指示胶带
立体显微镜	奥林巴斯	SZ61	用于显微外科手术过程中可视化的显微镜
Zeiss Stemi 508 立体显微镜	Zeiss	Stemi 508	用于高放大度手术可视化的立体显微镜
无菌生理盐水（0.9%）	科伦	25M14D07	用于维持手术区湿润的无菌灌洗液
链霉素	Gibco	1758-9319	用于细胞培养的抗生素
手术布	大方	001-2016	用于隔离手术区的无菌布
手术手套	Ansell Medi-grip	210-64005	用于手术过程中的无菌手套
三溴乙醇	Sigma-Aldrich	MKBH0634V	用于手术过程中的可注射麻醉剂