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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 su....

Results

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.......

Discussion

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.......}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10-0 microsurgical suture with needle	Lingqiao	2650798	Microsurgical suture for repair of the common carotid artery puncture site
100 µL microliter syringe	Bolige Tools	100UL	Microliter syringe for tumor cell delivery during interlock pulsatile injection
100 µL pipette	Eppendorf	3123-000250	Pipette for saline irrigation of the surgical field
32G 1 mL insulin syringe	Kindly	YY0497	Insulin syringe used to create the initial arterial puncture site
4-0 silk suture	Ethicon	W501	Suture material for closure of the skin incision
4T1 breast cancer cell line	Obtained from Laboratory Shengdian Wang, Institute of Biophysics, Chinese Academy of Sciences	CRL-2539	Tumor cell line used for intracarotid brain metastasis modeling
75% Ethanol	Annjet	Q/371402AAJ008	Disinfectant for cleaning the injection site
7–8-week-old male C57BL/6J mice	Charles River Laboratories	1.10011E+17	Experimental animals used for the B16F10 brain metastasis model
8-week-old female BALB/c mice	Charles River Laboratories	1.10011E+17	Experimental animals used for the 4T1 brain metastasis model
Angled-tip forceps	Kewo Tools	—	Microsurgical forceps for tissue dissection and vessel manipulation
B16F10 melanoma cell line	Obtained from Laboratory Shengdian Wang, Institute of Biophysics, Chinese Academy of Sciences	CRL-6475	Melanoma cell line used for intracarotid brain metastasis modeling
Bioluminescence imaging analysis software	Living Image (version 4.7.2)	—	Software for analysis of bioluminescence imaging data
DMEM	Xigong Bio Tech	M1805	Cell culture medium for tumor cell maintenance
Enrofloxacin (antibiotic)	MCE	HY-B0502	Antibiotic used for postoperative infection prophylaxis
Eosin Y solution	Beyotime	E301879	Histological staining reagent for eosin staining
Eye ointment	Baiyunshan	H44023089	Ophthalmic ointment for protection of the eyes during anesthesia
Fetal bovine serum (FBS)	Pan Sera Tech	ST30-3302	Serum supplement for cell culture medium
GraphPad Prism	Version 4.7.2	—	Statistical analysis software for survival curves and quantitative data analysis
Hematoxylin solution	Beyotime	C0105S	Histological staining reagent for hematoxylin staining
Isoflurane	H.F.Q. Biotechnology	101357015	Inhalation anesthetic used during imaging procedures
IVIS Spectrum in vivo imaging system	Lumina Series III	—	Imaging system for in vivo bioluminescence imaging
Meloxicam (analgesic)	Qilu Animal Health	150252467	Non-steroidal anti-inflammatory drug used for postoperative analgesia
Microsurgical scissors	Kewo Tools	—	Microsurgical scissors for tissue dissection
Mini vessel clip	Zheyong Tools	200521	Microvascular clip for temporary occlusion of the common carotid artery
Needle holder	Jiangxin Tools	1420 20Cr13	Surgical instrument for handling microsurgical sutures
Paraformaldehyde (4%)	Zhongsheng Wanda Tech	20240411	Fixative for brain tissue preservation prior to histological analysis
Penicillin	Gibco	1758-9324	Antibiotic for cell culture
Povidone-iodine solution	Sihuanpai	Q/PGSHW0009	Antiseptic solution for surgical skin disinfection
RPMI-1640 medium	Xigong Bio Tech	12-115F	Cell culture medium for tumor cell growth
Scilogex mixers	Scilogex	MX-S	Vortex mixer for homogenizing tumor cell suspensions
Steam indicator tape	3M-Comply	1322-24MM	Sterilization indicator for autoclaved surgical instruments
Stereomicroscope	Olympus	SZ61	Microscope used for visualization during microsurgical procedures
Zeiss Stemi 508 stereomicroscope	Zeiss	Stemi 508	Stereomicroscope used for high-magnification surgical visualization
Sterile saline (0.9%)	Kelun	25M14D07	Sterile irrigation solution used to maintain moisture of the surgical field
Streptomycin	Gibco	1758-9319	Antibiotic for cell culture
Surgical drape	Dafang	001-2016	Sterile drape used to isolate the surgical field
Surgical gloves	Ansell Medi-grip	210-64005	Sterile gloves used during surgical procedures
Tribromoethanol	Sigma-Aldrich	MKBH0634V	Injectable anesthetic for surgical procedures
Ultrasound Doppler	Mindray S7 SCI	—	High-resolution ultrasound system for assessment of carotid artery blood flow