Generation of a Patient-Derived Brain Metastasis Breast Cancer Cell Line via Novel Orthotopic Injection Placement and Serial Mouse Transplantation to Develop PDX Mouse Model

摘要

Background: The incidence of brain metastasis appears to be increasing, potentially due to advanced technology that aids early diagnosis. Patient-derived xenografts (PDX) have high translational value, as these models retain key functional characteristics of the patient tumor. PDX models are useful to understand the molecular basis of tumorigenesis and to identify new treatment targets. However, generating a first-line PDX model is challenging as engraftment failure is high. Serial transplanting tumor tissue via mouse-to-mouse propagation increases engraftment rates and decreases PDX development time. Herein we report methods to generate a PDX cell line from patient-derived tumor tissue that includes the cerebral aqueduct as a novel intracranial orthotopic implantation site. Purpose: Develop human tumors in mouse models for therapeutic purpose. Methods: Patient-derived brain metastasis tumor tissue was enzymatically dissociated into a single cell suspension and maintained in neurocult media supplemented with human recombinant bFGF and EGF (20 ng/ml). The cells were seeded at a density of 1.0 × 104/cm2 on ultra-low attachment plates and maintained at 37°C with 5% CO2. PDX models were generated via orthotopic stereotactic surgeries. Athymic nude mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). The cerebral aqueduct was located using these coordinates from bregma: A: -5; L: +0.2; V: -2.4. Mice were injected with 5.0 × 104 cells in 2 μl of media at a rate of 0.4 ul/min. Mice were monitored daily for symptoms of tumor formation. Upon becoming symptomatic, mice were euthanized and tumor tissue was harvested for both culture and H&E stain for tumor verification. Results: Mice injected with primary patient cells (first-generation mice) developed tumors at 7 weeks (average: 6.77 weeks), second-generation mice yielded tumors at 2 weeks (average: 13.5 days), and third-generation mice replicated results from second-generation mice (average: 13 days). H&E stain revealed invasive tumor masses in the ventricular system that extended from the cerebral aqueduct to the lateral ventricles. Immunohistochemistry analysis confirmed the third-generation cell line retained key characteristics of the patient tumor. Conclusion: These methods successfully generated a PDX cell line from patient-derived brain metastasis that demonstrates reliable tumor formation and phenotypic stability. Importantly, our unique intracranial implantation site revealed several distinct masses, a hallmark of brain metastasis in patients.