Mice were anaesthetized with an intraperitoneal injection (ip.) of 0.1 ml of a stock solution containing ketamine HCl (25 mg/ml), xylazine (2.5 mg/ml) and 14.25 %25 % ethyl alcohol (diluted 1:3 in 0.9 % NaCl). non-toxic and rational therapeutic approach for the successful treatment Mevalonic acid of HER2 overexpressing BCBM, which now warrants further preclinical and clinical investigation. Keywords: Neural stem cells, Breast cancer brain metastasis, Human epidermal growth factor receptor 2, HER2 overexpressing breast cancer, Trastuzumab, Blood brain barrier, Monoclonal antibody therapy Graphical Abstract Introduction Brain metastases are a fatal complication of breast cancer with a median survival time of 4-12 months [1]. Surgical resection in addition to whole brain / stereotactic radiation therapy are the only available options which provide limited survival benefits. Moreover, some lesions, such as diffuse micro-metastases and those located in eloquent cortex or critical structures, are not amenable to surgical resection. At present, there is a dearth of targeted treatment modalities for the treatment of breast cancer brain metastases (BCBM), warranting the development of novel therapies in this domain. Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that is overexpressed in about 30% of breast cancer patients and is associated with advanced disease and poor prognosis [2]. This overexpression of HER2 in breast cancer patients increases the propensity for CNS metastases, which ranges from 30.7% – 53% in various cohorts [3, 4]. Therefore, HER2 protein is a suitable target for the treatment of BCBM. Trastuzumab (trade name Herceptin?) is a humanized monoclonal antibody which is Rabbit polyclonal to FOXRED2 effective in the treatment of systemic metastatic disease [5]. However, it is futile in treatment of BCBM because of poor drug penetration through Mevalonic acid the blood-brain barrier (BBB)[6]. Furthermore, trastuzumab treatment is strongly associated with increased incidence of brain metastases, an observation that has been documented in a number of reports [4, 6-8]. This is attributable to the systemic control of the disease through trastuzumab, which prolongs survival, allowing the outgrowth of cancer cells at a sanctuary site i.e. brain. A recent clinical trial of Herceptin? (HERA) documents that, of all the patients who died, 53% had CNS involvement [9]. The BBB is a major obstacle in the treatment of brain malignancies. To overcome this limitation, our group has developed neural stem cells (HB1F3), which are tumor tropic and can cross the BBB when injected systemically [10, 11]. These NSCs have been employed as carriers of therapeutic molecules and oncolytic viruses [12-15] to achieve significant survival benefits for brain malignancies. Moreover, NSCs (HB1.F3.CD) are FDA approved for phase-I clinical studies in patients with glioblastoma (completed safety study NCT01172964; phase I dose escalation study in progress NCT02015819). Our group has previously demonstrated the ability of modified NSCs to deliver functional, anti-HER2-antibody to non-CNS, orthotopic breast cancer cells [16]. However the potential therapeutic implication of NSCs secreting anti-HER2Ab in a brain metastatic breast cancer model has not been evaluated. In this report, NSCs secreting stable and high amount of anti-HER2 antibody (HER2Ab-NSCs) were generated. Using these genetically modified NSCs, we performed intracranial Mevalonic acid xenograft studies using HER2 overexpressing, human brain metastatic cells. Our results demonstrate significant improvement in the survival of mice injected with HER2Ab-NSCs. Hence our work provides compelling evidence for the use of HER2Ab-NSCs to treat HER2 overexpressing BCBM. Materials and Methods Cell culture The HB1.F3 human NSC line was derived from primary cultures of fetal telencephalon by immortalization with an amphotropic, replication incompetent retrovirus encoding the v-gene as previously described [17, 18]. NSC, BT474 (ATCC, Manassas, VA), BT474M1BrM3 [19] (will be referred to as BT474Br), Lenti-X 293T cells (Clonetech, Mountain View, CA), MCF7 cells (Dr. Suzanne Conzen, University of Chicago) were maintained in DMEM Mevalonic acid supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) in a humidified (5%) CO2 incubator. MDA-MB-361 (Dr. Seungpyo Hong, University of Illinois.
