br In this study we
In this study, we demonstrate that BCF treatment significantly sup-presses the onset of metastatic growth in the MK-2206 by activating cellular stress pathways. Notably, BCF inhibition was found to be mediated by the activation of CACNA1H, which increased intracellular influx of cal-cium, leading to p38 MAPK activation. We also found that BCF reduces the self-renewal of cancer stem cells (CSCs) through modulation of High Mobility Group AT-2 (HMGA2) gene expression. Furthermore, BCF suppressed angiogenesis in the tumour microenvironment by de-creasing exosomal secretion of miR-1246. These results strongly dem-onstrate that BCF is a promising novel modality of treatment for breast cancer brain metastasis.
2. Materials and methods
2.1. Cell culture and reagents
2.2. Animal experiments
All animal experiments were conducted in compliance with the pro-tocol approved by the Laboratory Animal Care and Use Committee of Wake Forest University. Intracranial injections were performed as previously described. Briefly, 5–6 weeks SCID mice (Harlan) were anesthesised by intraperitoneal injection of ketamine/xylazine (90–120/7–10 mg/kg). The hair was removed using clippers (ChroMini chordless clippers, Harvard apparatus) followed by shaving the hair (2 mm breadth and 8 mm length) with the razor. The area of incision was cleaned using sterile cotton swab. Then the mouse was positioned into a Kopf stereotactic frame. With the mouse secured in the stereotac-tic frame, we swabbed the forehead (between eyes back to ears) with betadine via sterilised cotton swab, and then used a scalpel to make a 5–6 mm caudal-rostral incision slightly to the right of midline while stretching skin with thumb and forefinger and avoiding the prefrontal sinus. We then used the wood end of cotton swab to scrape away fascial tissues covering the skull, and dry the skull well with the cotton end to
help locate midline and coronal sutures. A small burr hole was made by using sterilised Dremmel cordless drill (#76 drill bit) at the desired co-ordinates. A sterile 25-gauge needle attached to the syringe was intro-duced through the calvarium and into the brain at a depth of 4 mm. The cells were injected (volume of 5uL, 20,000 for SKBrM3 and 25,000 for 231-BrM cells). After one minute, the syringe was pulled up and a small amount of bone wax was applied to occlude the hole. The mouse was then removed from the frame and wound clips were used to close the skin. The tumour progression in the brain was monitored by bioluminescence imaging. Mice received Sham or BCF treatment one day after tumour implantation.
For intracranial injection of PDX2147 and PDX1435, PDXs were dis-sociated to single cell suspension using human tumour dissociation kit (Miltenyi Biotech). Dead cells were removed by using dead cell removal kit (Miltenyi Biotech) and 250,000 live cells were intracranially im-planted to NOD/SCID mice. Tumour growth in brain was examined by MRI at day 30. Mice received Sham or BCF treatment one day after tu-mour implantation.
For intracardiac injections, 5–6 weeks SCID mice (Harlan) were injected into the left cardiac ventricle of the mice (105 SKBrM3 cells; 2 × 10  231-BrM cells). The cell growth and development of metastasis were monitored by bioluminescence imaging (BLI). Mice received Sham or BCF treatment one day after tumour implantation. For combination of radiation and BCF, R2G2 mice were intracranially
injected with 20,000 SKBrM3 cells labelled with luciferase and tumour growth was examined by BLI. When BLI reached 1 × 106, tumours were irradiated using precision X-Ray XRAD 320 Orthovoltage X-ray Unit with custom-made collimators (b5 mm diameter) and irradiation jigs housed in a shielded irradiator room. 40 gy (5 gy × 2 fractions/day for 4 days) radiation was delivered through positioning devices that en-sured target-beam alignment with rodents positioned in the lateral or sternal recumbency position. Mice received Sham or BCF treatment after irradiation schedule was completed.
Cell lines were exposed to 27.12 MHz radiofrequency electromag-netic fields using exposure systems designed to replicate clinical expo-sure levels. Experiments were conducted at an SAR of 30 and 400 mW/kg. Cells were exposed for three hours daily, seven days in a row. Cells were exposed to tumour-specific modulation frequencies that were previously identified by changes in pulse pressure in patients with a diagnosis of breast cancer or modulation frequencies never iden-tified in patients with a diagnosis of cancer . Specifically, the ran-domly chosen frequencies have been selected at random in the range of 500 Hz to 22 kHz, i.e. within the same range as the hepatocellular carcinoma-specific and breast cancer-specific frequencies. The only se-lection criterion within this range was for frequencies to be at least 5 Hz higher or lower than any hepatocellular carcinoma or breast cancer-specific frequency identified in patients with the corresponding diagnoses [15–17]. We have previously reported that the primary method for identification of tumour-specific frequencies is an increase in the amplitude of the pulse for one or more beats during scanning of frequencies . Using the same method, we monitored variations in the amplitude of the radial pulse in thirty patients with a diagnosis of cancer, and did not observe any change in pulse amplitude during expo-sure to the randomly chosen frequencies.