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  • br Immunohistochemistry br Tissue sections from Sham or

    2020-08-28


    2.13. Immunohistochemistry
    Tissue sections from Sham or BCF treated mice were baked at 60 °C for 1 h, deparaffinized in xylene, and rehydrated and fixed in 10% neu-tral buffered formalin. The sections were stained with MK-2206 spe-cific to human HMGA2 (Abcam) and mouse CD31 (Cell signalling) and visualised using the EnVision Plus system (Dako).
    Ninety six-well plates were coated with 50-mL growth factor-reduced Matrigel (BD) and incubated at 37 °C for 30 min to solidify. HBMECs (2 × 10 [4] cells) were suspended in EGM-2 growth medium or serum-free conditioned medium from cancer cells, and they were 
    seeded on top oggrowth factor-reduced Matrigel. For exosome treat-ment, exosomes were added to the EGM-2 media (100 μl total volume). After 6 h, photos were taken under a microscope, and the number of tubes structures per field was counted.
    2.16. Exosome isolation
    Exosomes were isolated by differential centrifugation as described previously. Cells were grown on extracellular vesicle-depleted 10% FBS DMEM media for 48 h and conditioned medium (CM) was collected for exosome isolation. CM was centrifuged at 300g for 30 min to remove cells. The supernatant was then centrifuged at 2000g for 20 min to re-move apoptotic bodies. The supernatant from this step was further cen-trifuged at 16,500g for 20 min to remove microvesicles, and passed through 0.2 μM filter (Sarstedt, Numbrecht, Germany) to remove parti-cles that are larger than 200 nm. Final centrifugation was done at 120,000g for 70 min to pellet down the exosomes. The isolated exosomes were analysed by electron microscopy with negative staining. Blood eoxsomes were isolated as previously described [20]. Briefly, 1 ml of serum was loaded to the qEV size exclusion colums (Izon), and exosomes were concentrated by centrifugation using the Amicon® Ultra-4 10 kDa column.
    2.17. Knockdown and overexpression
    Knockdown of Cav T-type channels was accomplished using Human lentiviral shRNA plasmids in p-GFP-c-shLenti vector from Origene, CACNA1g (Locus ID 8913; Catalog # TL305680), CACNA1H (Locus ID 8912; Catalog # TL314243) and CACNA1I (Locus ID 8911; Catalog # TL314242). Lentivirus was derived from four different shRNAs for each individual gene, infected to cancer cells and knockdown was verified by qRT-PCR. The shRNA infected cells that showed highest knockdown were selected for further experiments. HMGA2 gene knockdown was performed using two human lentiviral shRNA plasmids in pLKO.1 vector from Sigma (Catalog number: TRCN0000021964 and TRCN0000021965). HMGA2 cDNA plasmid (Myc-DDK-HMGA2) was obtained from Origene (Catalog #: RC214629), and HMGA2 gene was subcloned to pSIN vector. pSIN-HMGA2 plasmid was then used to make the virus, which was infected to cancer cells and cells were further selected with puromycin. pCDNA3-β-catenin plasmid and control vec-tor were obtained from Addgene and transfected to MDA-MB-231 and SKBr3 cells.
    2.18. Statistical analysis
    Results were reported as mean ± SEM. For in vitro experiments, the Student's t-test or one-way analysis of variance was applied. For in vivo experiments, group comparisons were performed using the nonpara-metric Mann-Whitney test or unpaired Student's t-test. Kaplan-Meier curve comparison was performed with the log-lank test.
    2.19. Ethical statement
    The Wake Forest Institutional Animal Care and Use Committee (IACUC) approved all animal experiments performed in this study. The patient was offered compassionate treatment with the TheraBionic P1 device, written informed consent was obtained, authorization to publish the patient's clinical data was granted and compassionate use was approved by the Ethics Committee of the Cabinet Médical de l'Avenue de la Gare 6, 1003 Lausanne, Switzerland. Additionally, a com-passionate use request for use a non-conforming medical device was submitted to and approved by swissmedic: https://www.swissmedic. ch/swissmedic/en/home/medical-devices/market-access/exemptions-for-non-conforming-medical-devices.html Exemptions for non-conforming medical devices.
    3. Results
    3.1. Breast cancer-specific AM RF EMF (BCF) suppress growth of brain metastasis
    The remarkable efficacy of BCF in a previous feasibility study [10] prompted us to examine MK-2206 the effect of BCF in vivo. We implanted Her2 positive breast cancer brain metastatic variant line SKBrM3 cells via in-tracardiac route in NOD-SCID mice followed by the treatment of animals with BCF for 3 h daily. As shown in Fig. 1A-C, BCF significantly reduced tumour metastasis in the brain and improved brain metastasis-free sur-vival. However, treatment of mice with randomly chosen AM RF EMF (RCF) did not affect the tumour growth and metastasis-free survival of