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  • br B br C br ShamSham

    2020-08-28


    B
    C
    ShamSham ShamBCF1BCF2BCF3
    3000 Blood exosome
    Sham BCF Sham BCF
    Sham BCF
    D TCGA
    Overallsurvival
    Time (Months)
    E
    Primary Brain- Other-
    No- Brain-
    met met
    met met
    I
    HBMEC Control J
    Sham
    Numberoftubes/field
    G Intracellular Exosomes
    BCF
    Numberofmicrovessels/field
    Sham 
    H
    HBMEC
    oftubes/field 10 *
    Number
    Sham BCF Sham BCF
    CM Exosomes
    BCF
    rol
    Cont
    HM
    Fig. 5. BCF inhibits angiogenesis in U-46619 tumour microenvironment. (A) Exosomes were isolated from SKBrM3 cells that were treated with Sham or BCF, and small RNAs were extracted and subjected to microRNA array analysis. Heat map shows the miRNAs that were significantly up- or down-regulated. (B) The expression level of miR-1246 in the exosomes prepared from 231-BrM and SKBrM3 cells that were treated with Sham or BCF for 7 days was examined by Taqman PCR. (C) Blood exosomes were isolated from mice treated with Sham or BCF and the expression level of miR-1246 was examined by Taqman qRT-PCR (n = 5 for Sham and n = 8 for BCF). (D) Relationship between miR-1246 expression and overall survival of patients in TCGA dataset was examined using kmplot website. (E) miR-1246 expression was examined in GEO37407 consisting of patients with no metastasis (n = 17), brain-metastasis (n = 14) and other metastasis including lymph node-, liver-, skin- and ovarian-metastasis. (F) miR-1246 expression was examined in serum exosomes isolated from patients with no metastasis (n = 12) or brain-metastasis (n = 8) patients. (G) miR-1246 expression in exosomes isolated from endogenous or exosomal fractions of MDA-MB-231 and 231-BrM were examined by Taqman qRT-PCR. (H) HBMEC were treated with CM or exosomes isolated from 231-BrM cells that were treated with or without BCF for 48 h, and the tube formation assay was performed by seeding cells in growth factor-reduced matrigel. Number of tubes were counted 6 h after the treatment with CMs. (I) Exosomes isolated from 231 or 231-miR-1246 cells were used to treat HBMEC cells for 48 h followed by seeding cells in growth factor reduced matrigel, and the number of tube formation were counted 6 h post seeding. (J-K) SKBrM3 tumours treated with BCF stained with mouse-specific CD31 antibody and the number of microvessel per 40× field was counted (K). Representative images are shown in J. (L) miR-1246 level was examined by Taqman PCR in 231 cells overexpressed with HMGA2 or β-catenin or control vector. *, P-valueb.05, ** P-valueb.01 and ***,
    proteins, which act as an antenna [42,43]. We have shown that both Ca2 + and CACNA1H are key mediators of the tumour cell response, capable of distinguishing breast cancer-specific AM RF EMF. Therefore, we pro-pose that CACNA1H is selectively sensitive for tumour-specific AM RF EMF frequencies in epithelial malignancies. The notion that CACNA1H acts as the bioantenna for tumour specific AM RF EMF is strongly sup-ported by our own findings showing that the same CACNA1H mediates the antiproliferative effects and repress CSCs in hepatocellular carci-
    noma following exposure to hepatocellular carcinoma-specific AM RF EMF [13]. We have previously reported that variation in pulse amplitude constitutes the primary method for identification of tumour-specific modulation frequencies [15], which have subsequently been shown to target cancer cell proliferation in a tumour and tissue-specific 
    fashion [16]. CACNA1H is expressed in both endothelial cells and vas-cular smooth muscle cells of small arteries, which in turn directly af-fect pulse amplitude [44]. Hence, CACNA1H is the most probable link between the vascular system, crucial to the tumour-specific frequency identification process [15] and the anticancer effects observed in vitro, in vivo and in patients. Tumour-specific AM RF EMF appear to have a broad therapeutic window as tumour shrinkage was observed in humans [15,17], in human xenografts as presented in this report, and in vitro 16 at SARs ranging from 0.02 mW/kg to 400 mW/kg. In all conditions studied, treatment complies with the two standards for human exposure to RF EMF, the ICNIRP [45] and the IEEE [11]. Use of Pulsed Electromagnetic Fields (PEMF) has been associated with changes in Ca2+ metabolism but the mechanism is mediated by L-type, not T-type VGCCs [46,47].
    A
    C
    Sham
    Numberofcolonies
    3 BCF * %CSCpopulation
    -Thymidine(CPM*10
    SKBrM3
    SKBrM3-RR
    Gy
    D
    ** (normalizedtoSham)
    Numberofcolonies 3H-ThymidineCPM
    HMGA2
    SKBrM3 SKBrM3
    G
    Sham