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  • br mice Supplementary Fig A C In

    2020-08-28

    
    mice (Supplementary Fig. 1A-C). In addition, BCF decreased the tumour growth in Paclitaxel when SKBrM3 cells were orthotopically implanted in the brain (Fig. 1D). We also implanted triple negative brain metastatic variant 231-BrM cells both systemically (Fig. 1E-F) and orthotopically (Fig. 1G), and found significant suppression of tumour growth and me-tastasis in the brain by BCF, similar to that observed for SKBrM3 cells. These results strongly indicate that BCF is able to suppress brain metas-tasis of breast cancer in vivo.
    To accurately represent the cancer heterogeneity, we examined the effect of BCF using two patient derived xenograft (PDX) models. PDX2147 was established from the primary tumour of a patient with brain metastasis [21], and PDX1435 was established at our institution by implanting tumour from a brain metastatic lesion arising from
    A SKBrM3 B C SKBrM3
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    Fig. 1. Breast-specific BCF suppresses brain metastatic breast cancer cells in vivo. (A) SKBrM3 cells were injected through the intracardiac route followed by treatment of mice with Sham or BCF. Brain photon flux was examined by bioluminescence (n = 11/group). (B) Ex vivo bioluminescence was quantified for experiment performed in fig. A (C) Brain-metastasis free survival was plotted for Sham and BCF treated mice in fig. A. (D) SKBrM3 cells were implanted in the brain by intracranial injection and mice were exposed to Sham or BCF daily till day 36 (n = 10/ group). Tumour growth was monitored by bioluminescence (n = 8/group). Inset panel shows representative bioluminescence images of Sham and BCF treated groups. (E) 231-BrM cells were intracardially implanted in mice followed by treating mice with BCF for 27 days (n = 9/group). Tumour growth in brain was quantified by bioluminescence imaging. (F) Ex vivo bioluminescence was quantified for experiment performed in fig. E (G) Brain metastasis-free survival was plotted for Sham and BCF treated mice in fig. E. (H) 231-BrM cells were intracranially implanted in brain and mice were treated with Sham or BCF everyday till day 42. Tumour growth in the brain was quantified ex vivo by using bioluminescence imaging at day 42. Right panel shows representative ex vivo bioluminescence image for BCF and Sham group. *, P-valueb.05 and ** P-valueb.01.
    triple negative breast cancer. PDXs were dissociated into single cell suspension and implanted into the brain of NOD/SCID mice. As shown in Fig. 2A-C and supplementary fig. 1D-E), BCF significantly reduced PDX growth in brain. These promising results led us to offer compassionate treatment to a patient with metastatic disease (Supplementary Fig. 1F). Importantly, BCF daily treatment resulted in the remarkable reduction of the brain metastasis as shown in the MRI (Fig. 2D-G). The effect of treatment persisted for 
    11 months, while the average survival rate of patients with brain metastasis is b4 months [22].
    3.2. Tumour suppressive effect of BCF is mediated through T-type voltage gated calcium channel and p38 MAPK signalling
    To examine the direct inhibitory effect of BCF in vitro, we treated
    231-BrM and SKBrM3 cells with BCF daily for 3 h, the same exposure
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    Fig. 2. Effect of BCF in a brain-met PDX model and patient. (A) PDX2147 was dissociated into single cell suspension and 250,000 live cells were intracranially injected into the brain. Mice were treated with Sham (n = 6) or BCF (n = 6) for 30 days and MRI was performed to visualise and quantify the tumour volume. (B) PDX1435 Paclitaxel was implanted in brain treated with Sham (n = 4) or BCF (n = 5) and quantified for tumour volume at day 30 as in A. (C) Brain tumours from PDX2147 were stained for Ki67 expression by immunohistochemistry. (D-G). Treatment of brain metastasis (metastatic calvarial lesion) arising from triple negative breast cancer. Serial T1-weighted post-contrast magnetic resonance imaging (MRI) in sagittal plane, including magnified views (E and G) dated 8/30/2013 (D and E) and 8/8/2014 (F and G). Images D and E demonstrate an aggressive ovoid 2.7 cm (AP) x 3.5 cm (ML) x 1.6 cm (CC) enhancing mass centered in the parasagittal high left parietal calverium that extends into the scalp and intracranially, abutting and displacing the adjacent superior sagittal sinus. Images F and G demonstrate a marked interval decrease in size of the enhancing mass, now primarily localized in the diploic space and measuring 1.7 cm (AP) x 3.2 cm (ML) x 0.6 cm (CC) in size, with resolution of intracranial mass effect. AP = anteroposterior; ML = mediolateral; CC = craniocaudal; *, P-valueb.05 and **, P-valueb.01.