br Collectively we have demonstrated that BCF effectively
Collectively, we have demonstrated that BCF effectively blocks the growth of breast cancer cells as well as breast CSCs through targeted ac-tivation of breast cancer cell CACNA1H resulting in increased intracellu-lar calcium followed by activation of the CAMKII-p38 MAPK pathway and downregulation of HMGA2 expression. Therefore, BCF is a promis-ing therapeutic approach to treat breast cancer metastatic disease, espe-cially in the brain.
The results of our preclinical studies reported here and the example of a long-lasting response in a patient with Adriamycin metastasis together with the class IIa low risk designation of the device lay a strong founda-tion to test this promising novel approach in clinical trials that include patients with brain metastasis.
SS and KW designed the study and wrote the manuscript. SS con-ducted experiments and acquired, analysed and interpreted the data. SYW performed PDX experiment. HJ treated cells and animals with BCF. FX and SYW derived the radiation resistant cells and performed in-tracardiac injections. SYW, DZ, YL, LMB assisted with intracranial injec-tions and bioluminescence imaging. K Wu and AT isolated exosomes and performed tube formation assay. HWL assisted with Immunohisto-chemistry. PS, DJB, MDC and A Th provided various advices on experimental procedures. CTW and VK assisted with MRI image inter-pretation. RD assisted with study design, data analysis, data interpreta-tion and review of manuscript. CB and BP provided the EMF exposure device, reviewed and edited manuscript and interpreted the data. KW supervised the study.
Competing financial interests
Boris Pasche and Alexandre Barbault hold stocks in TheraBionic Inc. and TheraBionic GmbH. Carl Blackman has provided a loan to
Fig. 6. Combination treatment of radiation and BCF: (A) 231-BrM-RR and SKBrM3-RR cells were seeded into six-well plate and exposed to 2-, 5- or 10-Gy of radiation and number of colonies formed at Day 7 was counted. (B\\C) 231-BrM-RR and SKBrM3-RR cells were treated with Sham or BCF for 7 days, followed by quantification of cell proliferation by thymidine incorporation assay (n = 6/group) (B) and CSC population by FACS (C). (D) HMGA2 expression was examined by western blot for 231-BrM-RR and SKBrM3 –RR cells after 7 days of exposure to BCF. (E) SKBr3 and SKBrM3 cells were treated with Sham or BCF for 7 days and 500 cells were seeded into each well of six-well plate followed by exposure to 5 gy of radiation. Number of colonies was counted at Day 7 (n = 6/group). (F) 231-BrM-RR cells were exposed to BCF or Sham daily for 7 days in the presence of ethosuximide or vehicle, and cell proliferation was quantified by thymidine incorporation at day 7 (n = 6/group). (G) SKBrM3 cells were injected in the brain of R2G2 mice. When bioluminescence signal reached 1*e , mice were irradiated with 40Gy (in 4 doses) followed by treating mice with Sham or BCF for 30 days. No irradiation mice were used as controls (n = 8/group) Representative images are shown in right panel. (H) Scheme of mechanistic action of BCF. *, P-value b .05, **, P-value b .01 and ***, P-value b .0001.
TheraBionic Inc. No other authors have any particular competing interests.
We thank Dr. Joan Massagué for providing the 231-BrM cell line. We also thank Dr. Michael T. Lewis for providing the PDX2147. This work was supported by NIH grant R01CA173499, R01CA185650 and R01CA205067 (to KW). The Tumour Tissue and Pathology Shared Re-sources, and Biostatistics/Bioinformatics Shared Resource are supported by the Comprehensive Cancer Center of Wake Forest University and, National Institutes of Health Grant (P30CA012197). The funding agency had no role in study design, data collection, data analysis, interpretation and writing of the report. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Na-tional Cancer Institute.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
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