Brain infiltration of breast cancer stem cells is facilitated by paracrine signaling by inhibitor of differentiation 3 to nuclear respiratory factor 1

Abstract

Treatment options for brain metastatic breast cancer are limited because the molecular mechanism for how breast cancer cells infiltrate the brain is not fully understood. For breast tumors to metastasize to the brain first, cells need to detach from the primary tumor, enter in the blood circulation, survive within the microvascular niche, and then cross the blood-brain barrier (BBB) to colonize into the brain. It is critical to understand how breast cancer cells transmigrate through the BBB to prevent brain metastasis. Nuclear respiratory factor 1 (NRF1) transcription factor has been reported to be highly active in several human cancers and its aberrant expression facilitates in the acquisition of breast cancer stem cells (BCSCs). Inhibitor of differentiation protein 3 (ID3), a transcription regulating protein, induces pluripotent endothelial stem cells (ESCs). Herein, we investigated if NRF1-induced BCSCs could cross a BBB model and guiding of BCSCs by ID3-induced ESCs across the BBB. BCSCs and ESCs were subjected to functional gain/loss experiments to determine if NRF1/ID3 contributed to lineage-specific BCSCs organ entry. First, we tested whether NRF1 promoted migration of breast cancer using a BBB model consisting of BCSCs or MDA-MB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BCSCs and NRF1-induced MDA-MB231 cells to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1-induced BCSCs to ESCs^ID3 was detected. NRF1-induced BCSCs crossed through the BBB model and this was promoted by ESCs^ID3. We also showed that environmental relevant exposure to PCBs (PCB153 and PCB77) produced differential effects. Treatment with PCB153 showed increased growth of NRF1-induced BCSCs tumor spheroids and increased in vivo migration of ESCs^ID3. Exosomal ID3 released from endothelial cells also supported the growth of NRF1-induced BCSCs and provide the basis for paracrine effects by ESCs^ID3 associated with breast tumors. Xenograft experiments showed that ID3 overexpressing brain ESCs not only supported the growth of BCSC tumor spheroids but guided them to the neural crest in zebrafish. These findings show for the first time a novel role for ID3 and NRF1 by which ESCs^ID3 help guide BCSCs^NRF1 to distant metastatic sites where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents to prevent the spread of breast cancer to the brain.

Keywords: Breast cancer metastasis; Cancer stem cells; ID3; NRF1.

Fig. 1

Exposure to PCB congeners increased spheroid formation of BCSCs co-cultured with ESCs and astrocyte cells. Tumor spheroid assay 16 days after exposure to PCB congeners. A Wildtype brain microvascular endothelial cells shown by endothelial CD34 marker (in blue). B NRF1-induced BCSCs expressing GFP (in green). C NRF1-induced BCSCs co-cultured with endothelial cells. D–E Astrocyte cells (AC) co-cultured with BCSCs and wildtype ECs. Astrocyte marker GFAP is shown in red. F Astrocyte cells co-cultured with BCSCs and ID3-induced ESCs (in green). Representative confocal microscopy images from three separate experiments. 200×. PCB77 [60 µM]; PCB153 [60 µM]

Fig. 2

ID3-induced endothelial stem cells support the growth of BCSC spheroids. Spheroid assay of BCSCs^NRF1 co-cultured with wildtype ECs or ESCs^ID3. Cells were treated with vehicle control, PCB77 or PCB153 and grown for 8–16 days. A Wild-type brain microvascular endothelial cells. B NRF1-induced BCSCs. C NRF1-induced BCSCs cultured with wildtype ECs. D NRF1-induced BCSCs cultured with ECs and astrocyte cells (ACs). E NRF1-induced BCSCs cultured with ESCs and astrocyte cells. Representative light microscopy images from three separate experiments. F Graphs of spheroid diameter for groups A–E PCB congener treatment exhibited a significant increase in spheroid diameter at days 8 and 16 when compared to vehicle control. Error bars represent the mean sizes of fifteen spheroids ± SD. *p < 0.05 **p < 0.01 vs. Control. Data analyzed by ANOVA; Tukey HSD test for multiple comparisons

Fig. 3

NRF1 increased adhesion and migration of breast cancer cells. A BCSCs^NRF1 and NRF1-induced MDA-MB231 were seeded on top of a brain barrier model in serum-free media for 3 days. Adhesion to the endothelial layer is shown by CD34 marker (in blue) and CD133 marker (in red) for BCSCs and NRF1-induced mesenchymal MDA-MB231 cells. Pink, fluorescent cells show where both CD133 and CD34 markers overlap. B Invasion of BCSCs^NRF1 and NRF1-induced mesenchymal MDA-MB231 through the brain barrier. C 3D brain barrier model composed of NRF1-induced breast cancer cells (CD133 in red) surrounded by a Matrigel of endothelial cells (CD34 in blue) at day 0 (D0). BCSCs^NRF1 top panel and NRF1-induced MDAMB231 cells lower panel. Migration of CD133 positive cells away from the tumor spheroid shown in magnified images at day 9 (D9). Representative confocal microscopy images from three separate experiments. Representative images by Nikon confocal microscopy

Fig. 4

ID3-induced endothelial stem cells increase BCSCs migration. A BCSCs tumor spheroids seeded on a monolayer of wildtype ECs or ESCs^ID3. BCSCs were induced by either NRF1 or NRF1 + E2. Wildtype ECs stained with CD34 in blue. ESCs^ID3 stained with anti-ID3 in red. B BCSCs^NRF1 spheroids were seeded on an endothelial monolayer for 24 h in serum-free media in the upper chamber of a transwell insert with FBS as a chemoattractant in lower chamber. NRF1 overexpressing cells shown by GFP in green were photographed in the lower chamber. Representative confocal microscopy images from three separate experiments

Fig. 5

Endothelial exosome treatment increases ID3 protein level in BCSCs. BCSCs were treated with endothelial exosomes from ESCs^ID3 for 24 h. BCSCs showed increased ID3 protein levels from exosome treatment. Representative Western blot of three separate experiments. ID3 levels shown in top panel with loading control on bottom

Fig. 6

Flow cytometry analysis of BCSCs treated with endothelial exosome. FACS analysis using ID3 biotin-FITC (pink) and NRF1 conjugated PE (blue). Co-expression of ID3 and NRF1 shown in green. Cells not expressing either marker shown in red. A Wildtype endothelial cells. B NRF1-induced BCSCs. C NRF1 + E2-induced BCSCs. D ESCs ^ID3 (positive control)

Fig. 7

Endothelial exosome treatment BCSCs tumor spheroids. Spheroid assay of NRF1 induced BCSCs co-cultured with wildtype ECs. Cells were treated with endothelial exosome from ESCs in serum free media for 7–10 days. BCSCs are shown by CD133 marker in red and WT-EC are shown by CD34 marker in blue

Fig. 8

PCB153 increased in vivo migration of lung ESCs. Top panel show lung ESCs^ID3 xenotransplanted in zebrafish embryo (2dpf). Bottom panel showing PCB153 [60 µM] treated lung ESCs xenotransplanted into the zebrafish. Total number of injected cells is shown by red fluorescent dye CellTracker™ CM-DiI and ID3 overexpressing cells indicated by GFP (in green). Representative images from three individual experiments

Fig. 9

BCSCs form xenograft tumor in neural crest of zebrafish embryo. Total number of injected cells is shown by red fluorescent dye CellTracker™ CM-DiI and NRF1 overexpressing BCSCs indicated by GFP (in green). Top panel represents ID3-induced ESCs (GFP expressing) xenotransplanted in zebrafish embryo (2dpf). Row two represents NRF1 induced BCSCs. Row three represents NRF1 + E2-induced BCSCs. Bottom panel represents NRF1 + E2-induced BCSCs and ESCs. Representative images of three individual experiment