Figure 4: HBB expression enhances the ability of breast CTCs to form distant metastases.

(a) Orthotopic inoculation of 200,000 BRx50 CTCs into the mammary fat pad showing transiently delayed primary tumour formation upon HBB-depletion. Top: representative images of mice at day 1 and day 27 following tumour inoculation (luminescence by IVIS imaging). For each mouse, the mammary fat pad seen in the upper left of the image received control CTCs, while the other three fat pads received CTCs expressing three different shRNA constructs targeting HBB. Bottom: Primary tumour growth curves from mice orthotopically implanted with BRx50 CTCs expressing either control or HBB shRNAs. The transient but significant reduction in tumour size at day 27 is resolved in subsequent time points. (b) Intravenous injection of BRx50 cultured CTCs into the mouse tail vein, showing impaired metastatic potential following depletion of endogenous HBB. Top: representative IVIS-luminescence images of isolated whole lungs from mice, at day 33 following intravenous injection of CTCs expressing either control or HBB-targeting shRNAs. Bottom: longitudinal monitoring of mice for emergence of lung metastases, following intravascular injection of either control BRx50 cells or BRx50 cells expressing HBB shRNAs. (c) Bar graph showing normalized lung photon counts from mice, representing metastatic tumour burden, 5 days after they were intravenously injected with either control BRx50 cells or HBB-depleted BRx50 cells. Where indicated, mice were pretreated with the antioxidant NAC (200 mg kg⁻¹ day⁻¹) for 3 days before tumour cell inoculation, and then treated daily at the same dose. Total lung photon counts at day 5 were normalized to the photon counts at day 1. Data (a–c) are represented as mean±s.e.m.; n=4 in (a), n=3 in b,c; *denotes a statistical significance at P<0.05 (t-Test).