For FLO-1 LM cell line xenografts, 5 x 10⁶ cells suspended in 100 ul of 1:1 PBS and Matrigel (BD Biosciences) were subcutaneously injected into the right flank of ~6 week-old female nonobese diabeticsevere combined immunodeficient interleukin-2RKO (NSG) mice. PDXs were established and implanted into a dorsal intramuscular pocket of NSG mice as previously described. Mice were randomized to SG deplete or control chow ad libitum (AIN93G rodent diet, Specialty Feeds, Australia) and dosed with eprenetapopt (100 mg/kg) or 0.9% saline, intraperitoneally injected daily, once tumors reached 100 mm³. Tumor volume was assessed blinded to treatment group with caliper measurements every 3 to 4 days and calculated using the formula (length x weight2)/2. Metastatic spread was determined by bioluminescence imaging as previously described involving weekly monitoring using the Xenogen IVIS 100 Imaging System (Caliper Life Science). At experimental end point (tumor volume > 1400 mm³), the whole mouse and its organs were imaged to determine the extent and distribution of metastases. Tumors were weighed and tumor growth inhibition was calculated with the formula [1 - (Tf- Ti)/mean(Cf - Ci)] x 100, where Tf, Ti, Cf, and Ci represent final (f) and initial (i) tumor volume of drug treated (T) and control (C) animals, respectively.

Tumours were initiated in 4-8-week-old female NOD. CB17 Scid/J mice. Orthotopically in the mouse mammary gland, by implantation of 500,000 cells in 25 ul 33% Matrigel into the fourth mouse mammary fat pad; subcutaneously, by injection of 500,000 cells in 100 ul 33% Matrigel into the left or right flank of the mouse; via tail vein by injection of 500,000 cells in 150 ul RPMI into the mouse tail vein; and via intratracheal instillation by instilling 200,000 cells in 50 ul 2 mM EDTA as described. Cancer cells were transduced with viral shRNAs, selected for 3 days with puromycin, and allowed to recover for one day before introduction into mice. For experiments comparing subcutaneous and lung tumour formation, shRNA transduced cells were prepared at the same time and injected on the same day. Animals were imaged by IVIS (Perkin Elmer) 15 min following injection subcutaneously into the neck scruff with XenoLight d-Luciferin (165 mg per kg body weight, Perkin Elmer). Average luminescence was quantified per mouse from equal sized bins covering the mouse thorax. For experiments in which tumour growth was measured upon drug treatment, MDA-MB-231 cells, implanted as described above, were allowed to form palpable tumours (~4 mm diameter) and mice were sorted into treatment groups as described below. PEG-Cyst(e)inase was delivered via intraperitoneal injection at 50 mg per kg body weight every 3 days, SSA was delivered by daily intraperitoneal injection at 250 mg per kg body weight, and BSO was delivered in the drinking water at 20 mM with 5 mg ml-1 sucralose.

Tumours were initiated in 4-8-week-old female NOD. CB17 Scid/J mice. Orthotopically in the mouse mammary gland, by implantation of 500,000 cells in 25 ul 33% Matrigel into the fourth mouse mammary fat pad; subcutaneously, by injection of 500,000 cells in 100 ul 33% Matrigel into the left or right flank of the mouse; via tail vein by injection of 500,000 cells in 150 ul RPMI into the mouse tail vein; and via intratracheal instillation by instilling 200,000 cells in 50 ul 2 mM EDTA as described. Cancer cells were transduced with viral shRNAs, selected for 3 days with puromycin, and allowed to recover for one day before introduction into mice. For experiments comparing subcutaneous and lung tumour formation, shRNA transduced cells were prepared at the same time and injected on the same day. Animals were imaged by IVIS (Perkin Elmer) 15 min following injection subcutaneously into the neck scruff with XenoLight d-Luciferin (165 mg per kg body weight, Perkin Elmer). Average luminescence was quantified per mouse from equal sized bins covering the mouse thorax. For experiments in which tumour growth was measured upon drug treatment, MDA-MB-231 cells, implanted as described above, were allowed to form palpable tumours (~4 mm diameter) and mice were sorted into treatment groups as described below. PEG-Cyst(e)inase was delivered via intraperitoneal injection at 50 mg per kg body weight every 3 days, SSA was delivered by daily intraperitoneal injection at 250 mg per kg body weight, and BSO was delivered in the drinking water at 20 mM with 5 mg ml-1 sucralose.