Figure 3.

ASCT2 knockdown in vivo represses tumour growth and improves survival. (a) HCC1806 cells expressing an additional mCherry-luciferase reporter construct (pHIV1SDm-CMV-mCherry-P2A-luc⁴⁷) and doxycycline-inducible shRNAs (see Figure 2h legend), shControl or shASCT2 (pFH1tUTG³⁴) were sorted for mCherry^hi/GFP⁺ cells using fluorescence-activated cell sorting and then 4 × 10⁶ cells suspended in a solution of 50% Matrigel in RPMI (100 μl) were implanted orthotopically into the left and right abdominal mammary glands of 8–12-week-old female athymic nu/nu mice (Animal Resource Centre, Perth, Western Australia, Australia), five mice per group from two separate experiments (n=10). Sample size was estimated on the basis of previous publications²¹ with no randomization or blinding. Doxycycline (200 μg/ml) was administered ad libitum in drinking water from Day 0 and tumour growth was measured twice weekly until individual shControl tumours reached ~1000 mm³, when all the mice were humanely euthanized in accordance with ethics approval protocol 2013/030 A from Sydney Local Health District Animal Welfare Committee. (b) Bioluminescence was detected twice weekly using a Xenogen IVIS Lumina II following intraperitoneal injection of 150 mg/kg d-luciferin. At end point, all the mice were euthanized and tumours collected for the measurement of size (c) and weight (d; error bars represent mean±s.d.). (e) Tumour RNA was extracted from shCont and shASCT2 (n=4 each) tumours using TRI Reagent, then reverse transcribed using SuperScript III (Life Technologies, Carlsbad, CA, USA) and random hexamers. Relative gene expression (GAPDH/ACTB) was measured by RT-qPCR using a custom Taqman Low-Density Array (Taqman, Life Technologies) run on a QuantStudio 12 K Flex Real-Time PCR System. Heat map of relative expression (compared with the first shControl lane) for each gene was generated in Gene-E (Broad Institute). Average fold-change between the shControl and shASCT2 groups are indicated to the right of the heat-map, asterisks indicate significant difference (P<0.05, n=4; Mann–Whitney U-test). (f), HCC1806-mCherry^hi/GFP⁺ cells (2 × 10⁶) were injected orthotopically as in a and allowed to establish (tumour size ~100 mm³) before the administration of doxycycline (200 μg/ml). Tumour size was measured twice weekly using digital calipers to calculate mean tumour volume per mouse (g) and individual mice were euthanized when they reached ethical end point (individual tumour size >1000 mm³; h). Data in b represents mean±s.e.m., n=9 (shCont; one mouse was euthanized owing to ulceration at tumour site and is not included in the analyses) or n=10 (shASCT2) mice (with two tumours per mouse), five mice per group in two independent experiments. Data in g represents mean, n=10 (shCont) or n=10 (shASCT2) mice (with two tumours per mouse), five mice per group in two independent experiments. Asterisks denote P-values as follows: *P⩽0.05; **P⩽0.01; ***P⩽0.001; ****P⩽0.0001, NS, not significant; two-way analysis of variance (b), Mann–Whitney U-test (d and e), or log-rank Mantel–Cox test (h). Data in c are representative of two independent experiments.