Evaluation of Hedgehog pathway activity

Initially, we attempted to evaluate the activity of the Hh pathway using both gene expression and protein expression. However, all commercially available Hh antibodies showed non-specific binding in both immunohistochemical staining and western blots of our PDXs to the extent that assessing protein expression was abandoned. This non-specific binding of antibodies to GLI1/2, PTCH1, SHH and SMO across all samples was consistent with those reported by other groups[6].

For gene expression, primers were designed for mouse and human SHH, IHH, PTCH1, PTCH2, GLI1, SMO, and the housekeeping genes ACTB, HSP90AB1 and YWHAZ (S1 Table). Separate human and mouse-specific primers were desirable in order to differentiate transcriptional changes in the human tumour epithelium versus murine stroma. Housekeeping genes were chosen for expression stability after irradiation. The species specificity of each primer was tested by qRT-PCR on normal mouse liver and the human EAC cell line, OE33. Primers that cross-amplified in both species, or that produced doublet dissociation curves were redesigned. Two to three xenograft mice from each treatment group were sacrificed at multiple time points, including 24 hours after irradiation, during the plateau phase of the curve following treatment, and during the re-growth phase. Volume- or time-matched control tumours were sacrificed for comparison, depending on the experiment. Biopsied normal esophagus served as a control to determine baseline Hh expression in untreated EAC tumours. PDX tumours were immediately excised and frozen at -80°C in Optimized Cutting Temperature compound (Sakura Finetek). RNA was isolated from frozen whole tissue sections, and additional sections stained with hematoxylin and eosin were assessed by a pathologist. RNA was extracted using the RNeasy Mini Kit (Qiagen) with on-column DNA digestion, quantified using a Nanodrop spectrophotometer, and evaluated for quality using the Agilent 2100 Bioanalyzer. qRT-PCR experiments used RNA samples with RNA integrity numbers above 8.

RNA samples were reverse-transcribed (iScript™ cDNA Synthesis Kit (Bio Rad); RT² SYBR® Green ROX™ qPCR Mastermix (Qiagen); ABI 7900HT (Life Technologies)) in triplicate wells, and quantified by qRT-PCR (Sequence Detection System v2.3). For each sample, a ΔC_t was calculated by normalizing to the geometric mean of the three housekeeping genes. ΔΔC_t was the difference between the ΔC_t of each irradiated sample and its matched control; fold differences were calculated as 2^-ΔΔCt.