Hedgehog inhibition mediates radiation sensitivity in mouse xenograft models of human esophageal adenocarcinoma

<div><p>Background</p><p>The Hedgehog (Hh) signaling pathway is active in esophageal adenocarcinoma (EAC). We used a patient-derived murine xenograft (PDX) model of EAC to evaluate tumour response to conventional treatment with radiation/chemoradiation with or without Hh inhibition. Our goal was to determine the potential radioresistance effects of Hh signaling and radiosensitization by Hh inhibitors.</p><p>Methods</p><p>PDX models were treated with radiation, chemotherapy or combined chemoradiation. Tumour response was measured by growth delay. Hh transcript levels (qRT-PCR) were compared among frozen tumours from treated and control mice. 5E1, a monoclonal SHH antibody, or LDE225, a clinical SMO inhibitor (Novartis®) inhibited Hh signaling.</p><p>Results</p><p>Precision irradiation significantly delayed xenograft tumour growth in all 7 PDX models. Combined chemoradiation further delayed growth relative to either modality alone in three of six PDX models. Following irradiation, two of three PDX models demonstrated sustained up-regulation of Hh transcripts. Combined LDE225 and radiation, and 5E1 alone delayed growth relative to either treatment alone in a Hh-responsive PDX model, but not in a non-responsive model.</p><p>Conclusion</p><p>Hh signaling mediates the radiation response in some EAC PDX models, and inhibition of this pathway may augment the efficacy of radiation in tumours that are Hh dependent.</p></div

Radiation significantly delays PDX tumour growth across multiple passages.

<p>Three representative PDX models (rows <b>A</b>-<b>C</b>) are shown. Arrows indicate time of irradiation.</p

Chemoradiation growth delays vary across PDX models.

<p>Arrows indicate time of irradiation. Growth delays that are significant versus chemotherapy alone and versus radiation alone are marked with an asterisk and circle, respectively.</p

Xenograft experimental design.

<p>A radiation experiment is shown as an example. Similar protocols were used for chemoradiation and for hedgehog inhibitor experiments, albeit with larger numbers of mice and without RT-PCR. *Up to 90 mice were used for large hedgehog inhibitor experiments to ensure sufficient numbers remained at the conclusion of the experiment.</p

Baseline Hh expression in untreated EAC tumours versus normal esophagus using RT-PCR.

<p>Error bars represent standard error of the fold change. SHH was expressed 168-fold higher in untreated tumours. Fold changes in expression of IHH, GLI1 and PTCH1 were 0.75, 0.12 and 0.23, respectively.</p

Hedgehog inhibition increases growth delay in some PDX models.

<p>Arrows indicate day of irradiation. <b>(A)</b> 5E1 used on a Hh-nonresponsive model. <b>(B)</b> 5E1 used on a Hh-responsive model. <b>(C)</b> LDE225 used on the same Hh-responsive model.</p

Baseline expression of Hedgehog transcripts in untreated EAC tumours from six PDX models.

<p>“Human” represents the patient-derived epithelium. “Mouse” represents the host stroma. Transcript levels are normalized to the highest expressed gene per species per model.</p

Radiation up-regulates Hedgehog transcription in some EAC tumours relative to controls.

<p>Significant (p<0.05) fold changes are underlined. Fold changes that are both ≥1.5 and statistically significant are shaded in grey. A lemniscate indicates that transcripts were undetectable in controls but detectable in treated tumours (infinite fold change). N/A indicates undetectable transcripts in treated tumours. (A-C) Fold changes in transcript levels in models 8, 6 and 7.</p