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

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

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

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

Selected molecular marker expression by immunohistochemistry (IHC).

<p>P53 in Line A and Ki-67 in Line H are examples of similar expression between patient, early passage (P1) and latest passage (P_{<i>latest</i>}) xenografts. P16 in Line H was selected to demonstrate the heterogeneity detected in the same tissue (P_<i>early</i> showing both positive and negative expression). EGFR expression in Line E exhibited an increase in intensity from patient to xenografts while Her-2/<i>neu</i> expression in Line A showed a decrease in intensity. These examples were included to demonstrate that the differences exhibited between patient tissue, early passage and latest passage xenografts were due to intrinsic heterogeneity and not to any specific patterns of expression.</p

Multivariate analysis of Clinicopathological and Immunohistochemical Characteristics of Patient Tumors.

<p>The final multivariate models are shown. Factors assessed included age, gender, stage, differentiation, location, neo-adjuvant chemo-radiation, heartburn, Barrett’s esophagus and expression of p16, p53, Her-2/<i>neu</i>, EGFR and Ki-67.</p><p>*Age was modeled as a continuous variable in the logistic regression analysis; the odds ratio is reported for every increase in 10 years. For example, this is the odds ratio comparing someone aged 70 vs 60 years old; or 65 vs 55 years old.</p><p>Multivariate analysis of Clinicopathological and Immunohistochemical Characteristics of Patient Tumors.</p