P2-09-05: Molecular Imaging of Hedgehog Induced Epithelial-Stromal Interactions.
Authors: Sims-Mourtada J , Yang DJ , Tworowska I , Larson R , Smith D , Mourtada F , Woodward W 
Published: June 2012

Introduction: Epithelial-stromal Hedghehog (HH) pathway signaling, originating from Sonic Hedgehog (SHH) ligand secreted by tumor cells, promotes breast cancer growth and metastasis. Furthermore, high tumor SHH expression is associated with poor prognosis in invasive ductal carcinoma. Studies have also shown high expression of Patched (PTCH) mRNA in SHH expressing tumors as well as cancer stem-like cells. This is in part due to a feedback loop that results in increased PTCH gene expression upon HH signaling. We sought to exploit this feedback loop by using radiolabeled derivative of SHH to identify tumors that have a high level of hedgehog signaling. These agents can potentially be used for molecular imaging of HH induced changes in PTCH expression originating from tumor expressed SHH.

Methods: Recombinant N-terminal SHH derivatives were radioiodinated with iodine-131 using the idogen method. Iodinated proteins were obtained with radiolabeling yields of 50–60 % and specific activity of 9.3E10Ci/mol. Receptor binding studies were performed on breast cancer cell lines with varying SHH expression. Studies were also conducted on cells cultured in stem cell promoting conditions (mammospheres), after exposure to ionizing radiation or treatment with the hedgehog inhibitor cyclopamine. Scatchard plots were generated using Graph pad software. Pilot studies were conducted with 131I-SHH to test the potential of imaging PTCH receptor expression in a rat model of breast cancer. Fisher rats bearing breast cancer xenografts were injected with approximately 250 microCi of 131I-SHH. Planar scinitigraphy was conducted at 2, 4 and 24 hours. Studies were performed with iodinated BSA as a control for extracellular perfusion and retention. Tissue biodistribution studies were also performed using this model.

Results: Receptor binding of 131I-SHH was significantly increased in cell lines with high endogenous HH pathway activation. Receptor binding was also increased after exposure to ionizing radiation and significantly decreased following treatment with hedgehog inhibitors. Scatchard analysis revealed up to a 14 fold increase in PTCH receptor sites in mammospheres compared to the entire breast cancer population. A 10-fold increase in PTCH receptor binding sites was also observed in cells after exposure to ionizing radiation. In vivo imaging and biodistribution studies revealed significant accumulation of 131I-SHH within tumor tissue as compared to normal organs. Tumor:muscle ratios were approximately 8:1 at 4 hours, while tumor to blood and tumor to bone were 2:1 and 5:1 respectively. Significant uptake was also observed in liver tissue, as a result of protein degradation and excretion and endogenous PTCH expression.

Conclusions: These studies show that expression of the PTCH receptor is increased in cells with active HH signaling and in breast cancer mammospheres. Our findings also show that PTCH receptor expression is decreased upon treatment with HH signaling inhibitors. Preliminary imaging studies show that 131I-SHH is capable of in vivo detection of breast tumors with high hedgehog signaling. Our data suggests that radiolabeled SHH derivatives may provide a method to follow epithelial-stromal HH interactions and determine response to SHH targeted therapies.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-09-05.