The Role of Radiation Therapy in Perineural Invasion: A Translational Approach

Abstract

Perineural invasion (PNI) is a frequent finding in head and neck, pancreatic, prostate, and other cancers, which portends a poor prognosis. Recent studies have demonstrated that PNI involves a complex signaling cascade involving tumor, nerve, and stromal cells through autocrine and paracrine mechanisms. The mechanism through which radiation (RT) may improve local control in patients with PNI has never been established. We explored how RT alters the pathogenesis of PNI in order to further improve our treatment designs. We optimized an in-vitro model of PNI that co-cultures cancer cells adjacent to nerve cells from explanted dorsal root ganglia (DRG) from newborn mice. We studied the effects of RT on 1) a neuroinvasive cancer cell line, MiaPaCa and 2) DRG. MiaPaCa were radiated to single fraction doses up to 8 Gy and 24-hours later plated in a culture divider 500 microns away from a DRG in Matrigel. Alternatively, DRG were radiated up to doses of 16 Gy and grown in a similar manner adjacent to a colony of non-radiated MiaPaCa. Nerve invasion was measured by calculating the area of nerve invasion (microns2) at 4 and 8 days after divider removal. Boyden chamber migration assays were performed to characterize the migration of radiated and non-irradiated MiaPaCa to various chemoattractants. Radiated and non-radiated cells were plated and counted in parallel to these studies to control for effects on cellular proliferation. ELISA and cytokine arrays were performed on conditioned media from radiated and non-radiated DRG. In the DRG model of PNI, RT of MiaPaCa between 2 - 8 Gy significantly decreased area of nerve invasion by at least 80% in a dose-dependent manner. In Boyden chamber assays, RT of MiaPaCa inhibited cancer cell migration towards DRG and Glial Derived Neurotrophic Factor (GDNF) in a dose-dependent manner. There were dose-dependent decreases in cancer cell number with radiation, while DRG remained viable up to doses of 20 Gy. RT of DRG alone, but not the cancer cells, between 4 - 16 Gy significantly decreased area of invasion by 86% across all doses without DRG death or morphologic change. At these respective doses, GDNF secretion by the DRG was suppressed and cytokine production altered. RT of MiaPaCa impairs PNI partly through cellular death and loss of reproductive potential. However, low doses of RT to the DRG significantly impaired nerve invasion potentially through altered nerve chemokine and cytokine secretion. RT along nerve pathways may interrupt paracrine mechanisms of PNI thereby conveying a therapeutic benefit beyond just cancer cell death. The data lends support to prophylactic irradiation of the nerve in patients at risk for PNI; however, further study is needed.