Radiation Induced Salivary Gland Damage is Driven by Immunologic Damage

Objectives: Many head and neck cancer patients develop Radiation-induced xerostomia (RIX). To better understand the molecular alterations induced by radiation to the salivary glands, we used transcriptional profiling assess acute and long-term alterations. Based on evidence that immunologic changes are induced by radiation, we used immune-depletion experiments to investigate the role of B-cells and macrophages in RIX.
Methods: The submandibular gland (SMG) of male-C57/Bl6-mice was irradiated (15Gy) micewere followed for 90 days. Saliva was collected throughout the time-course. SMGs were collected 3-, 7-, 60-, and 90-days following radiation. Tissues were analyzed by Nanostring Digital-Spatial-Profiling (DSP) utilizing the murine whole-transcriptome panel and by immunohistochemical staining for fibrosis (masons trichrome), α-amylase, F4/80, Lgr5, Sox2, and MIST1 to evaluate glandular structure, salivary proteins, immune-microenvironment and pro-acinar salivary-stem-cell presence. Specific immune populations were depleted using anti-CD20 and clodronate-liposome prior to radiation and salivary function and histology was assessed at 3-, 7-, and 60-days following radiation.
Results: Radiation resulted in a significant decrease in salivary production (p-value = 0.03) in immune competent models. Transcriptional pathway analysis identified early alterations in signal-transduction and innate immune-response. Late timepoints identified alterations in B-cell activity and Wnt signaling pathways. Histological evaluation of the tissues showed increased disorganization of the gland, with decreases in size and distribution of the acinar compartments (mean area stained: No RT=25.81% vs 90d RT=19.98%, p-value=0.005). Acutely, there was an increase in expression of MIST1 and macrophages (mean area stained: No RT=14.8% vs 7d RT=18.76%, p-value=0.0004) within the SMG that resolved by the 60- and 90-day time-points. Results of immune depletion studies will be presented.
Conclusions: Results implicate alterations in macrophages and B-cells in radiation-induced damage. These data along with the data from our immune-depletion experiments will provide valuable mechanistic insight guiding modulation of cell-therapies for treatment of RIX which can be further tested in preclinical models.