*3.10. Fibrosis*

SMG biopsies from patients with advanced stage oropharyngeal cancer who received fractionated radiotherapy (1.8–2 Gy per fraction, ~35 fractions) showed glandular atrophy and periductal and parenchymal fibrosis that correlates with the degree of sialadenitis (i.e., lymphocytic infiltration of the gland). Destruction of salivary gland parenchyma with progressive replacement of functional tissue by extracellular matrix proteins impairs saliva production [33]. In rodent models, the development of fibrosis following irradiation is inconsistent, but has been reported to develop between 4 and 6 months post-IR [1,44,71]. In minipigs, IR-induced fibrosis has been reported at 30 days post-fractionated IR (200 cGy per fraction, 70 Gy total dose) [57]. Extensive fibrosis, measured by collagen deposition, was reported in CH3 mice and minipigs after 300 days and 16 weeks post-IR, respectively. In this study, CH3 mice received fractionated IR (5 × 6 Gy doses), whereas minipigs were exposed to a single 15 Gy dose of IR [54]. RNA-seq analysis of mouse SMGs 300 days post-IR revealed upregulation of genes involved in extracellular matrix remodeling and fibrosis (i.e., *Col23a1, Mmp2, Mmp3, Serping1*), whereas *Serping1* and *Mmp2* were also upregulated in minipigs at 16 weeks post-IR [54]. In a partial gland resection model utilized to investigate the mechanisms of salivary gland regeneration in the absence of confounding external stimuli, such as irradiation, genes involved in fibrotic development, ECM remodeling and the innate and adaptive immune system were similarly upregulated at days 3 and 14 post-resection in the murine SMG [89].

While humans [33,36,90] as well as mice and minipigs [1,44,54,57] show significant fibrotic damage to the salivary glands following irradiation, there is insufficient evidence to determine whether fibrosis is a cause or consequence of gland dysfunction. TGF-β, a known mediator of fibrogenesis in several tissues [91–94] is elevated in HNC patients following radiotherapy [95] and in murine models of IR-induced hyposalivation [96]. We have previously shown that TGF-β is upregulated in a mouse model of fibrosis caused by SMG excretory duct ligation and that in vivo administration of TGF-β inhibitors reduces duct ligation-induced salivary gland fibrosis [97]. TGF-β inhibition also efficiently reduces IR-induced lung [98,99] and rectal [100] fibrosis in mouse models. Further investigation is needed on the relationship of TGF-β and fibrosis to IR-induced hyposalivation and whether this pathway plays a significant role in chronic salivary gland dysfunction in RT.
