Pharmacological Activation of the EDA/EDAR Signaling Pathway Restores Salivary Gland Function following Radiation-Induced Damage

Radiotherapy of head and neck cancers often results in collateral damage to adjacent salivary glands associated with clinically significant hyposalivation and xerostomia. Due to the reduced capacity of salivary glands to regenerate, hyposalivation is treated by substitution with artificial saliva, rather than through functional restoration of the glands. During embryogenesis, the ectodysplasin/ectodysplasin receptor (EDA/EDAR) signaling pathway is a critical element in the development and growth of salivary glands. We have assessed the effects of pharmacological activation of this pathway in a mouse model of radiation-induced salivary gland dysfunction. We report that post-irradiation administration of an EDAR-agonist monoclonal antibody (mAbEDAR1) normalizes function of radiation damaged adult salivary glands as determined by stimulated salivary flow rates. In addition, salivary gland structure and homeostasis is restored to pre-irradiation levels. These results suggest that transient activation of pathways involved in salivary gland development could facilitate regeneration and restoration of function following damage

Increased vacuolization in irradiated salivary glands is reversed with post-therapy mAbEDAR1.

<p>H&E images from submandibular salivary glands were analyzed for cellular vacuoles as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112840#s2" target="_blank">materials and methods</a>. Vacuoles were defined as an unstained, acellular area greater than 7.35 µm in diameter. (A) Representative annotated image of vacuole measurement compared to the unmarked image is shown. ImagePro software was utilized to calculate the area annotated as a vacuole compared to the total area in the image. (B) Quantification of the percent area occupied by cellular vacuoles (mean+/−SEM) is graphed. Significant differences (p<0.05) were determined using an ANOVA followed by a Bonferroni post-hoc test. Treatment groups with the same lower case letters are not significantly different from each other. All error bars represent standard error of the mean (SEM). N = 4 mice per group.</p

Reduced compensatory proliferation in mice treated with post-therapy mAbEDAR1.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112840#pone-0112840-g002" target="_blank">Figure 2</a> and parotid salivary glands were collected at days 30, 60, and 90 for analysis of PCNA levels. (A) Representative images from each treatment group are shown. The total number of PCNA positive cells in the acinar compartment was graphed as a percentage of total number of cells in the acinar compartment at days 30 (B), 60 (C) and 90 (D). Significant differences (p<0.05) were determined using an ANOVA followed by a Bonferroni post-hoc test. Treatment groups with the same lower case letters are not significantly different from each other. All error bars represent standard error of the mean (SEM). N = 4 mice per group.</p

Histological analysis of salivary glands.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112840#pone-0112840-g002" target="_blank">Figure 2</a> and submandibular salivary glands were collected at day 60 for histological analysis. Representative H&E images from unirradiated control (A), mAbEDAR1 alone (B), irradiated with 5Gy (C) and 5Gy+mAbEDAR are shown (D). Areas of focal inflammation denoted with asterisks and vacuoles are delineated by an arrowhead.</p

mAbEDAR1 administration following radiation restores stimulated salivary flow rates to pretreatment levels.

<p>On day 0, the head and neck region of mice was exposed to a single dose of 5 Gy radiation. On day 4, mice received a single dose injection of mAbEDAR or vehicle control. Stimulated salivary flow rates were determined on days 14, 30, 60, 90 post-irradiation. Flow rates (mean+/−SEM) were calculated by determining the average µg/min value for the unirradiated controls (CTRL) for each collection day followed by the percent change in individual experimental animals. Unirradiated controls averaged ∼27 µg/min across all time points and were set to 100% (normalized value of 1) for each time point. Significant differences (p<0.05) were determined using an ANOVA followed by a Student-Newman-Keuls post-hoc test. Treatment groups with the same lower case letters are not significantly different from each other. All error bars represent standard error of the mean (SEM). N>16 mice per group.</p

Restoration of amylase positive area in parotid glands of mice treated with post-therapy mAbEDAR1.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112840#pone-0112840-g002" target="_blank">Figure 2</a> and parotid salivary glands were collected at days 30, 60, and 90 for analysis of amylase area. (A) Representative images from each treatment group are shown. Quantification of the mean area of parotid tissue staining positive for amylase was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112840#s2" target="_blank">materials and methods</a> at days 30 (B), 60 (C) and 90 (D). Significant differences (p<0.05) were determined using an ANOVA followed by a Bonferroni post-hoc test. Treatment groups with the same lower case letters are not significantly different from each other. All error bars represent standard error of the mean (SEM). N = 4 mice per group.</p

Analysis of <i>Edar</i> expression following radiation and experimental design of study.

<p>(A) On day 0 the head and neck region of mice was exposed to a single dose of 5 Gy radiation. <i>Edar</i> expression (mRNA) in parotid salivary glands was determined on days 4–10 post-radiation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112840#s2" target="_blank">materials and methods</a> and graphed as fold over unirradiated (UT). Significant differences (p<0.05) were determined using an ANOVA followed by a Bonferroni post-hoc test. Treatment groups with the same lower case letters are not significantly different from each other. N = 4 mice per group. (B) Experimental design timeline. (C) Three days post-radiation stimulated salivary flow rates were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112840#s2" target="_blank">materials and methods</a>. Irradiated flow rates were normalized to corresponding unirradiated (UT) controls. There were 60 animals in the control (UT) and 62 animals in the irradiated group (5Gy). Significant difference (p<0.05) was determined by t-test and designated by an asterisk (*). All error bars represent standard error of the mean (SEM).</p

The Rapalogue, CCI-779, Improves Salivary Gland Function following Radiation

<div><p>The standard of care for head and neck cancer typically includes surgical resection of the tumor followed by targeted head and neck radiation. However depending on tumor location and stage, some cases may not require surgical resection while others may be treated with chemoradiation. Unfortunately, these radiation treatments cause chronic negative side effects for patients. These side effects are associated with damage to surrounding normal salivary gland tissue and include xerostomia, changes in taste and malnutrition. The underlying mechanisms of chronic radiation-induced salivary gland dysfunction are unknown, however, in rodent models persistently elevated proliferation is correlated with reduced stimulated salivary flow. The rapalogue, CCI-779, has been used in other cell systems to induce autophagy and reduce proliferation, therefore the aim of this study was to determine if CCI-779 could be utilized to ameliorate chronic radiation-induced salivary gland dysfunction. Four to six week old <i>Atg5^f/f; Aqp5-Cre</i>, <i>Atg5^+/+; Aqp5-Cre</i> and FVB mice were treated with targeted head and neck radiation. FVB mice were treated with CCI-779, chloroquine, or DMSO post-radiation. Stimulated salivary flow rates were determined and parotid and submandibular salivary gland tissues were collected for analyses. Mice with a defect in autophagy, via a conditional knockout of <i>Atg5</i> in the salivary glands, display increased compensatory proliferation in the acinar cell compartment and hypertrophy at 24-72 hours following radiation. FVB mice treated with post-therapy CCI-779 have significant improvements in salivary gland physiology as determined by stimulated salivary flow rates, proliferation indices and amylase production and secretion. Consequently, post-radiation use of CCI-779 allows for improvement of salivary gland function and reestablishment of glandular homeostasis. As CCI-779 is already FDA approved for other uses, it could have a secondary use to alleviate the chronic side effects in head and neck cancer patients who have completed anti-tumor therapy.</p></div

Radiation plus post-therapy CCI-779 improves parotid acinar cell proliferation indices and amylase production levels similar to unirradiated mice.

<p>The head and neck region of FVB mice was exposed to a single 5-Gy dose of radiation and mice received injections of vehicle or CCI-779 on days 4-8 following initial radiation treatment. Parotid salivary glands were then collected 30 days following treatment. Significant differences (<i>p</i><0.05) were determined using an ANOVA followed by a post-hoc Bonferroni multiple-comparison test. Letters above treatment groups are used to signify statistical significance; treatment groups with the same letters are not significantly different from each other. Data are presented as the mean ±SEM. UT: unirradiated. <b>A.</b>) Serial sections were stained for PCNA, a marker of proliferation, and the graph represents the number of acinar cells with positive PCNA staining in the parotid glands as a percentage of the total number of acinar cells. n = 4 per genotype/per treatment. <b>B.</b>) Representative images of positive PCNA staining. <b>C.</b>) Serial sections were stained to determine positive amylase area of the parotid glands. The graph represents the positive amylase area as a percentage of the parotid area as a whole. n = 4 per genotype/per treatment. <b>D.</b>) Stimulated saliva was collected from mice 30 days after treatment with a single 5Gy dose of targeted head and neck radiation and analyzed for total protein content as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113183#s2" target="_blank">Materials and Methods</a>. The graph represents the percentage of amylase protein (ranging from 50–57 kD); n = 10 per genotype/per treatment.</p

<i>Atg5^f/f;Aqp5-Cre</i> autophagy-deficient mice display increased hyperplasia and elevated compensatory proliferation of the parotid acinar cell population following targeted head and neck irradiation.

<p><i>Atg5^+/+;Aqp5-Cre</i> and <i>Atg5^f/f;Aqp5-Cre</i> (autophagy-deficient) mice were treated with a single 5-Gy dose of targeted head and neck radiation on day 0. At 24, 48, 72 hours and day 30 following treatment, salivary tissue was collected and serial sections were stained. Letters above treatment groups are used to signify statistical significance; treatment groups with the same letters are not significantly different from each other. Significant differences (<i>p</i><0.05) were determined using an ANOVA followed by a post-hoc Bonferroni multiple-comparison test. Data are presented as the mean ±SEM. <b>A.</b>) Representative image of H&E staining of unirradiated <i>Atg5^+/+;Aqp5-Cre</i> control-mice. <b>B.</b>) Representative image of H&E staining of <i>Atg5^f/f</i>;<i>Aqp5-Cre</i> unirradiated mice. <b>C.</b>) Representative image of H&E staining of irradiated <i>Atg5^+/+;Aqp5-Cre</i> mice 30 days after radiotherapy. <b>D.</b>) Representative image of H&E staining of irradiated <i>Atg5^f/f</i>;<i>Aqp5-Cre</i> mice 30 days after radiotherapy. <b>E.</b>) Total acinar cell counts taken from 3–5 images per mouse 30 days after radiotherapy. <i>p</i><0.05; n≥4 per genotype. UT: unirradiated. <b>F.</b>) Serial sections were stained for PCNA, a marker of proliferation, and the graph represents the number of acinar cells with positive PCNA staining in the parotid glands as a percentage of the total number of acinar cells. <i>p</i><0.05, n = 4 per genotype/per treatment. <b>G.</b>) Representative images of positive PCNA staining in the parotid glands.</p