Ionocytes are distinct epithelial sensory cells scattered throughout the ductal system of the salivary glands. These cells are distinguished by their unusual morphology, as well as by a specific transcriptomic signature that includes the expression of the forkhead boxI1 (Foxi1) and achaete scute-like 3 (Ascl3) transcription factors. Currently little is known about the biology or function of ionocytes in the salivary glands. To facilitate the characterization of these cells, we produced an inducible Cre mouse allele driven by the Ascl3 promoter. This strain was crossed with a reporter to fluorescently label Ascl3+ ionocytes, highlighting that they are the site of enriched cystic fibrosis transmembrane conductance regulator (Cftr) expression in the salivary glands and demonstrating the proximity of these cells to neurons. Conditional Cre-mediated cell ablation, using diphtheria toxin (DTA), removed Ascl3+ ionocytes from the salivary glands and resulted in an altered pH of total saliva, supporting a function for ionocytes in transepithelial ion flow. Finally Cre-mediated expression of the calcium indicator GCaMP6f revealed thatAscl3+ ionocytes exhibit unique properties not observed in the acinar or surrounding duct cells, including elevated basal [Ca2+]i, spontaneous blinking in the absence of stimulation and a rapid loss of [Ca2+]i after nerve stimulation. These unique properties distinguish ionocytes as a specialized subset of salivary gland duct cells. KEY POINTS: Ionocytes are epithelial cells originally identified in marine teleosts and subsequently reported to be present in pulmonary epithelia, olfactory epithelium and salivary and lachrimal glands. We produced an inducible Cre mouse allele driven by the Ascl3 promoter to study the distribution and function of Ascl3+ ionocytes in salivary glands. By crossing this strain with a fluorescent reporter, we demonstrated the prominent localization of Ascl3+ ionocytes in salivary gland ducts. Cre-mediated expression of the genetically encoded Ca2+ indicator GCamp6f in Ascl3+ ionocytes revealed unique Ca2+ signalling properties of this cell type distinct from acinar cells and other ductal cell types. Cre-mediated ablation of Ascl3+ ionocytes altered the pH of the final saliva. We conclude that ionocytes resident in salivary gland ducts function to modify the primary saliva contributing to the composition of the final saliva entering the oral cavity.

Most head and neck cancer patients treated with ionizing radiation loose salivary gland function. Patients with decreased saliva have trouble eating, speaking and are predisposed to oral infections and tooth decay. Amifostine is the only FDA approved drug to prevent radiation-induced hyposalivation. However, it has intolerable side-effects that limit its use, motivating the discovery of alternative therapeutics. We leveraged our salivary gland tissue chip platform for high-content drug discovery that we developed using submandibular gland tissue from female SKH1 hairless mice, backcrossed 6 generations with C57BL/6 J mice. We developed in-chip assays to quantify reduced glutathione and cellular senescence, which are accepted biomarkers of radiation damage. We validated radioprotection using WR-1065, the active form of Amifostine and tested other reported radioprotective drugs including Edaravone, Tempol, N-acetylcysteine, Rapamycin, Ex-Rad, and Palifermin. Next, a Selleck Chemicals library of FDA-approved drugs was screened for radioprotection. Lead hits were tested in mouse models. We identify 25 candidate compounds and down-select them using EC50 values and published pharmacologic data. This lead us to test Phenylbutazone (an anti-inflammatory), Enoxacin (a fluoroquinolone antibiotic), and Doripenem (a carbapenem antibiotic) for in vivo radioprotection in mice. Results confirm that these three drugs exhibit radioprotection equivalent to Amifostine but with superior EC50 values, ranging from 140 to 6900-fold lower values. This body of work demonstrates the development and validation of assays using a tissue chip platform for high-content drug screening and the successful discovery and in vivo validation of candidate radioprotective drugs with non-antioxidant primary modes of action. These results point to possible unknown mechanisms of radioprotection. These drugs can be developed to improve radioprotection efficacy and clinical administration without adverse side-effects. Salivary glands are often damaged in patients undergoing radiation therapy to treat head and neck cancers. Lack of saliva results in a poor quality of life for these patients. Currently there are no good drugs that patients can take to protect their salivary glands from radiation damage. We used a system that models the salivary gland called a tissue chip to test a large panel of drugs to see if they can prevent damage by radiation. We identified several drug candidates that could protect mouse salivary glands from radiation. These drugs should now be further investigated as they could potentially be used to protect the salivary glands of patients undergoing radiation therapy. Piraino, Chen, Mereness et al use a salivary gland tissue chip for high-content radioprotective drug discovery. Three drugs are identified that exhibit higher potency and equivalent radioprotection to Amifostine, the only FDA approved drug that is rarely used clinically due to severe side effects and lack of efficacy.

The key role of CFTR in secretory epithelia has been extensively documented. Additionally, CFTR plays a significant role in ion absorption in exocrine glands, including salivary and sweat glands. Most of the knowledge about CFTR expression comes from animal models such as the mouse or the rat, but there is limited information about CFTR expression in human tissues. In the present study, we assessed the expression of CFTR in human submandibular and parotid glands. Consistent with findings in rodent salivary glands, our immunolocalization studies show that CFTR is expressed in duct cells. However, CFTR expression in human salivary glands differs from that in rodents, as immunolocalization and single-cell RNA sequencing analysis from a previous study performed in the human parotid gland revealed the presence of CFTR protein and transcripts within a distinct cell cluster. Based on cell marker expression, this cluster corresponds to acinar cells. To obtain functional evidence supporting CFTR expression, we isolated human parotid acinar cells through collagenase digestion. Acinar cells displayed an anion conductance that was activated in response to cAMP-increasing agents and was effectively blocked by CFTR(Inh)172, a known CFTR blocker. This study provides novel evidence of CFTR expression within acinar cells of human salivary glands. This finding challenges the established model positioning CFTR exclusively in duct cells from exocrine glands. NEW & NOTEWORTHY This study addresses the uncertainty about the impact of CFTR on human salivary gland function. We found CFTR transcripts in a subset of duct cells known as ionocytes, as well as in acinar cells. Isolated human parotid acinar cells exhibited Cl- conductance consistent with CFTR activity. This marks the first documented evidence of functional CFTR expression in human salivary gland acinar cells.

Saliva is vital to oral health, fulfilling multiple functions in the oral cavity. Three pairs of major salivary glands and hundreds of minor salivary glands contribute to saliva production. The secretory acinar cells within these glands include two distinct populations. Serous acinar cells secrete a watery saliva containing enzymes, while mucous acinar cells secrete a more viscous fluid containing highly glycosylated mucins. Despite their shared developmental origins, the parotid gland (PG) is comprised of only serous acinar cells, while the sublingual gland (SLG) contains predominantly mucous acinar cells. The instructive signals that govern the identity of serous versus mucous acinar cell phenotypes are not yet known. The homeobox transcription factor Nkx2.3 is uniquely expressed in the SLG. Disruption of the Nkx2.3 gene was reported to delay the maturation of SLG mucous acinar cells. To examine whether Nkx2.3 plays a role in directing the mucous cell phenotype, we analyzed SLG from Nkx2.3-/- mice using RNAseq, immunostaining and proteomic analysis of saliva. Our results indicate that Nkx2.3, most likely in concert with other transcription factors uniquely expressed in the SLG, is a key regulator of the molecular program that specifies the identity of mucous acinar cells.

During head and neck cancer treatment, off-target ionizing radiation damage to the salivary glands commonly causes a permanent loss of secretory function. Due to the resulting decrease in saliva production, patients have trouble eating, speaking and are predisposed to oral infections and tooth decay. While the radioprotective antioxidant drug Amifostine is approved to prevent radiation-induced hyposalivation, it has intolerable side effects that limit its use, motivating the discovery of alternative therapeutics. To address this issue, we previously developed a salivary gland mimetic (SGm) tissue chip platform. Here, we leverage this SGm tissue chip for high-content drug discovery. First, we developed in-chip assays to quantify glutathione and cellular senescence (β-galactosidase), which are biomarkers of radiation damage, and we validated radioprotection using WR-1065, the active form of Amifostine. Following validation, we tested other reported radioprotective drugs, including, Edaravone, Tempol, N-acetylcysteine (NAC), Rapamycin, Ex-Rad, and Palifermin, confirming that all drugs but NAC and Ex-Rad exhibited robust radioprotection. Next, a Selleck Chemicals library of 438 FDA-approved drugs was screened for radioprotection. We discovered 25 hits, with most of the drugs identified with mechanisms of action other than antioxidant activity. Hits were down-selected using EC 50 values and pharmacokinetics and pharmacodynamics data from the PubChem database leading to testing of Phenylbutazone (anti-inflammatory), Enoxacin (antibiotic), and Doripenem (antibiotic) for in vivo radioprotection in mice using retroductal injections. Results confirm that Phenylbutazone and Enoxacin exhibited equivalent radioprotection to Amifostine. This body of work demonstrates the development and validation of assays using a SGm tissue chip platform for high-content drug screening and the successful in vitro discovery and in vivo validation of novel radioprotective drugs with nonantioxidant primary indications pointing to possible, yet unknown novel mechanisms of radioprotection.

We recently developed a salivary gland tissue mimetic (SGm), comprised of salivary gland cells encap-sulated in matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) hydrogels within arrays of & SIM;320 & mu;m diameter spherical cavities molded in PDMS. The SGm provides a functional and physiolog-ically relevant platform well-suited to high-throughput drug screening for radioprotective compounds. However, the utility of the SGm would benefit from improved retention of acinar cell phenotype and function. We hypothesized that tuning biochemical cues presented within the PEG hydrogel matrix would improve maintenance of acinar cell phenotype and function by mimicking the natural extracellular matrix microenvironment of the intact gland. Hydrogels formed using slower-degrading MMP-sensitive peptide crosslinkers showed > 2-fold increase in sphere number formed at 48 h, increased expression of acinar cell markers, and more robust response to calcium stimulation by the secretory agonist, carbachol, with reduced SGm tissue cluster disruption and outgrowth during prolonged culture. The incorporation of ad-hesive peptides containing RGD or IKVAV improved calcium flux response to secretory agonists at 14 days of culture. Tuning the hydrogel matrix improved cell aggregation, and promoted acinar cell phenotype, and stability of the SGm over 14 days of culture. Furthermore, combining this matrix with optimized media conditions synergistically prolonged the retention of the acinar cell phenotype in SGm.Statement of significanceSalivary gland (SG) dysfunction occurs due to off-target radiation due to head and neck cancer treatments. Progress in understanding gland dysfunction and developing therapeutic strategies for the SG are ham-pered by the lack of in vitro models, as salivary gland cells rapidly lose critical secretory function within 24 hours in vitro . Herein, we identify properties of poly(ethylene glycol) hydrogel matrices that enhance the secretory phenotype of SG tissue mimetics within the previously-described SG-microbubble tissue chip environment. Combining slow-degrading hydrogels with media conditions optimized for secretory marker expression further enhanced functional secretory response and secretory marker expression.& COPY; 2023 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

ABSTRACT Many patients treated for head and neck cancers experience salivary gland hypofunction due to radiation damage. Understanding the mechanisms of cellular damage induced by radiation treatment is important in order to design methods of radioprotection. In addition, it is crucial to recognize the indirect effects of irradiation and the systemic responses that may alter saliva secretion. In this study, radiation was delivered to murine submandibular glands (SMGs) bilaterally, using a 137Cs gamma ray irradiator, or unilaterally, using a small-animal radiation research platform (SARRP). Analysis at 3, 24 and 48 h showed dynamic changes in mRNA and protein expression in SMGs irradiated bilaterally. Unilateral irradiation using the SARRP caused similar changes in the irradiated SMGs, as well as significant off-target, bystander effects in the non-irradiated contralateral SMGs.

Progress in the development of salivary gland regenerative strategies is limited by poor maintenance of the secretory function of salivary gland cells (SGCs) in vitro. To reduce the precipitous loss of secretory function, a modified approach to isolate intact acinar cell clusters and intercalated ducts (AIDUCs), rather than commonly used single cell suspension, is investigated. This isolation approach yields AIDUCs that maintain many of the cell-cell and cell-matrix interactions of intact glands. Encapsulation of AIDUCs in matrix metalloproteinase (MMP)-degradable PEG hydrogels promotes self-assembly into salivary gland mimetics (SGm) with acinar-like structure. Expression of Mist1, a transcription factor associated with secretory function, is detectable throughout the in vitro culture period up to 14 days. Immunohistochemistry also confirms expression of acinar cell markers (NKCC1, PIP and AQP5), duct cell markers (K7 and K5), and myoepithelial cell markers (SMA). Robust carbachol and ATP-stimulated calcium flux is observed within the SGm for up to 14 days after encapsulation, indicating that secretory function is maintained. Though some acinar-to-ductal metaplasia is observed within SGm, it is reduced compared to previous reports. In conclusion, cell-cell interactions maintained within AIDUCs together with the hydrogel microenvironment may be a promising platform for salivary gland regenerative strategies.

Radiation therapy for head and neck cancers causes salivary gland dysfunction leading to permanent xerostomia. Limited progress in the discovery of new therapeutic strategies is attributed to the lack of in vitro models that mimic salivary gland function and allow high-throughput drug screening. We address this limitation by combining engineered extracellular matrices with microbubble (MB) array technology to develop functional tissue mimetics for mouse and human salivary glands. We demonstrate that mouse and human salivary tissues encapsulated within matrix metalloproteinase-degradable poly(ethylene glycol) hydrogels formed in MB arrays are viable, express key salivary gland markers, and exhibit polarized localization of functional proteins. The salivary gland mimetics (SGm) respond to calcium signaling agonists and secrete salivary proteins. SGm were then used to evaluate radiosensitivity and mitigation of radiation damage using a radioprotective compound. Altogether, SGm exhibit phenotypic and functional parameters of salivary glands, and provide an enabling technology for high-content/throughput drug testing.

Salivary gland dysfunction resulting in xerostomia occurs in the majority of head and neck cancer patients treated with radiation therapy. Progress in determining the mechanism of radiation damage and methods for prevention has been hampered by the lack of in vitro salivary gland models. We recently developed a salivary gland tissue mimetic (SGm), comprised of salivary gland tissue clusters encapsulated in matrix metalloproteinase (MMP)–degradable poly(ethylene glycol) hydrogels within arrays of ~320 m diameter spherical cavities molded in PDMS. The SGm provides a functional and physiologically relevant salivary gland model well suited to high-throughput drug screening for radioprotective compounds. However, the utility of the SGm would significantly benefit from improved retention of acinar phenotype and function over longer culture durations. We hypothesize that tuning biochemical cues presented within the PEG hydrogel matrix will improve maintenance of acinar cell phenotype and function by mimicking the natural extracellular matrix microenvironment of the intact gland. Hydrogels formed using slower-degrading MMP-sensitive peptide crosslinkers showed >2-fold increase in sphere number formed at 48 hr, increased expression of acinar cell markers, and more robust response to calcium stimulation by the secretory agonist, carbachol, with reduced SGm tissue cluster disruption and outgrowth during prolonged culture. The incorporation of adhesive peptides containing RGD or IKVAV improved calcium flux response to secretory agonists at 14 days of culture. Tuning the hydrogel matrix improved cell aggregation, and promoted acinar cell phenotype, and stability of the SGm in long-term culture. Furthermore, combining this matrix with optimized media conditions synergistically improved retention of acinar cells in SGm.

The maintenance of balanced oral homeostasis depends on saliva. A readily available and molecularly rich source of biological fluid, saliva fulfills many functions in the oral cavity, including lubrication, pH buffering, and tooth mineralization. Saliva composition and flow can be modulated by different factors, including circadian rhythm, diet, age, drugs, and disease. Recent events have revealed that saliva plays a central role in the dissemination and detection of the SARS-CoV-2 coronavirus. A working knowledge of saliva function and physiology is essential for dental health professionals.

Purpose: In a combined retrospective and prospective study, human salivary glands were investigated after radiation treat-ment for head and neck cancers. The aim was to assess acinar cell loss and morphologic changes after radiation therapy and to determine whether irradiated salivary glands have regenerative potential. Methods and Materials: Irradiated human submandibular and parotid salivary glands were collected from 16 patients at a range of time intervals after completion of radiation therapy (RT). Control samples were collected from 14 patients who had not received radiation treatments. Tissue sections were analyzed using immunohistochemistry to stain for molecular markers. Results: Human submandibular and parotid glands isolated less than 1 year after RT showed a near complete loss of acinar cells. However, acinar units expressing functional secretory markers were observed in all samples isolated at later intervals after RT. Significantly lower acinar cell numbers and increased fibrosis were found in glands treated with combined radiation and chemotherapy, in comparison to glands treated with RT alone. Irradiated samples showed increased staining for duct cell keratin markers, as well as many cells coexpressing acinar-and duct cellespecific markers, in comparison to nonirradiated control samples. Conclusions: After RT, acinar cell clusters are maintained in human submandibular glands for years. The surviving acinar cells retain proliferative potential, although significant regeneration does not occur. Persistent DNA damage, increased fibrosis, and altered cell identity suggest mechanisms that may impair regeneration. (C) 2020 Elsevier Inc. All rights reserved.

To develop treatments for salivary gland dysfunction, it is important to understand how human salivary glands are maintained under normal homeostasis. Previous data from our lab demonstrated that murine salivary acinar cells maintain the acinar cell population through self‐duplication under conditions of homeostasis, as well as after injury. Early studies suggested that human acinar cells are mitotically active, but the identity of the resultant daughter cells was not clear. Using markers of cell cycle activity and mitosis, as well as an ex vivo 5‐Ethynyl‐2´‐deoxyuridine assay, we show that human salivary gland acinar cells divide to generate daughter acinar cells. As in mouse, our data indicate that human salivary gland homeostasis is supported by the intrinsic mitotic capacity of acinar cells.

In the effort to develop cell-based therapies to treat salivary gland dysfunction, many different populations of cells in the adult salivary glands have been proposed as stem cells. These cell populations vary, depending on the assay used, and are often nonoverlapping, leading to the conclusion that salivary glands harbor multiple stem cells. The goal of this review is to critically appraise the assays and properties used to identify stem cells in the adult salivary gland, and to consider the caveats of each. Re-evaluation of the defining criteria may help to reconcile the many potential stem cell populations described in the salivary gland, in order to increase comparability between studies and build consensus in the field. Stem Cells 2019;37:1144-1150.

Salivary gland function is severely disrupted by radiation therapy used to treat patients diagnosed with head and neck cancer and by Sjögren's syndrome. The resulting condition, which results in xerostomia or dry mouth, is due to irreversible loss of the secretory acinar cells within the major salivary glands. There are presently no treatments for the resolution of xerostomia. Cell-based approaches could be employed to repopulate acinar cells in the salivary gland but investigations into potential therapeutic strategies are limited by the challenges of maintaining and expanding acinar cells in vitro. We investigate the encapsulation of salivary gland cell aggregates within PEG hydrogels as a means of culturing secretory acinar cells. Lineage tracing was used to monitor the fate of acinar cells isolated from murine submandibular gland (SMG). Upon initial formation in vitro, SMG aggregates comprise both acinar and duct cells, with the majority cells of acinar origin. With longer culture times, acinar cells significantly decreased the expression of specific markers and activated the expression of keratins normally found in duct cells. A similar acinar-to-duct cell transition was also observed in vivo, following duct ligation injury. These results indicate that under conditions of stress (mechanical and enzymatic isolation from glands) or injury (duct ligation), salivary gland acinar cells exhibit plasticity to adopt a duct cell phenotype.

RNA-Seq was used to better understand the molecular nature of the biological differences among the three major exocrine salivary glands in mammals. Transcriptional profiling found that the adult murine parotid, submandibular, and sublingual salivary glands express greater than 14,300 protein-coding genes, and nearly 2,000 of these genes were differentially expressed. Principle component analysis of the differentially expressed genes revealed three distinct clusters according to gland type. The three salivary gland transcriptomes were dominated by a relatively few number of highly expressed genes (6.3%) that accounted for more than 90% of transcriptional output. Of the 912 transcription factors expressed in the major salivary glands, greater than 90% of them were detected in all three glands, while expression for ~2% of them was enriched in an individual gland. Expression of these unique transcription factors correlated with sublingual and parotid specific subsets of both highly expressed and differentially expressed genes. Gene ontology analyses revealed that the highly expressed genes common to all glands were associated with global functions, while many of the genes expressed in a single gland play a major role in the function of that gland. In summary, transcriptional profiling of the three murine major salivary glands identified a limited number of highly expressed genes, differentially expressed genes, and unique transcription factors that represent the transcriptional signatures underlying gland-specific biological properties.

OBJECTIVE:The objective of this study was to characterize the mechanism by which salivary gland cells (SGC) aggregate in vitro.DESIGN:Timelapse microscopy was utilized to analyze the process of salivary gland aggregate formation using both primary murine and human salivary gland cells. The role of cell density, proliferation, extracellular calcium, and secretory acinar cells in aggregate formation was investigated. Finally, the ability of cells isolated from irradiated glands to form aggregates was also evaluated.RESULTS:Salivary gland cell self-organization rather than proliferation was the predominant mechanism of aggregate formation in both primary mouse and human salivary gland cultures. Aggregation was found to require extracellular calcium while acinar lineage cells account for ∼80% of the total aggregate cell population. Finally, aggregation was not impaired by irradiation.CONCLUSIONS:The data reveal that aggregation occurs as a result of heterogeneous salivary gland cell self-organization rather than from stem cell proliferation and differentiation, contradicting previous dogma. These results suggest a re-evaluation of aggregate formation as a criterion defining salivary gland stem cells.

Radiotherapy for head and neck cancers commonly causes damage to salivary gland tissue, resulting in xerostomia (dry mouth) and numerous adverse medical and quality-of-life issues. Amifostine is the only Food and Drug Administration–approved radioprotective drug used clinically to prevent xerostomia. However, systemic administration of amifostine is limited by severe side effects, including rapid decrease in blood pressure (hypotension), nausea, and a narrow therapeutic window. In this study, we demonstrate that retroductal delivery of amifostine and its active metabolite, WR-1065, to murine submandibular glands prior to a single radiation dose of 15 Gy maintained gland function and significantly increased acinar cell survival. Furthermore, in vivo stimulated saliva secretion was maintained in retrograde-treated groups at levels significantly higher than irradiated-only and systemically treated groups. In contrast to intravenous injections, retroductal delivery of WR-1065 or amifostine significantly attenuated hypotension. We conclude that localized delivery to salivary glands markedly improves radioprotection at the cellular level, as well as mitigates the adverse side effects associated with systemic administration. These results support the further development of a localized delivery system that would be compatible with the fractionated dose regimen used clinically.

Nanoparticles (NPs) interact with complex protein milieus in biological fluids, and these interactions have profound effects on NP physicochemical properties and function. Surprisingly, most studies neglect the impact of these interactions, especially with respect to NP-mediated siRNA delivery. Here, the effects of serum on colloidal stability and siRNA delivery of a pH-responsive micellar NP delivery system were characterized. Results show cationic NP-siRNA complexes aggregate in ≥2% serum in buffer, but are stable in serum-free media. Furthermore, nonaggregated NP-siRNA delivered in serum-free media result in 4-fold greater siRNA uptake in vitro, compared to aggregated NP-siRNA. Interestingly, pH-responsive membrane lysis behavior, which is required for endosomal escape, and NP-siRNA dissociation, necessary for gene knockdown, are significantly reduced in serum. Consistent with these data, nonaggregated NP-siRNA in serum-free conditions result in highly efficient gene silencing, even at doses as low as 5 nM siRNA. NP-siRNA diameter was measured at albumin and IgG levels mimicking biological fluids. Neither albumin nor IgG alone induces NP-siRNA aggregation, implicating other serum proteins in NP colloidal instability. Finally, as a proof-of-principle that stability is maintained in established in vivo models, transmission electron microscopy reveals NP-siRNA are taken up by ductal epithelial cells in a nonaggregated state when injected retroductally into mouse salivary glands in vivo. Overall, this study shows serum-induced NP-siRNA aggregation significantly diminishes efficiency of siRNA delivery by reducing uptake, pH-responsive membrane lysis activity, and NP-siRNA dissociation. Moreover, these results highlight the importance of local NP-mediated drug delivery and are broadly applicable to other drug delivery systems.