Murine Salivary Gland Research Articles

Follistatin expressed in mechanically-damaged salivary glands of male mice induces proliferation of CD49f+ cells

Salivary glands (SGs) are very important for maintaining the physiological functions of the mouth. When SGs regenerate and repair from various damages, including mechanical, radiological, and immune diseases, acinar and granular duct cells originate from intercalated duct cells. However, the recovery is often insufficient because of SGs' limited self-repair function. Furthermore, the precise repair mechanism has been unclear. Here, we focused on CD49f, one of the putative stem cell markers, and characterized CD49f positive cells (CD49f+ cells) isolated from male murine SGs. CD49f+ cells possess self-renewal ability and express epithelial and pluripotent markers. Compared to CD49f negative cells, freshly isolated CD49f+ cells highly expressed inhibin beta A and beta B, which are components of activin that has anti-proliferative effects. Notably, an inhibitor of activin, follistatin was expressed in mechanically-damaged SGs, meanwhile no follistatin was expressed in normal SGs in vivo. Moreover, sub-cultured CD49f+ cells highly expressed both Follistatin and a series of proliferative genes, expressions of which were decreased by Follistatin siRNA. These findings indicated that the molecular interaction between activin and follistatin may induce CD49f+ cells proliferation in the regeneration and repair of mouse SGs.

Characteristics of aquaporin 1, 3, and 5 expression during early murine salivary gland development.

Aquaporins (AQPs) are essential to coordinate the transit of water and ions through the cell membrane. In salivary glands (SGs), AQPs have been associated with saliva formation, facilitating water absorption through the epithelium during the formation of hypotonic saliva, which is then secreted into the oral cavity. Different members of the AQP family have been suggested to play distinct roles during embryonic development, highlighted by their specific expression patterns. Here, we have investigated the expression patterns of AQP-1, AQP-3 and AQP-5 by immunofluorescence at key stages of salivary gland development, utilising cultured mouse embryonic submandibular (SMG) and sublingual (SLG) glands. The expression of AQPs was compared to a mitotic marker, phospho-histone 3 (PH3), a myoepithelial marker, smooth muscle actin (SMA), and a vascular marker, CD31. Qualitative analysis revealed that AQP-1 and AQP-3 were primarily expressed during the earlier phases of SG morphogenesis and were associated with cells undergoing mitotic processes (PH3-positive). AQP-5, in contrast, was not associated to mitotic figures, but was predominantly expressed during late stages of SG morphogenesis. Our results highlight that AQPs are expressed from early stages of SG morphogenesis and exhibit complimentary expression patterns that may contribute to the morphogenesis of salivary glands.

Ano6 disruption impairs acinar cell regulatory volume decrease and protein secretion in murine submandibular salivary glands.

The widely expressed Anoctamin 6 (Ano6) supports different Ca2+ -dependent functions, but little is known about its role in salivary glands. Mouse submandibular gland (SMG) acinar cells exhibited a robust regulatory volume decrease (RVD) following cell swelling that was reduced approximately 70% in Ano6-/- mice. Ca2+ -free conditions nearly eliminated the RVD response suggesting that Ano6 is an obligatory component of thecell volume-activated, Ca2+ -dependent RVD pathway in salivary gland acinar cells. Ex vivo agonist-stimulated secretion of water and ions was unaffected by Ano6 disruption under both isotonic and hypotonic conditions suggesting that Ano6 does not play a major role in fluid and electrolyte secretion. In contrast, the total amount of β-adrenergic-dependent protein secretion by the SMG was significantly reduced in Ano6-/- mice. Closer inspection of these latter results revealed that protein secretion was affected only in the female SMG by Ano6 disruption. These results indicate that Ano6 modulates the RVD response and protein secretion by salivary gland acinar cells.

Role of CD44 in tumor-initiating cells of salivary gland pleomorphic adenoma: More than a surface biomarker.

CD44, a cell-surface glycoprotein, functions as a receptor for hyaluronic acid. Our research group has previously shown that CD44 is a biomarker for the CD44hi cells (tumor-initiating cells; TICs) in murine salivary gland tumors. However, little is known concerning the biological roles of CD44 in the tumorigenesis of pleomorphic adenoma. The present study is aimed to investigate the effects of CD44 on the proliferation, invasive capability, and apoptosis of TICs in vitro, as well as the tumorigenicity of TICs in vivo. The results demonstrated that knockdown of CD44 attenuated the malignant phenotype of TICs. Furthermore, in vivo xenograft studies indicated that CD44 knockdown inhibited tumorigenesis of pleomorphic adenoma. In addition, neither the CD44low cells nor the CD44-modified CD44low cells developed neo-tumors, which indicated that overexpression of CD44 did not enable the CD44low cells to be transformed into TICs. Taken together, these data demonstrate that CD44 not only acts as a biomarker, but also functions as a key player in the tumor-initiating capacity of TICs. These results shed light on the pathogenesis of salivary gland tumors and provide a potential therapeutic target for treating pleomorphic adenoma.

VDR in salivary gland homeostasis and cancer

The vitamin D receptor (VDR) and its ligand 1,25(OH)2D3 (1,25D) impact differentiation and exert anti-tumor effects in many tissues, but its role in salivary gland has yet to be defined. Using immunohistochemistry (IHC), we have detected strong VDR expression in murine and human salivary gland ducts. Compared to normal gland, VDR protein expression was retained in differentiated human pleomorphic adenoma (PA) but was undetectable in undifferentiated PA and in carcinomas, suggesting deregulation of VDR during salivary cancer progression. To gain insight into the potential role of VDR in salivary cancer, we assessed the effects of vitamin D in vivo and in vitro. Despite the presence of VDR in salivary gland, chronic dietary vitamin D restriction did not alter morphology of the salivary epithelium in C57/Bl6 mice. The localization of VDR in ductal epithelium prompted us to examine the effects of 1,25D in an established cell line (mSGc) derived from normal murine submandibular gland (SMG). This previously characterized cell line consists of multiple stem, progenitor and differentiated cell types as determined by mutually exclusive cellular expression of basal, ductal and myoepithelial markers. We demonstrated VDR expression and regulation of VDR target genes Vdr and Postn by 1,25D in mSGc, indicating functional ligand-mediated transcriptional activity. The effect of VDR signaling on epithelial differentiation markers was assessed by qPCR and IHC in mSGc cells treated with 1,25D. We found that 1,25D reduced mRNA expression of the basal cell progenitor marker keratin 5 (K5) and increased expression of the differentiated ductal cell marker keratin 7 (K7). Further, we found that 1,25D significantly decreased the number of proliferating cells, including proliferating K5+ cells. Characterization of cell cycle by Muse cytometry indicated 1,25D treatment decreased cells in S, G2, and M phase. The inhibition of K5+ cell proliferation by 1,25D is of particular interest because K5+ basal cells contribute to a wide variety of salivary tumor types. Our studies suggest that 1,25D alters cancer-relevant progenitor and differentiation markers in the salivary gland.

Regional Differences following Partial Salivary Gland Resection

Regenerative medicine aims to repair, replace, or restore function to tissues damaged by aging, disease, or injury. Partial organ resection is not only a common clinical approach in cancer therapy but also an experimental injury model used to examine mechanisms of regeneration and repair in organs. We performed a partial resection, or partial sialoadenectomy, in the female murine submandibular salivary gland (SMG) to establish a model for investigation of repair mechanisms in salivary glands (SGs). After partial sialoadenectomy, we performed whole-gland measurements over a period of 56 d and found that the gland increased slightly in size. We used microarray analysis and immunohistochemistry (IHC) to examine messenger RNA and protein changes in glands over time. Microarray analysis identified dynamic changes in the transcriptome 3 d after injury that were largely resolved by day 14. At the 3-d time point, we detected gene signatures for cell cycle regulation, inflammatory/repair response, and extracellular matrix (ECM) remodeling in the partially resected glands. Using quantitative IHC, we identified a transient proliferative response throughout the gland. Both secretory epithelial and stromal cells expressed Ki67 that was detectable at day 3 and largely resolved by day 14. IHC also revealed that while most of the gland underwent a wound-healing response that resolved by day 14, a small region of the gland showed an aberrant sustained fibrotic response characterized by increased levels of ECM deposition, sustained Ki67 levels in stromal cells, and a persistent M2 macrophage response through day 56. The partial submandibular salivary gland resection model provides an opportunity to examine a normal healing response and an aberrant fibrotic response within the same gland to uncover mechanisms that prevent wound healing and regeneration in mammals. Understanding regional differences in the wound-healing responses may ultimately affect regenerative therapies for patients.

Natriuretic peptide system expression in murine and human submandibular salivary glands: a study of the spatial localisation of ANB, BNP, CNP and their receptors

The natriuretic peptide (NP) system comprises of three ligands, the Atrial Natriuretic Peptide (ANP), Brain Natriuretic peptide (BNP) and C-type Natriuretic peptide (CNP), and three natriuretic peptide receptors, NPRA, NPRB and NPRC. Here we present a comprehensive study of the natriuretic peptide system in healthy murine and human submandibular salivary glands (SMGs). We show CNP is the dominant NP in mouse and human SMG and is expressed together with NP receptors in ducts, autonomic nerves and the microvasculature of the gland, suggesting CNP autocrine signalling may take place in some of these glandular structures. These data suggest the NP system may control salivary gland function during homeostasis through the regulation of electrolyte re-absorption, neural stimulation and/or blood vessel wall contraction/relaxation. We also show abnormal expression of NPRA in the stroma of a subset of human SMGs resected from patients diagnosed with oral squamous cell carcinoma (OSCC) of non-salivary gland origin. This finding warrants further research to investigate a possible correlation between early OSCC invasion and NPRA overexpression.

Establishment of a Murine Pro-acinar Cell Line to Characterize Roles for FGF2 and \u03b13\u03b21 Integrins in Regulating Pro-acinar Characteristics

Radiation therapy for head and neck cancers results in permanent damage to the saliva producing acinar compartment of the salivary gland. To date, a pure pro-acinar cell line to study underlying mechanisms of acinar cell differentiation in culture has not been described. Here, we report the establishment of a pro-acinar (mSG-PAC1) and ductal (mSG-DUC1) cell line, from the murine submandibular salivary gland (SMG), which recapitulate developmental milestones in differentiation. mSG-DUC1 cells express the ductal markers, keratin-7 and keratin-19, and form lumenized spheroids. mSG-PAC1 cells express the pro-acinar markers SOX10 and aquaporin-5. Using the mSG-PAC1 cell line, we demonstrate that FGF2 regulates specific steps during acinar cell maturation. FGF2 up-regulates aquaporin-5 and the expression of the α3 and α6 subunits of the α3β1 and α6β1 integrins that are known to promote SMG morphogenesis and differentiation. mSG-DUC1 and mSG-PAC1 cells were derived from genetically modified mice, homozygous for floxed alleles of the integrin α3 subunit. Similar to SMGs from α3-null mice, deletion of α3 alleles in mSG-PAC1 cells results in the up-regulation of E-cadherin and the down-regulation of CDC42. Our data indicate that mSG-DUC1 and mSG-PAC1 cells will serve as important tools to gain mechanistic insight into salivary gland morphogenesis and differentiation.

Dual Sympathetic Input into Developing Salivary Glands

Neuronal signaling is known to be required for salivary gland development, with parasympathetic nerves interacting with the surrounding tissues from early stages to maintain a progenitor cell population and control morphogenesis. In contrast, postganglionic sympathetic nerves arrive late in salivary gland development to perform a secretory function; however, no previous report has shown their role during development. Here, we show that a subset of neuronal cells within the parasympathetic submandibular ganglion (PSG) express the catecholaminergic marker tyrosine hydroxylase (TH) in developing murine and human submandibular glands. This sympathetic phenotype coincided with the expression of transcription factor Hand2 within the PSG from the bud stage (E12.5) of mouse embryonic salivary gland development. Hand2 was previously associated with the decision of neural crest cells to become sympathetic in other systems, suggesting a role in controlling neuronal fate in the salivary gland. The PSG therefore provides a population of TH-expressing neurons prior to the arrival of the postganglionic sympathetic axons from the superior cervical ganglion at E15.5. In culture, in the absence of nerves from the superior cervical ganglion, these PSG-derived TH neurons were clearly evident forming a network around the gland. Chemical ablation of dopamine receptors in explant culture with the neurotoxin 6-hydroxydopamine at early stages of gland development resulted in specific loss of the TH-positive neurons from the PSG, and subsequent branching was inhibited. Taken altogether, these results highlight for the first time the detailed developmental time course of TH-expressing neurons during murine salivary gland development and suggest a role for these neurons in branching morphogenesis.

Short-term RANKL exposure initiates a neoplastic transcriptional program in the basal epithelium of the murine salivary gland

Although salivary gland cancers comprise only ∼3–6% of head and neck cancers, treatment options for patients with advanced-stage disease are limited. Because of their rarity, salivary gland malignancies are understudied compared to other exocrine tissue cancers. The comparative lack of progress in this cancer field is particularly evident when it comes to our incomplete understanding of the key molecular signals that are causal for the development and/or progression of salivary gland cancers. Using a novel conditional transgenic mouse (K5:RANKL), we demonstrate that Receptor Activator of NFkB Ligand (RANKL) targeted to cytokeratin 5-positive basal epithelial cells of the salivary gland causes aggressive tumorigenesis within a short period of RANKL exposure. Genome-wide transcriptomic analysis reveals that RANKL markedly increases the expression levels of numerous gene families involved in cellular proliferation, migration, and intra- and extra-tumoral communication. Importantly, cross-species comparison of the K5:RANKL transcriptomic dataset with The Cancer Genome Atlas cancer signatures reveals the strongest molecular similarity with cancer subtypes of the human head and neck squamous cell carcinoma. These studies not only provide a much needed transcriptomic resource to mine for novel molecular targets for therapy and/or diagnosis but validates the K5:RANKL transgenic as a preclinical model to further investigate the in vivo oncogenic role of RANKL signaling in salivary gland tumorigenesis.

Neurturin-containing laminin matrices support innervated branching epithelium from adult epithelial salispheres

Cell transplantation of autologous adult biopsies, grown ex vivo as epithelial organoids or expanded as spheroids, are proposed treatments to regenerate damaged branching organs. However, it is not clear whether transplantation of adult organoids or spheroids alone is sufficient to initiate a fetal-like program of branching morphogenesis in which coordinated branching of multiple cell types including nerves, mesenchyme and blood vessels occurs. Yet this is an essential concept for the regeneration of branching organs such as lung, pancreas, and lacrimal and salivary glands. Here, we used factors identified from fetal organogenesis to maintain and expand adult murine and human epithelial salivary gland progenitors in non-adherent spheroid cultures, called salispheres. These factors stimulated critical developmental pathways, and increased expression of epithelial progenitor markers such as Keratin5, Keratin14, FGFR2b and KIT. Moreover, physical recombination of adult salispheres in a laminin-111 extracellular matrix with fetal salivary mesenchyme, containing endothelial and neuronal cells, only induced branching morphogenesis when neurturin, a neurotrophic factor, was added to the matrix. Neurturin was essential to improve neuronal survival, axon outgrowth, innervation of the salispheres, and resulted in the formation of branching structures with a proximal-distal axis that mimicked fetal branching morphogenesis, thus recapitulating organogenesis. Epithelial progenitors were also maintained, and developmental differentiation programs were initiated, showing that the fetal microenvironment provides a template for adult epithelial progenitors to initiate branching and differentiation. Further delineation of secreted and physical cues from the fetal niche will be useful to develop novel regenerative therapies that instruct adult salispheres to resume a developmental-like program in vitro and to regenerate branching organs in vivo.

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics.

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Sexual dimorphisms in the transcriptomes of murine salivary glands.

Transcriptional profiling identified 933 sexually dimorphic genes out of the 14 371 protein‐coding genes expressed in the three major murine salivary glands: parotid, sublingual, and submandibular. Most (89%) sex‐specific genes were enriched in a single gland, while only 0.5% of the sexually dimorphic genes were enriched in all glands. The sublingual gland displayed a strong male sex bias (94% of sex‐enriched genes), while a sex preference was not obvious in the parotid or submandibular glands. A subset of transcription factor genes was correlated with the expression of gland‐specific, sex‐enriched genes. Higher expression of Cftr chloride and Scnn1 sodium channels in the male submandibular correlated with greater NaCl reabsorption. In conclusion, adult salivary glands display sex‐ and gland‐specific differences in gene expression that reflect their unique functional properties.

Induction of squamous cell carcinoma after MAP3K8 overexpression in murine salivry gland epithelial cells.

Salivary gland neoplasms are relatively rare and comprise only 1%-4% of all human neoplasms. Salivary gland neoplasms also show an extremely wide range of morphological diversity. Currently, the genetic alterations and corresponding molecular mechanisms underlying salivary gland neoplasms development remain largely unknown. We generated an inducible Tet-MAP3K8::MMTV-rTA mouse model by crossing the MAP3K8 transgenic mice with MMTR-rTA transgenic mice to express MAP3K8 in the salivary gland. MAP3K8 overexpression in the murine salivary glands of Tet-MAP3K8::MMTR-rTA transgenic mice induces tumorigenesis. Pathological investigations reveal partial fibrosis and adenosis of salivary glands, and foci of atypical squamoid cellular proliferation, which represent invasive squamous cell carcinoma (SCC). MAP3K8 overexpression is associated with SCC development in murine salivary glands. It provides an in vivo framework for the understanding of molecular mechanisms underlying SCC development in the salivary glands and also for the development of a future therapeutic strategy targeting this tumor type.

3D Organoid Formation from the Murine Salivary Gland Cell Line SIMS.

Salivary glands consist of multiple phenotypically and functionally unique cell populations, such as the acinar, ductal, and myoepithelial cells that help produce, modify, and secrete saliva (Lombaert et al., 2011). Identification of mechanisms and factors that regulate these populations has been of key interest, as salivary gland-related diseases have detrimental effects on these cell populations. A variety of approaches have been used to understand the roles different signaling mechanisms and transcription factors play in regulating salivary gland development and homeostasis. Differentiation assays have been performed with primary salivary cells in the past (Maimets et al., 2016), however this approach may sometimes be limiting due to tissue availability, labor intensity of processing the tissue samples, and/or inability to long-term passage the cells. Here we describe in detail a 3D differentiation assay to analyze the differentiation potential of a salivary gland cell line, SIMS, which was immortalized from an adult mouse submandibular salivary gland (Laoide et al., 1996). SIMS cells express cytokeratin 7 and 19, which is characteristic for a ductal cell type. Although adult acinar and myoepithelial cells were found in vivo to preserve their own cell population through self-duplication (Aure et al., 2015; Song et al. 2018), in some cases duct cells can differentiate into acinar cells in vivo, such as after radiation injury (Lombaert et al., 2008; Weng et al., 2018). Thus, utilization of SIMS cells allows us to target and analyze the self-renewal and differentiation effects of ductal cells under specific in vitro controlled conditions.

Lack of DNA Damage Response at Low Radiation Doses in Adult Stem Cells Contributes to Organ Dysfunction.

Radiotherapy for head and neck cancer may result in serious side effects, such as hyposalivation, impairing the patient's quality of life. Modern radiotherapy techniques attempt to reduce the dose to salivary glands, which, however, results in low-dose irradiation of the tissue stem cells. Here we assess the low-dose sensitivity of tissue stem cells and the consequences for tissue function. Postirradiation rat salivary gland secretory function was determined after pilocarpine induction. Murine and patient-derived salivary gland and thyroid gland organoids were irradiated and clonogenic survival was assessed. The DNA damage response (DDR) was analyzed in organoids and modulated using different radiation modalities, chemical inhibition, and genetic modification. Relative low-dose irradiation to the high-density stem cell region of rat salivary gland disproportionally impaired function. Hyper-radiosensitivity at doses <1 Gy, followed by relative radioresistance at doses ≥1 Gy, was observed in salivary gland and thyroid gland organoid cultures. DDR modulation resulted in diminished, or even abrogated, relative radioresistance. Furthermore, inhibition of the DDR protein ATM impaired DNA repair after 1 Gy, but not 0.25 Gy. Irradiation of patient-derived salivary gland organoid cells showed similar responses, whereas a single 1 Gy dose to salivary gland-derived stem cells resulted in greater survival than clinically relevant fractionated doses of 4 × 0.25 Gy. We show that murine and human glandular tissue stem cells exhibit a dose threshold in DDR activation, resulting in low-dose hyper-radiosensitivity, with clinical implications in radiotherapy treatment planning. Furthermore, our results from patient-derived organoids highlight the potential of organoids to study normal tissue responses to radiation.

Salivary gland cell aggregates are derived from self-organization of acinar lineage cells

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.

Cooperation Between Pten and Smad4 in Murine Salivary Gland Tumor Formation and Progression

Salivary gland tumor (SGT) is a rare tumor type, which exhibits broad-spectrum phenotypic, biological, and clinical heterogeneity. Currently, the molecular mechanisms that cause SGT pathogenesis remain poorly understood. A lack of animal models that faithfully recapitulate the naturally occurring process of human SGTs has hampered research progress on this field. In this report, we developed an inducible keratin 5-driven conditional knockout mouse model to delete gene(s) of interest in murine salivary gland upon local RU486 delivery. We have deleted two major tumor suppressors, Pten, a negative regulator of the PI3K pathway, and Smad4, the central signaling mediator of TGFβ pathway, in the murine salivary gland. Our results have shown that deletion of either Pten or Smad4 in murine salivary gland resulted in pleomorphic adenomas, the most common tumor in human SGT patients. Deletion of both Pten and Smad4 in murine salivary gland developed several malignancies, with salivary adenoid cystic carcinoma (SACC) being the most frequently seen. Molecular characterization showed that SACC exhibited mTOR activation and TGFβ1 overexpression. Examination of human SGT clinical samples revealed that loss of Pten and Smad4 is common in human SACC samples, particularly in the most aggressive solid form, and is correlated with survival of SACC patients, highlighting the human relevance of the murine models. In summary, our results offer significant insight into synergistic role of Pten and Smad4 in SGT, providing a rationale for targeting mTOR and/or TGFβ signaling to control SGT formation and progression.

Aldehyde dehydrogenase 3A1 activation prevents radiation-induced xerostomia by protecting salivary stem cells from toxic aldehydes

Xerostomia (dry mouth) is the most common side effect of radiation therapy in patients with head and neck cancer and causes difficulty speaking and swallowing. Since aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in mouse salivary stem/progenitor cells (SSPCs), we sought to determine the role of ALDH3A1 in SSPCs using genetic loss-of-function and pharmacologic gain-of-function studies. Using DarkZone dye to measure intracellular aldehydes, we observed higher aldehyde accumulation in irradiated Aldh3a1-/- adult murine salisphere cells and in situ in whole murine embryonic salivary glands enriched in SSPCs compared with wild-type glands. To identify a safe ALDH3A1 activator for potential clinical testing, we screened a traditional Chinese medicine library and isolated d-limonene, commonly used as a food-flavoring agent, as a single constituent activator. ALDH3A1 activation by d-limonene significantly reduced aldehyde accumulation in SSPCs and whole embryonic glands, increased sphere-forming ability, decreased apoptosis, and improved submandibular gland structure and function in vivo after radiation. A phase 0 study in patients with salivary gland tumors showed effective delivery of d-limonene into human salivary glands following daily oral dosing. Given its safety and bioavailability, d-limonene may be a good clinical candidate for mitigating xerostomia in patients with head and neck cancer receiving radiation therapy.

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy.

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.