Salivary Gland Tissue Research Articles

Novel Cross-Species Salivary Gland-Parasympathetic Neuron Coculture System.

The parasympathetic nervous system is essential for salivary gland development and functionality. Parasympathetic neuron (parasymN) innervation is the main neural network that controls salivary secretion. Therefore, an exclusive model to study parasympathetic neurons and salivary gland tissue circuitry will significantly improve the understanding of the role of parasymN activation on salivary regulation. Harvesting primary rodent parasymNs is challenging due to their body-wide disbursed location. Similarly, the salivary glands are distributed in various locations around and within the oral cavity. Here, we present a coculture model system using human pluripotent stem cell (hPSC)-derived parasymNs and primary mouse von Ebner's gland cells. We previously reported the first protocol to robustly generate human parasymNs from hPSCs through the Schwann cell precursor (SCP) lineage. The hPSC-parasymNs are functional and have been applied to model several autonomic disorders. We also used a Sox10-Cre::tdTomato (hereafter referred to as RFP) reporter mouse line, which labeled von Ebner's glands, a type of minor salivary gland connected to the trough of circumvallate and foliate taste papillae. This labeling allowed for visualization and efficient isolation of primary tissues in young adult mice (8-10 weeks). By coculturing the two tissues, human parasymNs control mouse salivary gland cell growth and activation. Both parasymNs and primary salivary gland cells can be frozen and stocked at early stages of differentiation and isolation, making applications easier. This novel coculture model system could also be used to model and study related human diseases in the future, such as dry mouth syndrome. Key features • Differentiation of human parasymNs from hPSCs. • Dissecting RFP+ mouse Ebner's gland tissues for primary culture. • Protocol to coculture human parasymNs with mouse primary salivary gland cells. • Allows developmental and functional assessments of salivary regulation by the parasympathetic nervous system.

Evaluation of immune microenvironment in IgG4-related sialadenitis.

IgG4-related sialadenitis is a systemic autoimmune disease that can lead to fibro-inflammatory conditions. This study aims to investigate the immune microenvironment and potential signaling pathways associated with IgG4-related sialadenitis. Datasets related to IgG4-related sialadenitis were retrieved from the GEO database. Immune cell infiltration analysis was conducted using the Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) method. Differentially immune-related expressed genes (DIEG) and immune-related functional enrichment were identified. Moreover, potential treatment targets for IgG4-related sialadenitis were predicted using The Connectivity Map. Only two datasets from GEO were included for further analysis. The CIBERSORT results indicated dominant immune cell populations in IgG4-related sialadenitis, including CD8+ T cells, resting NK cells, monocytes, and naïve B cells in peripheral blood mononuclear cells. Additionally, high abundance of plasma cells was observed in labial salivary gland tissues. Furthermore, a total of 42 DIEGs were identified, with tumor necrosis factor (TNF) signaling via the NF-Kappa B signaling pathway and the p53 signaling pathway being highly enriched. Autophagy inhibitors and DNA topoisomerase inhibitors were strongly associated with potential targets for the treatment of IgG4-related sialadenitis (P<0.05). This study reveals altered immune microenvironment in peripheral blood mononuclear cells and labial salivary gland tissues in IgG4-related sialadenitis. Autophagy inhibitors and DNA topoisomerase inhibitors show promise as potential targets for treating IgG4-related sialadenitis, providing a novel therapeutic strategy for this disease.

Differentiation of Salivary Gland and Salivary Gland Tumor Tissue via Raman Imaging Combined with Multivariate Data Analysis.

Salivary gland tumors (SGTs) are a relevant, highly diverse subgroup of head and neck tumors whose entity determination can be difficult. Confocal Raman imaging in combination with multivariate data analysis may possibly support their correct classification. For the analysis of the translational potential of Raman imaging in SGT determination, a multi-stage evaluation process is necessary. By measuring a sample set of Warthin tumor, pleomorphic adenoma and non-tumor salivary gland tissue, Raman data were obtained and a thorough Raman band analysis was performed. This evaluation revealed highly overlapping Raman patterns with only minor spectral differences. Consequently, a principal component analysis (PCA) was calculated and further combined with a discriminant analysis (DA) to enable the best possible distinction. The PCA-DA model was characterized by accuracy, sensitivity, selectivity and precision values above 90% and validated by predicting model-unknown Raman spectra, of which 93% were classified correctly. Thus, we state our PCA-DA to be suitable for parotid tumor and non-salivary salivary gland tissue discrimination and prediction. For evaluation of the translational potential, further validation steps are necessary.

Title Expression of FOXO3 and MAPK1 Genes in Patients with Benign Salivary Gland Tumors.

Pleomorphic adenomas (PAs) and Warthin tumors (WTs) are the most common benign tumors that occur in the salivary gland. PA has a tendency towards malignant transformation. Thus, searching for new methods to diagnose salivary gland tumors and treatment is important. The members of the class O forehead box transcription factor (FOXO3) and mitogen-activated protein kinase 1 (MAPK1) genes participate in the cellular processes, including in cell proliferation. The aim of this study was to analyze these genes' expression in the salivary gland tissues and in salivary gland tumors. The study group consisted of 50 patients treated for salivary gland tumors. For genetic tests, fresh samples of tissue collected during the surgery were used. The expression levels of the FOXO3 and MAPK1 genes were statistically significantly lower in PA tissue than in normal salivary gland tissue and WT tissue. This research revealed that the FOXO3 and MAPK1 genes are present in benign salivary gland tumors and also indicated a role of these genes in the development of benign salivary gland tumors. The cause of the development of pleomorphic adenomas may be apoptotic disorder and the activation of the inflammatory process. The examined genes may have potential to be new therapeutic targets for the treatment of pleomorphic adenomas.

Immunohistochemical Expression of Estrogen Receptors in Pleomorphic Adenoma and Normal Salivary Gland Tissue

Background: The expression of estrogen receptors (ERs) in salivary gland tissue has been observed in some studies. Considering the histological similarity of salivary glands and breast tissue, as well as the prominent role of this receptor in the pathogenesis, treatment, and prognosis of breast tumors, this study aimed to compare the immunohistochemical expression of ERs in pleomorphic adenoma (PA) and normal salivary gland tissue and assess its possible role in salivary gland tumors. Methods: In this descriptive-analytical study, 26 samples of PA (16 females, and 10 males) and 12 samples of normal salivary gland tissue were selected. Immunohistochemical staining was performed by the standard EnVision method for ERs. The results were evaluated semi-quantitively as the percentage of the nuclear and cytoplasmic staining were analyzed independently by Mann-Whitney, Kruskal-Wallis, and Fisher’s exact tests. Results: All PA samples showed negative nuclear staining for ERs. ER expression was observed in the cytoplasm of the ducts in 27% of tumors and 59% of normal salivary gland tissue samples, but the difference was not statistically significant. In addition, no association was found between ER expression and independent variables, such as age, sex, the type of stroma, or the degree of cellularity in PA, and the location of the specimen. Conclusion: It seems that the expression of ERs does not play an effective role in the development and progression of PA. However, its occurrence in the normal salivary gland ducts is considerable, and further studies in this field seem to be necessary.

Transcriptomic profiling and the first spatial expression analysis of candidate genes in the salivary gland of the East Asian medicinal leech, Hirudo nipponia

Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors—i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin—as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.

Salivary Gland Bioengineering.

Salivary gland dysfunction affects millions globally, and tissue engineering may provide a promising therapeutic avenue. This review delves into the current state of salivary gland tissue engineering research, starting with a study of normal salivary gland development and function. It discusses the impact of fibrosis and cellular senescence on salivary gland pathologies. A diverse range of cells suitable for tissue engineering including cell lines, primary salivary gland cells, and stem cells are examined. Moreover, the paper explores various supportive biomaterials and scaffold fabrication methodologies that enhance salivary gland cell survival, differentiation, and engraftment. Innovative engineering strategies for the improvement of vascularization, innervation, and engraftment of engineered salivary gland tissue, including bioprinting, microfluidic hydrogels, mesh electronics, and nanoparticles, are also evaluated. This review underscores the promising potential of this research field for the treatment of salivary gland dysfunction and suggests directions for future exploration.

Secretagogue effect of PDE4 inhibitor apremilast on human salivary gland organoids obtained from primary Sjögren's syndrome patients.

The aim of the study was to culture vital salivary gland organoids obtained through labial or parotid biopsy of primary Sjögren's syndrome (pSS) patients in order to evaluate their morphological and functional features in basal condition and after stimulation with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) activator forskolin and phosphodiesterase 4 (PDE4) inhibitor apremilast, their in vitro regenerative capacity and the immune-histological resemblance with original tissue. Salivary gland tissues from five pSS patients were processed to obtain vital organoids; swelling assay and cell proliferation tests were performed after forskolin and apremilast application. Immunochemistry evaluation on original salivary gland tissue and corresponding organoids was performed, and secretomics analysis was conducted to assess their functional status. After application of forskolin and apremilast, we observed organoid swelling after 30 minutes, compatible with a positive functional status and enhancement of saliva production. In 3 cases, apremilast induced organoid proliferation. All cases were positive for cytokeratin 14 (CK14) and most for cytokeratin 5 (CK5). All the cases were positive for amylase; its secretion, and thus functional status of organoids, was confirmed by its high concentration in the culture medium. A focal ductal differentiation was found in some cases, highlighted by epithelial membrane antigen (EMA) positivity. The more differentiated EMA positive areas were negative for the staminal marker CK14, showing a sort of "complementary staining". Our data highlighted that differentiated cells and vital functional organoids that recapitulate the development of original salivary glands can be obtained from pSS epithelium. For the first time, the direct stimulating effect of PDE4 inhibitor apremilast on pSS human salivary gland organoids is reported, opening new perspectives on targeting oral dryness with drugs that combine secretagogue and immunomodulatory effects.

Ehrlichia chaffeensis Co-Opts Phagocytic Hemocytes for Systemic Dissemination in the Lone Star Tick, Amblyomma americanum

Introduction: Hematophagous arthropods can acquire and transmit several pathogens of medical importance. In ticks, the innate immune system is crucial in the outcome between vector-pathogen interaction and overall vector competence. However, the specific immune response(s) elicited by the immune cells known as hemocytes remains largely undefined in Ehrlichia chaffeensis and its competent tick vector, Amblyomma americanum. Methods: We utilized injection of clodronate liposome to deplete tick granulocytes combined with infection with E. chaffeensis to demonstrate their essential role in microbial infection. Results: Here, we show that granulocytes, professional phagocytic cells, are integral in eliciting immune responses against commensal and pathogen infection. The chemical depletion of granulocytes led to decreased phagocytic efficiency of tissue-associated hemocytes. We demonstrate that E. chaffeensis can infect circulating hemocytes, and both cell-free plasma and hemocytes from E. chaffeensis-infected ticks can establish Ehrlichia infection in recipient ticks. Lastly, we provide evidence to show that granulocytes play a dual role in E. chaffeensis infection. Depleting granulocytic hemocytes increased Ehrlichia load in the salivary gland and midgut tissues. In contrast, granulocyte depletion led to a reduced systemic load of Ehrlichia. Conclusion: This study has identified multiple roles for granulocytic hemocytes in the control and systemic dissemination of E. chaffeensis infection.

Anti-LGALS3BP antibody-drug conjugate treatment induces durable and potent antitumor response in a preclinical model of adenoid cystic carcinoma

ObjectivesAdenoid cystic carcinoma (ACC) is a rare type of cancer that typically arises from glandular tissues, most commonly in the salivary glands. Although relatively rare, it represents a serious clinical issue as the management of the disease is highly complex being the only therapeutic options represented by invasive surgery and/or radiotherapy. In the present study, we have explored the potential of galectin-3 binding protein (LGALS3BP) as a novel target for antibody-drug conjugate (ADC) therapy in ACC. Materials and methodsRNAseq was conducted on a panel of 10 ACC patient-derived xenografts (PDX)s tissues and 6 normal salivary glands to analyze LGALS3BP gene expression. Protein expression was assessed in ACC PDX and primary tumor tissues using immunohistochemistry. Anti-LGALS3BP ADC named 1959-sss/DM4, was tested in high LGALS3BP expressing ACC PDX model ST1502B. ResultsRNAseq analysis revealed that LGALS3BP expression was highly expressed in ACC PDX tissues compared to normal salivary gland tissues. As evaluated by immunohistochemical analysis, LGALS3BP protein was found to be heterogeneously expressed in 10 ACC PDX and in tumor tissues derived from a cohort of 37 ACC patients. Further, treatment with 1959-sss/DM4 ADC led to durable tumor growth inhibition (TGI) in 100% of animals without observed toxicity. ConclusionsOur study provides strong evidence that LGALS3BP is a promising therapeutic target for ACC, warranting further expedited preclinical and clinical investigation.

Expression of Ubiquitin C-Terminal Hydrolase-L1 in Minor Normal Salivary Gland Tissues in Perilesional Area to OSCC

Ubiquitin C-terminal hydrolase L1 (UCH-L1), is a deubiquitinating enzyme. Certain changes in ubiquitination attribute to uncontrolled growth, leading to oncogenesis. The aim of this study was to characterize UCH-L1 expression patterns in nor mal looking human minor salivary gland tissues in the perilesional area of oral squamous cell carcinoma (OSCC). A retrospective study in which UCH-L1 immunohistochemical staining performed for 27 formalin fixed paraffin embedded blocks of surgically removed primary OSCC containing perilesional minor salivary gland tissues. The expression, subcellular localization, pattern of distribution, and stain intensity were assessed. UCH-L1 was immunopositive in minor salivary gland tissues of all sections. The immunoreactivity was (70.4%) detected in the cytoplasm and (29.6%) as mixed expression (cytoplasm and nucleus). UCH-L1 was predominately expressed in the ductal system (40.7%), followed by the myoepithelial cells (33.35%), while it was weakly expressed in the acinar cells; mucous (14.9%) and serous acini (11.1%). UCH-L1 expression is detectable in minor salivary glands in a perilesional area of OSCC, Although previously approved that it was absolutely absent in normal salivary gland tissues, it may activate during the course of oncogenesis and can be classified as a marker for predicting the future invasion of OSCC to adjacent minor salivary gland tissues. This study provides evidence for additional studies on the UCH-L1 expression in normal salivary glands in patients with maxillofacial tumorigenesis.

Metabolomics analysis of pathways underlying radiation-induced salivary gland dysfunction stages.

Salivary gland hypofunction is an adverse side effect associated with radiotherapy for head and neck cancer patients. This study delineated metabolic changes at acute, intermediate, and chronic radiation damage response stages in mouse salivary glands following a single 5 Gy dose. Ultra-high performance liquid chromatography-mass spectrometry was performed on parotid salivary gland tissue collected at 3, 14, and 30 days following radiation (IR). Pathway enrichment analysis, network analysis based on metabolite structural similarity, and network analysis based on metabolite abundance correlations were used to incorporate both metabolite levels and structural annotation. The greatest number of enriched pathways are observed at 3 days and the lowest at 30 days following radiation. Amino acid metabolism pathways, glutathione metabolism, and central carbon metabolism in cancer are enriched at all radiation time points across different analytical methods. This study suggests that glutathione and central carbon metabolism in cancer may be important pathways in the unresolved effect of radiation treatment.

Extracellular vesicles of iPS cells highly capable of producing HGF and TGF-β1 can attenuate Sjögren's syndrome via innate immunity regulation

Previous studies have demonstrated that extracellular vesicles (EVs) from dental pulp stem cells (DPSCs), which release abundant hepatocyte growth factor (HGF) and transforming growth factor-β1 (TGF-β1), contribute to the pathogenesis of Sjögren's syndrome (SS). However, depending on the condition of DPSCs, this effect is often not achieved. In this study, we established induced pluripotent stem (iPS) cells highly capable of releasing HGF and TGF-β1 and iPS cells barely capable of releasing them, and administered each EV to SS model mice to see if there was a difference in therapeutic effect. EVs were collected from each iPS cell and their characteristics and shapes were examined. When they were administered to SS model mice, the EVs from iPS cells with higher concentrations of HGF and TGF-β1 showed significantly reduced inflammatory cell infiltration in salivary gland tissues, increased saliva volume, and decreased anti-SS-A and anti-SS-B antibodies. A comprehensive search of microRNA arrays for differences among those EVs revealed that EVs from iPS cells with higher concentrations of HGF and TGF-β1 contained more of the let-7 family. Thereafter, we examined the expression of toll-like receptors (TLRs), which are said to be regulated by the let-7 family, by qPCR, and found decreased TLR4 expression. Focusing on MAPK, a downstream signaling pathway, we examined cytokine concentrations in mouse macrophage culture supernatants and Western blotting of murine splenic tissues and found higher concentrations of anti-inflammatory cytokines in the EVs-treated group and decreased TLR4, NF-κB and phosphorylation (p)-p-38 MAPK expression by Western blotting. Alternatively, p-Smad2/3 was upregulated in the EVs-treated group. Our findings suggest that the let-7 family in EVs may suppress the expression of TLR4 and NF-κB, which may be involved in the suppression of MAPK-mediated pro-inflammatory cytokine production.

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging.

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration. The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Hong Kong Guangdong Rheumatology Meeting

Sjögren’s syndrome is a chronic autoimmune disease that is estimated to affect 35 million people worldwide. Hallmarks of the disease are a loss of salivary and lacrimal gland function as well as lymphocytic infiltration, elevated proinflammatory cytokines, and circulating autoantibodies. The effective treatments are limited. However, new therapies targeting specific immune pathways associated with Sjögren’s syndrome are being developed. Gene transfer to the salivary glands has been considered a promising approach to treating the dysfunction. The application of this treatment on salivary gland injuries has been studied for decades, yet its clinical progress is delayed. This chapter provides a coup d’oeil into gene transfer methods and various gene/vector types for salivary glands. AAV2-LAMP3-treated mice developed an SS-like phenotype with progressive salivary hypofunction and autoantibody production. To confirm the role of TLR4 in the induction of BMP6 following stimulation, AAV2-LAMP3–treated mice with established salivary hypofunction were treated i.p. with the TLR4 antagonist TAK242 for 10 days, and the effect of the drug on salivary gland protein expression and function was tested. We observed that treatment with TAK242 significantly decreased BMP6 expression and increased AQP5 expression in the salivary gland tissues. In agreement with this observation, the saliva flow rate of AAV2-LAMP3–treated mice also increased compared with that in vehicle control–treated mice. Gene therapy using recombinant viruses as gene transfer vectors to deliver a water channel (aquaporin 1, AQP1) to restore membrane water permeability in the salivary gland was shown to be safe and resulted in some patients with radiation-induced xerostomia having a sustained increase in saliva flow. A clinical trial using AAV2 to transfer AQP1 to the salivary glands of radiation-induced xerostomia patients is ongoing (NCT02446249) and results from this study may support application of this treatment in Sjögren’s syndrome. Gene therapy could also be used to deliver immunomodulators locally to the salivary gland resulting in a higher local concentration within the gland that could deliver therapeutics levels of recombinant protein, while minimizing the side effects associated with the drugs. Another study investigated whether a neurturin-expressing adenovirus could be used for gene therapy in vivo to protect parasympathetic neurons and prevent gland hypofunction after irradiation. The results suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction.

Analysis of B7-H4 Expression Across Salivary Gland Carcinomas Reveals Adenoid Cystic Carcinoma–Specific Prognostic Relevance

B7-H4 (VTCN1), a member of the B7 family, is overexpressed in several types of cancer. Here we investigated the pattern of expression of B7-H4 in salivary gland carcinomas (SGC) and assessed its potential as a prognostic marker and therapeutic target. Immunohistochemistry (IHC) analyses were performed in a cohort of 340 patient tumors, composed of 124 adenoid cystic carcinomas (ACC), 107 salivary duct carcinomas (SDC), 64 acinic cell carcinomas, 36 mucoepidermoid carcinomas (MEC), 9 secretory carcinomas (SC), as well as 20 normal salivary glands (controls). B7-H4 expression was scored and categorized into negative (<5% expression of any intensity), low (5%–70% expression of any intensity or >70% with weak intensity), or high (>70% moderate or strong diffuse intensity). The associations between B7-H4 expression and clinicopathologic characteristics, as well as overall survival, were assessed. Among all tumors, B7-H4 expression was more prevalent in ACC (94%) compared with those of SC (67%), MEC (44%), SDC (32%), and acinic cell carcinomas (0%). Normal salivary gland tissue did not express B7-H4. High expression of B7-H4 was found exclusively in ACC (27%), SDC (11%), and MEC (8%). In SDC, B7-H4 expression was associated with female gender (P = .002) and lack of androgen receptor expression (P = .012). In ACC, B7-H4 expression was significantly associated with solid histology (P < .0001) and minor salivary gland primary (P = .02). High B7-H4 expression was associated with a poorer prognosis in ACC, regardless of clinical stage and histologic subtype. B7-H4 expression was not prognostic in the non-ACC SGC evaluated. Our comparative study revealed distinct patterns of B7-H4 expression according to SGC histology, which has potential therapeutic implications. B7-H4 expression was particularly high in solid ACC and was an independent prognostic marker in this disease but not in the other SGC assessed.

Optimization of Matrigel concentration for salivary gland organoid culture

Background: Research on salivary gland tissue is important, both for maintaining good oral health and for understanding the properties and functions of secretory cells with similar developmental mechanisms and functions. However, research on the pathogenesis of salivary gland disease and the development of therapeutic agents is very insufficient, and no studies have yet reported effective approaches to changes that occur according to the concentration of Matrigel, which is used to mimic the interactions between cells and the extracellular matrix, in three-dimensional organoid culture systems.Methods: We aimed to identify the most suitable concentration of Matrigel for analyzing the morphology and differentiation patterns of organoids prepared using salivary submandibular gland-derived cells after inducing differentiation with or without a specific chemical, Y-27632.Results: In both low and high Matrigel concentration conditions, morphological differences were found between the control group and the Y-27632 treatment group, and the budding structure was also significantly higher in the Y-27632 treatment group. However, more distinct patterns of differentiation appeared in the high-concentration condition.Conclusion: The concentration of Matrigel in the 3-dimensional organoid culture system had a significant effect on mimicking the actual in vivo environment.

Injectable decellularized extracellular matrix hydrogel promotes salivary gland regeneration via endogenous stem cell recruitment and suppression of fibrogenesis.

Saliva is key to the maintenance of oral homeostasis. However, several forms of salivary gland (SG) disorders, followed by hyposalivation, often result in dental caries, oral infection, and decreased taste, which dramatically affect the quality of patient's life. Functional biomaterials hold great potential for tissue regeneration in damaged or dysfunctional SGs and maintaining the good health of oral cavity. Herein, we prepared an injectable hydrogel derived from decellularized porcine submandibular glands (pDSG-gel), the material and biological properties of the hydrogel were systematically investigated. First, good biocompatibility and bioactivities of the pDSG-gel were validated in 2D and 3D cultures of primary submandibular gland mesenchymal stem cells (SGMSCs). Especially, the pDSG-gel effectively facilitated SGMSCs migration and recruitment through the activation of PI3K/AKT signaling pathway, suggested by transcriptomic analysis and immunoblotting. Furthermore, proteomic analysis of the pDSG revealed that many extracellular matrix components and secreted factors were preserved, which may contribute to stem cell homing. The recruitment of endogenous SG cells was confirmed in vivo, upon in situ injection of the pDSG-gel into the defective SGs in rats. Acinar and ductal-like structures were evident in the injury sites after pDSG-gel treatment, suggesting the reconstruction of functional SG units. Meanwhile, histological characterizations showed that the administration of the pDSG-gel also significantly suppressed fibrogenesis within the injured SG tissues. Taken together, this tissue-specific hydrogel provides a pro-regenerative microenvironment for endogenous SG regeneration and holds great promise as a powerful and bioactive material for future treatments of SG diseases. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrix (dECM) has been acknowledged as one of the most promising biomaterials that recapitalizes the microenvironment in native tissues. Hydrogel derived from the dECM allows in situ administration for tissue repair. Herein, a tissue-specific dECM hydrogel derived from porcine salivary glands (pDSG-gel) was successfully prepared and developed for functional reconstruction of defective salivary gland (SG) tissues. The pDSG-gel effectively accelerated endogenous SG stem cells migration and their recruitment for acinar- and ductal-like regeneration, which was attributed to the activation of PI3K/AKT signaling pathway. Additionally, the introduction of the pDSG-gel resulted in highly suppressed fibrogenesis in the defective tissues. These outcomes indicated that the pDSG-gel holds great potential in clinical translation toward SG regeneration through cell-free treatments.

Histomorphological Spectrum of Juvenile Salivary Gland Swelling: A Retrospective Case Series Study

Abstract Background: Salivary gland neoplasms are a rare group of disorders with diverse morphologies and are even rarer in pediatric population. Aims and Objectives: This aimed to explore our experience with the histomorphological spectrum of salivary gland tumors in a tertiary care pediatric referral center so that it can add to the cumulative knowledge to these tumors. Materials and Methods: The histopathology records from the department of pathology from January 2012 to December 2020 were reviewed in retrospect. Perisalivary gland region lesions were excluded from the study. Results: A total of 50 cases were included in the study. The most common location for salivary gland masses was parotid region (70%). Nonneoplastic lesions were seen in 3 (6%) patients and neoplastic lesions were seen in 47 (94%) patients. The most found benign lesion of salivary gland tissue origin was pleomorphic adenoma and 10 out of 15 cases (66.7%) of malignant tumors were found to be mucoepidermoid carcinoma on histology. Conclusion: Salivary gland tumors, rare in childhood and adolescence, differ in their incidence and prognosis from those in adult patients. The percentage of malignant salivary gland tumors compared with benign is much more frequent than in adults. The parotid is the most common site, and mucoepidermoid carcinoma is the most common malignant tumor.

Abstract PO-092: Molecular characterization of the salivary adenoid cystic carcinoma tumor immune landscape by anatomic subsite

Abstract Introduction: Adenoid cystic carcinoma (AdCC) is typically indolent, however tends to behave more aggressively and present with perineural invasion and distant metastasis. Despite an improved understanding of AdCC pathobiology, the impact of anatomic tumor subsite (e.g., major versus minor salivary glands) on survival and response to treatment is relatively understudied. We recently discovered that submandibular AdCC’s exhibit unique differences in prognosis and treatment response to adjuvant radiotherapy. However, the impact of anatomic subsites on gene expression and immune cell composition, has not been investigated. Materials and methods: We used 4 publicly available AdCC molecular datasets (n = 37 AdCCs of different origin: 27 primary and 10 metastatic; 7 parotid (PG), 5 submandibular (SMG), 4 sublingual (SLG), 21 minor salivary gland (mG) and 21 normal salivary gland tissue samples). Data were harmonized between datasets using identical quantification procedures, followed by filtering and normalization performed simultaneously on the pooled data from all cohorts. Gene set enrichment analysis (GSEA) was performed Human Molecular Signatures Database (MSigDB) Hallmark and Oncologic signatures. Tumor immune microenvironment (TIME) decomposition was performed using a non-negative matrix factorization-based approach. GSEA and TIME differences between AdCC subsites were evaluated using Wilcoxon rank-sum and nonparametric equality-of-medians tests. Results: We identified different levels of enrichment for several key tumorigenic pathways between AdCCs arising within different anatomic subsites. Among AdCCs, major glands overexpressed the HALLMARK_SPERMATOGENESIS signature (parotid and sublingual glands), while the HALLMARK_REACTIVE_OXYGEN_SPECIES_PATHWAY (ROS) signature was significantly underexpressed in SMG AdCCs. In addition, the HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION (EMT) signature was significantly underexpressed in both SMG and PG AdCCs. These pathway signatures were not seen in control tissue comparison samples, indicating that these features are AdCC-specific. Additionally, TIME decomposition identified differences in CD4-T cell populations (minor &amp;gt; major gland AdCC) and natural killer (NK) cells (increased in PG and SLG). Meanwhile, normal control comparisons revealed a significant increase in plasma cells only within SM glands. Conclusions: AdCC subsites exhibit survival and treatment-response differences, and in this study demonstrate that these anatomical sites are associated with distinct molecular features. Specifically, these different anatomical sites vary in the expression of spermatogenesis, ROS, and EMT signature-related genes. Also, CD4-T and NK cell populations vary by anatomical site, suggesting that the SMG AdCC tumor-intrinsic pathway differences observed may be responsible for influencing the TIME composition and increased prognosis associated with these tumors following adjuvant radiotherapy. Validation with additional cohorts of primary AdCCs with accurate clinical annotation are required. Citation Format: Jason Tasoulas, Travis Schrank, Steven Johnson, Kimon Divaris, Stamatios Theocharis, Trevor Hackman, Siddharth Sheth, Kedar Kirtane, Juan Hernandez-Prera, Christine Chung, Wendell Gray Yarbrough, Natalia Issaeva, Antonio Amelio. Molecular characterization of the salivary adenoid cystic carcinoma tumor immune landscape by anatomic subsite [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-092.