Biofabrication, biochemical profiling, and in vitro applications of salivary gland decellularized matrices via magnetic bioassembly platforms.

Trending three-dimensional tissue engineering platforms developed via biofabrication and bioprinting of exocrine glands are on the rise due to a commitment to organogenesis principles. Nevertheless, a proper extracellular matrix (ECM) microarchitecture to harbor primary cells is yet to be established towards human salivary gland (SG) organogenesis. By using porcine submandibular gland (SMG) biopsies as a proof-of-concept to mimic the human SG, a new decellularized ECM bioassembly platform was developed herein with varying perfusions of sodium dodecyl sulfate (SDS) to limit denaturing events and ensure proper preservation of the native ECM biochemical niche. Porcine SMG biopsies were perfused with 0.01%, 0.1%, and 1% SDS and bio-assembled magnetically in porous polycarbonate track-etched (PCTE) membrane. Double-stranded DNA (dsDNA), cell removal efficiency, and ECM biochemical contents were analyzed. SDS at 0.1% and 1% efficiently removed dsDNA (< 50 ng/mg) and preserved key matrix components (sulfated glycosaminoglycans, collagens, elastin) and the microarchitecture of native SMG ECM. Bio-assembled SMG decellularized ECM (dECM) perfused with 0.1-1% SDS enhanced cell viability, proliferation, expansion confluency rates, and tethering of primary SMG cells during 7 culture days. Perfusion with 1% SDS promoted greater cell proliferation rates while 0.1% SDS supported higher acinar epithelial expression when compared to basement membrane extract and other substrates. Thus, this dECM magnetic bioassembly strategy was effective for decellularization while retaining the original ECM biochemical niche and promoting SMG cell proliferation, expansion, differentiation, and tethering. Altogether, these outcomes pave the way towards the recellularization of this novel SMG dECM in future in vitro and in vivo applications.

Identifying potential immuno-oncology targets in salivary gland mucoepidermoid carcinoma based on inflammatory status and treatment response.

BACKGROUND: Mucoepidermoid carcinoma is a rare salivary gland malignant tumour. This study aimed to investigate inflammatory and immune signatures of mucoepidermoid carcinoma by identifying potential proteo-transcriptomic biomarkers towards the development of precision immuno-oncology treatment strategies. METHODS: A total of 30 biopsies obtained from patients diagnosed with mucoepidermoid carcinoma between 2013 and 2022 were analysed after H&E staining for scoring of histological inflammatory stroma subtypes and inflammatory hotspots with QuPath. Multiplex immunofluorescence staining and NanoString nCounter PanCancer IO 360™ panel were used to assess stroma and tumour inflammation signatures in high grade mucoepidermoid carcinoma cases in the tumour microenvironment via proteomics and transcriptomics, respectively. RESULTS: Inflammatory cells within the histological inflammatory stroma inflammatory (HIS-INF/hot) tumour neighbourhoods were greater compared to the histological inflammatory stroma-immune desert (HIS-ID/cold) (p = 0.001). A similar trend was observed between treatment non-responders and responders in stroma neighbourhoods (p = 0.0625) and in stroma-to-interface inflammatory hotspots (p = 0.0081), indicating an augmented inflammatory response in hot tumours and non-responders. Furthermore, there were striking differences in the expression of pan-immune leukocyte marker CD45 between responders and non responders particularly in the tumour neighbourhoods (p = 0.0341), but such were not robust for PD-1 and macrophage fractions. Additionally, transcriptomic analysis revealed key differences in leukocyte activation profiles between responders and non-responders. CONCLUSION: This preliminary report unveils the importance of assessing immune leukocyte cellular fractions and pathways for future prognostic biomarker discoveries in mucoepidermoid carcinoma as per the involvement of CD45-driven inflammatory and immune mediators in high grade mucoepidermoid carcinoma in non-responders to treatment. These findings will potentially contribute to the development of novel personalised immunotherapies.

Medication-related osteonecrosis of the jaw: Osteoclast profile in comparison with osteoradionecrosis of the jaw and osteomyelitis of the jaw.

BACKGROUND: Medication-related osteonecrosis of the jaw (MRONJ) is a pathology condition of jaw bone caused by a side effect of medications prescribed for skeletal disease. The mechanism of MRONJ is still unknown now. Osteoclasts are cells directly influenced by the medication and the modification in cells metabolisms by the drugs lead to MRONJ. Thus, this study aimed to investigate the osteoclasts morphology, quantity, and comparing with other necrotic diseases. METHODS: Thirty-eight (38) subjects, including cases with MRONJ (n = 11), osteoradionecrosis of the jaw (n = 9), osteomyelitis of the jaw (n = 9), and normal jaw bone (n = 9), were studied. Hematoxylin-and-eosin-stained slides of these diagnosed cases were used to evaluate osteoclasts' characteristics. Immunohistochemistry of TRAP was performed to observed the function of osteoclasts. These characteristics of osteoclasts were also evaluated in the relationship with the histological features using regression analysis. RESULTS: The results showed that osteoclasts in MRONJ enhance activity by increasing the size and the quantity (p < 0.05). The presence of osteoblasts, inflammatory cells, and bacterial colonies showed a strong correlation with the change in morphology and the number of osteoclasts (p < 0.05). However, the TRAP-positive mean number and the TRAP intensity of osteoclasts in MRONJ did not show a significant difference with those in other groups (p > 0.05). CONCLUSION: In conclusion, osteoclasts in MRONJ increase the number and become bigger with multi-nuclei which might relate to the presence of osteoblasts, inflammation, and microorganisms. This finding supports the idea osteoclasts might be the main key to investigate MRONJ.

Epigallocatechin-3-Gallate Protects Pro-Acinar Epithelia Against Salivary Gland Radiation Injury.

Antioxidant agents are promising pharmaceuticals to prevent salivary gland (SG) epithelial injury from radiotherapy and their associated irreversible dry mouth symptoms. Epigallocatechin-3-gallate (EGCG) is a well-known antioxidant that can exert growth or inhibitory biological effects in normal or pathological tissues leading to disease prevention. The effects of EGCG in the various SG epithelial compartments are poorly understood during homeostasis and upon radiation (IR) injury. This study aims to: (1) determine whether EGCG can support epithelial proliferation during homeostasis; and (2) investigate what epithelial cells are protected by EGCG from IR injury. Ex vivo mouse SG were treated with EGCG from 7.5-30 µg/mL for up to 72 h. Next, SG epithelial branching morphogenesis was evaluated by bright-field microscopy, immunofluorescence, and gene expression arrays. To establish IR injury models, linear accelerator (LINAC) technologies were utilized, and radiation doses optimized. EGCG epithelial effects in these injury models were assessed using light, confocal and electron microscopy, the Griess assay, immunohistochemistry, and gene arrays. SG pretreated with EGCG 7.5 µg/mL promoted epithelial proliferation and the development of pro-acinar buds and ducts in regular homeostasis. Furthermore, EGCG increased the populations of epithelial progenitors in buds and ducts and pro-acinar cells, most probably due to its observed antioxidant activity after IR injury, which prevented epithelial apoptosis. Future studies will assess the potential for nanocarriers to increase the oral bioavailability of EGCG.

The Akt/mTOR pathway is activated in verrucous carcinoma of the oral cavity.

BACKGROUND: The Akt/mTOR pathway is activated in many malignancies, including oral squamous cell carcinoma (OSCC). However, the role of the Akt/mTOR pathway in oral verrucous carcinoma (OVC), a low-grade variant of OSCC, remains unknown. Thus, the objective of this study was to investigate the activation level of important markers of the Akt/mTOR pathway in OVC and to compare the results with OSCC samples. METHODS: The expression of p-Akt (Thr308), p-Akt (Ser473), and p-RPS6 was evaluated by immunohistochemistry in 30 OSCC cases, 18 OVC cases, and 30 control cases (normal epithelium overlying fibromas). Statistical analysis was performed to determine the differences in protein expression between samples. RESULTS: All OVC cases were positive for p-Akt (Thr308), p-Akt (Ser473), and p-RPS6. There were significant differences in expression level of all studied proteins between OVC and control, as well as between OVC and OSCC. However, OVC showed significant lower staining scores than OSCC. CONCLUSIONS: Our findings demonstrate that the Akt/mTOR pathway is upregulated in OVC, indicating a role for this pathway in the development and progression of this malignancy.

The TSC2/mTOR pathway drives endothelial cell transformation induced by the Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor.

The Kaposi's sarcoma-associated herpesvirus (KSHV), the infectious causative agent of Kaposi's sarcoma (KS), encodes a G protein-coupled receptor (vGPCR) implicated in the initiation of KS. Here we demonstrate that Kaposi's sarcomagenesis involves stimulation of tuberin (TSC2) phosphorylation by vGPCR, promoting the activation of mTOR through both direct and paracrine mechanisms. Pharmacologic inhibition of mTOR with rapamycin prevented vGPCR sarcomagenesis, while overactivation of this pathway was sufficient to render endothelial cells oncogenic. Moreover, mice haploinsufficient for TSC2 are predisposed to vascular sarcomas remarkably similar to KS. Collectively, these results implicate mTOR in KS initiation and suggest that the sarcomagenic potential of KSHV may be a direct consequence of the profound sensitivity of endothelial cells to vGPCR dysregulation of the TSC2/mTOR pathway.

Expression of PD-L1 and p-RPS6 in epithelial dysplasia and squamous cell carcinoma of the oral cavity.

Introduction: Oral squamous cell carcinoma (OSCC) is often preceded by oral epithelial dysplasia (OED). The role of ribosomal protein S6 (RPS6) and programmed cell death ligand-1 (PD-L1) in the progression of OED to OSCC remains unclear. This study aimed to investigate the expression of phosphorylated RPS6 (p-RPS6) and PD-L1 in OSCC and OED and to examine its relationship with clinicopathological features. Methods: Fifty-two OSCC and 48 OED cases were recruited for immunohistochemical analysis of p-RPS6 and PD-L1 expression. The expression of markers was correlated with clinicopathological features of OSCC and OED. Results: We found p-RPS6 expression in all cases of OSCC and OED, whereas PD-L1 was expressed in 42/48 (87%) OED and in 28/52 (53%) OSCC. The patients with mild OED presented higher expression level of PD-L1 and p-RPS6 significantly, when compared to moderate-differentiated OSCC patients (p < 0.05). Moreover, we found a significant positive correlation between PD-L1 and p-RPS6 expression in OED and OSCC patients (p < 0.01). The PD-L1 expression was significantly related to more than 2 cm tumor size in OSCC patients (p = 0.007). Discussion: Our findings suggest the upregulation of PD-L1 may be related with activation of the mTOR pathway in the early events of tumor progression and the pathogenesis of OSCC.

Unveiling senescence-associated ocular pathogenesis via lacrimal gland organoid magnetic bioassembly platform and HMGB1-Box A gene therapy.

Dry eye disease (DED) is a multifactorial aging disorder leading to tear film insufficiency and instability. Yet, an important knowledge gap lingers in understanding senescence-associated ocular pathogenesis, due to limited in vitro translational lacrimal gland (LG) models. Consequently, this remains a major roadblock to discover effective therapies for the restoration of tear film secretion. Herein, the authors reported the magnetic bioassembly of two LG organoid platforms to recapitulate functional and aging states. Using a proof-of-concept approach, porcine primary LG cells were assembled into organoids via a magnetic 3D bioprinting (M3DB) platform. This platform could form reproducible LG organoids with epithelial hallmarks (AQP5+) and exhibit epithelial secretory functions (lysozyme activity). DNA damage-induced senescence and cell death was induced with etoposide, and LG organoid hypofunction and senescence-associated pathogenesis were observed. To confer DNA protection against aging, a novel gene therapy with Box A domain of high-mobility group box-1 (HMGB1-Box A) previously established by our group, was applied here to prevent LG cellular senescence for the first time. HMGB1-Box A transfection prevented LG organoids from senescence-associated pathogenesis at the transcriptomic, metabolomic and proteomic levels. Thus, M3DB platforms could generate functional and DNA damage-induced senescence LG organoids, and this latter damage could be prevented with HMGB1-Box A gene therapy.

Histological pattern of tumor inflammation and stromal density correlate with patient demographics and immuno-oncologic transcriptional profile in oral squamous cell carcinoma.

Introduction: Oral squamous cell carcinoma (OSCC) is the most prevalent oral malignancy, with emerging interest in the characterization of its tumor microenvironment. Herein, we present a comprehensive histological analysis of OSCC stromal density and inflammation and their relationship with patient demographics, clinicopathologic features and immuno-oncologic signatures. Materials-methods: Eighty-seven completely excised OSCC tissues were prospectively collected and scored for histopathologic inflammatory subtypes [HIS]-inflamed (INF), immune-excluded (IE) and immune-desert (ID), peritumoral stromal inflammation (PTSI), and peritumoral stromal fibrosis (PTSF). Scoring of inflammation was complemented by Semaphorin 4D immunohistochemistry. NanoString differential gene expression (DGE) analysis was conducted for eight OSCC cases representative of the inflammatory and stromal subtypes and the demographic groups. Results: PTSF correlated with male gender (p = 0.0043), smoking (p = 0.0455), alcohol consumption (p = 0.0044), increased tumor size (p = 0.0054), and advanced stage (p = 0.002). On the contrary, PTSI occurred predominantly in females (p = 0.0105), non-drinkers (p = 0.0329), and small tumors (p = 0.0044). Transcriptionally, decreased cytokine signaling, and oncogenic pathway activation were observed in HIS-IE. Smokers and males displayed decreased global immune-cell levels and myeloid-cell predominance. Conclusion: Our work describes OSCC stromal and inflammatory phenotypes in correlation with distinct patient groups and DGE, highlighting the translational potential of characterizing the tumor microenvironment for optimal patient stratification.

Immuno-oncologic signature of malignant transformation in oral squamous cell carcinoma.

OBJECTIVE: The purpose of this study is to identify the immuno-oncologic (IO) signature at the surgical tumor margin (TM) of oral squamous cell carcinoma (OSCC) that is involved in the process of malignant transformation. STUDY DESIGN: Under institutional review board approval, TM of 73 OSCC were investigated using immunohistochemistry for the immune biomarker, programmed death ligand-1 (PD-L1). NanoString 770 IO-focused gene set was analyzed in 5 pairs of TM and invasive tumor (T). PD-L1 regulation in response to interferon-gamma (IFN-γ) was investigated in an oral potentially malignant cell line (OPMC). RESULTS: Programmed death ligand-1 expression in the epithelial margin directly correlated with its expression in the underlying immune cells (P = .0082). Differential gene expression showed downregulation of PD-L1 and IFN-γ 6 gene signature in the TM relative to T pair.CD8 and macrophages were higher in TM. CNTFR, LYZ, C7, RORC, and FGF13 downregulation in T relative to TM. TDO2, ADAM12, MMP1, LAMC2, MB21D1, TYMP, OASL, COL5A1, exhausted_CD8, Tregs,and NK_CD56dim were upregulated in T relative to TM. Finally, IFN-γ induced upregulation of PD-L1 in the OPMC. CONCLUSIONS: Our work suggests a role for IFN-γ in PD-L1 upregulation in OPMC and presents novel IO transcriptional signatures for frankly invasive OSCC relative to TM.

Plant molecular farming-derived epidermal growth factor revolutionizes hydrogels for improving glandular epithelial organoid biofabrication.

Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 - 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 - 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38-62% Ki67 cells) with a large epithelial progenitor population (∼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids.

Secretory leukocyte protease inhibitor (SLPI) expression and tumor invasion in oral squamous cell carcinoma.

Differential expression of secretory leukocyte protease inhibitor (SLPI) impacts on tumor progression. SLPI directly inhibits elastase and other serine proteases, and regulates matrix metalloproteinases, plasminogen activation, and plasmin downstream targets to influence invasion. We examined tissues from human oral squamous cell carcinoma (OSCC) for SLPI expression in parallel with proteases associated with tumor progression and evaluated their relationships using tumor cell lines. Significantly decreased SLPI was detected in OSCC compared to normal oral epithelium. Furthermore, an inverse correlation between SLPI and histological parameters associated with tumor progression, including stage of invasion, pattern of invasion, invasive cell grade, and composite histological tumor score was evident. Conversely, elevated plasmin and elastase were positively correlated with histological parameters of tumor invasion. In addition to its known inhibition of elastase, we identify SLPI as a novel inhibitor of plasminogen activation through its interaction with annexin A2 with concomitant reduced plasmin generation by macrophages and OSCC cell lines. In an in vitro assay measuring invasive activity, SLPI blocked protease-dependent tumor cell migration. Our data suggest that SLPI may possess antitumorigenic activity by virtue of its ability to interfere with multiple requisite proteolytic steps underlying tumor cell invasion and may provide insight into potential stratification of oral cancer according to risk of occult metastasis, guiding treatment strategies.

A clinicopathological study of malignant odontogenic tumours.

AIMS: Malignant odontogenic tumours (MOTs) are rare neoplasms occurring primarily within the jaw. The objective of this study was to determine the incidence, demographics and clinicopathological features of the MOTs from two institutions. METHODS AND RESULTS: The records of the Department of Oral Pathology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand and the Department of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, USA were searched from 1991 to 2010; we identified 17 cases of previously diagnosed MOTs. All cases were reviewed independently of the previous diagnosis by two blinded oral pathologists and reclassified based on the 2005 World Health Organization classification of head and neck tumours. In this study we describe in detail these 17 cases which presented with an average age of 50.29 years and a male to female ratio of 2.4:1. These cases included five ameloblastic carcinomas, four atypical ameloblastomas, three primary intraosseous squamous cell carcinomas, three intraosseous mucoepidermoid carcinomas and two clear cell odontogenic carcinomas. All cases were treated by surgical resection and one patient with ameloblastic carcinoma received postoperative radiotherapy. CONCLUSIONS: Malignant odontogenic tumours are considered rare central odontogenic lesions. Awareness of their existence, rapid diagnosis and successful treatment using surgery, radiation and/or chemotherapy is critical to patient survival.

Association Between PD-L1 and Histatin1, 3 Expression in Advanced Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: The prognosis of advanced stage head and neck squamous cell carcinoma (HNSCC) has remained unimproved for the past decades. Therefore, novel diagnostic markers and treatment options are required. Recently, an inhibitor for immune checkpoint program death ligand-1 (PD-L1), was approved by the FDA, and used in HNSCC patients. Histatins (HTNs), one of the common antimicrobial peptides in saliva, have demonstrated wound healing and antifungal capabilities and other functions on the oral epithelium. Dysregulation of HTN1 and HTN3 has also been reported in HNSCC through genomic and proteomic studies. This study aimed to investigate the association between histatins (HTN1 and HTN3) and PD-L1 in advanced HNSCC. PATIENTS AND METHODS: Data of gene expression in HNSCC were collected from TCGA and analyzed using a data-mining platform website (https://ualcan.path.uab.edu/). Tissue microarrays containing 98 samples of HNSCC patients and non-neoplastic controls were immunolabeled against PD-L1, HTN1, and HTN3. The immunohistochemistry results were quantified using ImageJ. RESULTS: The expression of PD-L1 and HTN1 was significantly higher in tumors than normal tissues (p<0.001), but no significant difference was found regarding HTN3. Metastatic HNSCC samples exhibited significantly higher expression of PD-L1 (p<0.018), compared to the non-metastatic group. Association between HTN1 and HTN3 was found using Pearson correlation coefficient (r=0.603, p<0.001). No overall survival difference was evident among our samples. CONCLUSION: PD-L1 and HTN1 are associated with the progression of HNSCC. PD-L1 expression correlated with that of HTN3.

Development of high-throughput lacrimal gland organoid platforms for drug discovery in dry eye disease.

Dysfunction and damage of the lacrimal gland (LG) results in ocular discomfort and dry eye disease (DED). Current therapies for DED do not fully replenish the necessary lubrication to rescue optimal vision. New drug discovery for DED has been limited perhaps because in vitro models cannot mimic the biology of the native LG. The existing platforms for LG organoid culture are scarce and still not ready for consistency and scale up production towards drug screening. The magnetic three-dimensional (3D) bioprinting (M3DB) is a novel system for 3D in vitro biofabrication of cellularized tissues using magnetic nanoparticles to bring cells together. M3DB provides a scalable platform for consistent handling of spheroid-like cell cultures facilitating consistent biofabrication of organoids. Previously, we successfully generated innervated secretory epithelial organoids from human dental pulp stem cells with M3DB and found that this platform is feasible for epithelial organoid bioprinting. Research targeting LG organogenesis, drug discovery for DED has extensively used mouse models. However, certain inter-species differences between mouse and human must be considered. Porcine LG appear to have more similarities to human LG than the mouse counterparts. We have conducted preliminary studies with the M3DB for fabricating LG organoids from primary cells isolated from murine and porcine LG, and found that this platform provides robust LG organoids for future potential high-throughput analysis and drug discovery. The LG organoid holds promise to be a functional model of tearing, a platform for drug screening, and may offer clinical applications for DED.

Juvenile trabecular ossifying fibroma: Immunohistochemical expression of MDM2, CDK4 and p53 compared to conventional ossifying fibroma.

BACKGROUND: Juvenile ossifying fibroma (JOF) is an uncommon benign fibro-osseous lesion of the craniofacial skeleton; compared to conventional ossifying fibroma (OF), JOF is characterized by local aggressiveness and propensity for recurrence. The biologic basis for this different biologic behavior between JOF and OF remains elusive. The aim of this study was to evaluate the immunohistochemical expression of MDM2, CDK4 and p53, molecules associated with bone oncogenesis, in the trabecular variant of JOF. MATERIAL AND METHODS: The study material consisted of five cases of trabecular JOF, affecting three male and two female patients with a mean age of 11.8 years. Three cases arose in the maxilla and two in the mandible. All cases were initially treated by enucleation; two cases recurred necessitating more aggressive treatment. Immunohistochemical study of MDM2, CDK4 and p53 was performed in all cases, as well as in five control cases of conventional OF. RESULTS: CDK4 positivity was noted in all JOF cases; the staining pattern was diffuse and strong in 4 cases and focal and weak in one case. In contrast, 4 out of 5 cases of OF were weakly and focally CDK4 positive, the remaining one being negative. Immunostaining for MDM2 was observed in 3 JOF cases; all OF were MDM2 negative. All cases of OF and JOF were negative for p53, except for one focally positive JOF case. CONCLUSIONS: CDK4 and MDM2 expression in the trabecular variant of JOF is higher compared to conventional OF. In contrast, p53 expression is almost universally negative in JOF and OF. Despite some overlapping features, differential expression patterns of proteins involved in bone oncogenesis can elucidate the pathogenesis and may facilitate accurate diagnosis and prediction of behavior of bone tumors in the craniofacial region. Key words:Juvenile ossifying fibroma, trabecular variant, conventional ossifying fibroma, MDM2, CDK4, p53.

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Zoledronic acid directly suppresses cell proliferation and induces apoptosis in highly tumorigenic prostate and breast cancers.

BACKGROUND: Bisphosphonates (BPs) were designed for the prevention of skeletal-related events secondary to bone metastases. The purpose of this study was to show that zoledronic acid (ZA) directly eradicates highly tumorigenic and potentially metastatic cancer cells. MATERIALS AND METHODS: Human prostate and breast highly tumorigenic (PC3, MCF 7) and low- or non-tumorigenic (LNCaP, MCF 10a) cell lines, respectively, were exposed to different concentrations of ZA (0-10 µM). Reverse transcriptase double quantitative polymerase chain reaction was used for quantitative gene expression analysis. Apoptosis and cell proliferation were determined using microscopic observation and MTS assays. Western blot was used to confirm the translational effects of apoptotic genes on protein expression. RESULTS: Human prostate and breast highly tumorigenic (PC3, MCF 7) and low- or non-tumorigenic (LNCaP, MCF 10a) cell lines, respectively, showed multiple genes demonstrating differential expressions, including TRAF, TRADD, BCL2, CASPASES and IAP families. Increasing ZA concentrations showed a greater concentration-time response on cell proliferation and apoptosis in the highly tumorigenic cells. These results were confirmed by both reversing and enhancing the effect of ZA on cell proliferation with caspase 3, 7 or survivin siRNA, respectively. Pro-apoptotic proteins bax and caspase 2, 3, 7 and 9 were up-regulated, while the anti-apoptotic proteins bcl2, birc3 and survivin were down-regulated only in the highly tumorigenic cells. CONCLUSIONS: This explains the ability of ZA to inhibit bony metastasis in highly tumorigenic cells compared with the low- or non-tumorigenic cells through a significant decrease in cell proliferation and increase in apoptosis through gene-regulated and translational-mediated down-regulation of survivin coupled with the inhibition of caspase 3 or 7. This has significant implications toward understanding the pharmacophysiology of BPs in metastasis and supports the clinically observed effect of BPs when administered adjunctively with anticancer drugs such as cyclophosphamide/methotrexate/5-fluorouracil, epirubicin in combination with cyclophosphamide or docetaxel, and doxorubicin.

Soluble Sema4D in Plasma of Head and Neck Squamous Cell Carcinoma Patients Is Associated With Underlying Non-Inflamed Tumor Profile.

Semaphorin 4D (Sema4D) is a glycoprotein that is expressed by several tumors and immune cells. It can function as a membrane bound protein or as a cleaved soluble protein (sSema4D). We sought to investigate the translational potential of plasma sSema4D as an immune marker in plasma of patients with head and neck squamous cell carcinoma (HNSCC). Paired peripheral blood and tumor tissue samples of 104 patients with HNSCC were collected at the same time point to allow for real time analysis. Scoring of the histological inflammatory subtype (HIS) was carried out using Sema4D immunohistochemistry on the tumor tissue. sSema4D was detected in plasma using direct ELISA assay. Defining elevated sSema4D as values above the 95th percentile in healthy controls, our data showed that sSema4D levels in plasma were elevated in 25.0% (95% CI, 16.7-34.9%) of the patients with HNSCC and showed significant association with HIS immune excluded (HIS-IE) (p = 0.007), Sema4D+ve tumor cells (TCs) (p = 0.018) and PD-L1+ve immune cells (ICs) (p = 0.038). A multi-variable logistic regression analysis showed that HIS was significantly (P = 0.004) associated with elevated sSema4D, an association not explained by available patient-level factors. Using the IO-360 nanoString platform, differential gene expression (DGE) analysis of 10 HNSCC tumor tissues showed that patients with high sSema4D in plasma (HsS4D) clustered as IFN-γ negative tumor immune signature and were mostly HIS-IE. The IC type in the HsS4D paired tumor tissue was predominantly myeloid, while the lymphoid compartment was higher in the low sSema4D (LsS4D). The Wnt signaling pathway was upregulated in the HsS4D group. Further analysis using the IO-360, 770 gene set, showed significant non-inflamed profile of the HsS4D tumors compared to the LsS4D. In conclusion, our data reveals an association between sSema4D and the histological inflammatory subtype.

Ameloblastic Carcinoma in a 2-Year-Old Child: A Case Report and Review of the Literature.

Ameloblastic carcinoma (AC) is a rare malignant odontogenic tumor in pediatric patients, only 22 cases have been reported in literature since 1932. We present an extremely rare case in which AC occurred in a 2-year-old girl, who had a tumor in the right mandible. Radiographic findings showed a multilocular, poorly defined, and mixed radiolucent-radiopaque lesion in the region of teeth #84 to #85, with bone and tooth root resorption. Computed tomography revealed buccal cortex destruction, tumor infiltration of soft tissue, and enlarged nodes. Incisional biopsy showed histomorphological features of AC. Immunohistochemical analysis exhibited a positive result for Cytokeratin (CK) 19 and overexpression of p53 and Ki67. The patient underwent right hemimandibulectomy and neck dissection. The final pathology was consistent with the initial diagnosis of AC. The patient did not exhibit signs of recurrence or metastasis within 2 years postoperatively. Given the rarity of this disease and the age of the patient, this report constitutes a valuable contribution to the current literature.