Histological differences related to autophagy in the minor salivary gland between primary and secondary types of Sjögren's syndrome.

Some forms of Sjögren's syndrome (SS) follow a clinical course accompanied by systemic symptoms caused by lymphocyte infiltration and proliferation in the liver, kidneys, and other organs. To better understand the clinical outcomes of SS, here we used minor salivary gland tissues from patients and examine their molecular, biological, and pathological characteristics. A retrospective study was performed, combining clinical data and formalin-fixed paraffin-embedded (FFPE) samples from female patients over 60 years of age who underwent biopsies at Okayama University Hospital. We employed direct digital RNA counting with nCounter® and multiplex immunofluorescence analysis with a PhenoCycler™ on the labial gland biopsies. We compared FFPE samples from SS patients who presented with other connective tissue diseases (secondary SS) with those from stable SS patients with symptoms restricted to the exocrine glands (primary SS). Secondary SS tissues showed enhanced epithelial damage and lymphocytic infiltration accompanied by elevated expression of autophagy marker genes in the immune cells of the labial glands. The close intercellular distance between helper T cells and B cells positive for autophagy-associated molecules suggests accelerated autophagy in these lymphocytes and potential B cell activation by helper T cells. These findings indicate that examination of FFPE samples from labial gland biopsies can be an effective tool for evaluating molecular histological differences between secondary and primary SS through multiplexed analysis of gene expression and tissue imaging.

The Role of Hedgehog Signaling in the Melanoma Tumor Bone Microenvironment.

A crucial regulator in melanoma progression and treatment resistance is tumor microenvironments, and Hedgehog (Hh) signals activated in a tumor bone microenvironment are a potential new therapeutic target. The mechanism of bone destruction by melanomas involving Hh/Gli signaling in such a tumor microenvironment is unknown. Here, we analyzed surgically resected oral malignant melanoma specimens and observed that Sonic Hedgehog, Gli1, and Gli2 were highly expressed in tumor cells, vasculatures, and osteoclasts. We established a tumor bone destruction mouse model by inoculating B16 cells into the bone marrow space of the right tibial metaphysis of 5-week-old female C57BL mice. An intraperitoneal administration of GANT61 (40 mg/kg), a small-molecule inhibitor of Gli1 and Gli2, resulted in significant inhibition of cortical bone destruction, TRAP-positive osteoclasts within the cortical bone, and endomucin-positive tumor vessels. The gene set enrichment analysis suggested that genes involved in apoptosis, angiogenesis, and the PD-L1 expression pathway in cancer were significantly altered by the GANT61 treatment. A flow cytometry analysis revealed that PD-L1 expression was significantly decreased in cells in which late apoptosis was induced by the GANT61 treatment. These results suggest that molecular targeting of Gli1 and Gli2 may release immunosuppression of the tumor bone microenvironment through normalization of abnormal angiogenesis and bone remodeling in advanced melanoma with jaw bone invasion.

Ligneous periodontitis exacerbated by Behçet's disease in a patient with plasminogen deficiency and a stop-gained variant PLG c.1468C > T: a case report.

BACKGROUND: Plasminogen serves as the precursor to plasmin, an essential element in the fibrinolytic process, and is synthesized primarily in the liver. Plasminogen activation occurs through the action of plasminogen activator, converting it into plasmin. This conversion greatly enhances the fibrinolytic system within tissues and blood vessels, facilitating the dissolution of fibrin clots. Consequently, congenital deficiency of plasminogen results in impaired fibrin degradation. Patients with plasminogen deficiency typically exhibit fibrin deposits in various mucosal sites throughout the body, including the oral cavity, eyes, vagina, and digestive organs. Behcet's disease is a chronic recurrent systemic inflammatory disease with four main symptoms: aphthous ulcers of the oral mucosa, vulvar ulcers, skin symptoms, and eye symptoms, and has been reported worldwide. This disease is highly prevalent around the Silk Road from the Mediterranean to East Asia. We report a case of periodontitis in a patient with these two rare diseases that worsened quickly, leading to alveolar bone destruction. Genetic testing revealed a novel variant characterized by a stop-gain mutation, which may be a previously unidentified etiologic gene associated with decreased plasminogen activity. CASE PRESENTATION: This case report depicts a patient diagnosed with ligneous gingivitis during childhood, originating from plasminogen deficiency and progressing to periodontitis. Genetic testing revealed a suspected association with the PLG c.1468C > T (p.Arg490*) stop-gain mutation. The patient's periodontal condition remained stable with brief intervals of supportive periodontal therapy. However, the emergence of Behçet's disease induced acute systemic inflammation, necessitating hospitalization and treatment with steroids. During hospitalization, the dental approach focused on maintaining oral hygiene and alleviating contact-related pain. The patient's overall health improved with inpatient care and the periodontal tissues deteriorated. CONCLUSIONS: Collaborative efforts between medical and dental professionals are paramount in comprehensively evaluating and treating patients with intricate complications from rare diseases. Furthermore, the PLG c.1468C > T (p.Arg490*) stop-gain mutation could contribute to the association between plasminogen deficiency and related conditions.

Reproduction of the Antitumor Effect of Cisplatin and Cetuximab Using a Three-dimensional Spheroid Model in Oral Cancer.

Background/Aim: Cancer research has been conducted using cultured cells as part of drug discovery testing, but conventional two-dimensional culture methods are unable to reflect the complex tumor microenvironment. On the other hand, three-dimensional cultures have recently been attracting attention as in vitro models that more closely resemble the in vivo physiological environment. The purpose of this study was to establish a 3D culture method for oral cancer and to verify its practicality. Materials and Methods: Three-dimensional cultures were performed using several oral cancer cell lines. Western blotting was used for protein expression analysis of the collected cell masses (spheroids), and H-E staining was used for structural observation. The cultures were exposed to cisplatin and cetuximab and the morphological changes of spheroids over time and the expression changes of target proteins were compared. Results: Each cell line formed spheroidal cell aggregates and showed enhancement of cell adhesion molecules over time. H-E staining showed tumor tissue-like structures specific to each cell line. Cisplatin showed concentration-dependent antitumor effects due to loss of cell adhesion and spheroid disruption in each cell line, while cetuximab exhibited antitumor effects that correlated with EGFR expression in each cell line. Conclusion: Spheroids made from oral cancer cell lines appeared to have tumor-like characteristics that may reflect their clinical significance. In the future, it may become possible to produce tumor spheroids from tissue samples of oral cancer patients, and then apply them to drug screening and to develop individualized diagnostic and treatment methods.

Homeobox transcription factor engrailed homeobox 1 is a possible diagnostic marker for adenoid cystic carcinoma and polymorphous adenocarcinoma.

Diagnostic utility of a homeobox transcription factor, engrailed homeobox 1 (En1) in the histopathology of salivary gland neoplasms was studied. The expression of En1 was immunohistochemically examined in 51 cases of adenoid cystic carcinoma (AdCC) and 143 cases of other salivary gland neoplasms. In all 51 AdCCs, En1 was expressed in 30-100% of tumor cells. In eight of nine polymorphous adenocarcinomas (PACs), En1 was expressed in 40-100% of tumor cells. Less than 5% of tumor cells expressed En1 in three of 12 epithelial-myoepithelial carcinomas, one of 17 basal cell adenomas (BCAs), and one of 34 pleomorphic adenomas (PAs). Among 55 other carcinoma cases, 1-30% of tumor cells expressed En1 in three salivary duct carcinomas (SDCs) ex PA. None of the myoepitheliomas and Warthin tumors expressed En1. When the cut-off value of the percentage of En1-expressing cells was set to 25%, all 51 AdCCs, eight of nine PACs and one SDC ex PA were En1-positive and the others were En1-negative. En1 is expressed consistently in AdCCs, frequently in PACs, but rarely in other salivary gland neoplasms. En1 is a possible diagnostic marker for AdCC and PAC in the histopathology of salivary gland neoplasms.

Potential role of myeloid-derived suppressor cells in transition from reaction to repair phase of bone healing process.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with immunosuppressive functions; these cells play a key role in infection, immunization, chronic inflammation, and cancer. Recent studies have reported that immunosuppression plays an important role in the healing process of tissues and that Treg play an important role in fracture healing. MDSCs suppress active T cell proliferation and reduce the severity of arthritis in mice and humans. Together, these findings suggest that MDSCs play a role in bone biotransformation. In the present study, we examined the role of MDSCs in the bone healing process by creating a bone injury at the tibial epiphysis in mice. MDSCs were identified by CD11b and GR1 immunohistochemistry and their role in new bone formation was observed by detection of Runx2 and osteocalcin expression. Significant numbers of MDSCs were observed in transitional areas from the reactionary to repair stages. Interestingly, MDSCs exhibited Runx2 and osteocalcin expression in the transitional area but not in the reactionary area. And at the same area, cllagene-1 and ALP expression level increased in osteoblast progenitor cells. These data is suggesting that MDSCs emerge to suppress inflammation and support new bone formation. Here, we report, for the first time (to our knowledge), the role of MDSCs in the initiation of bone formation. MDSC appeared at the transition from inflammation to bone making and regulates bone healing by suppressing inflammation.

General rules for clinical and pathological studies on oral cancer (2nd edition): a synopsis.

For doctors and other medical staff treating oral cancer, it is necessary to standardize the basic concepts and rules for oral cancer to achieve progress in its treatment, research, and diagnosis. Oral cancer is an integral part of head and neck cancer and is treated in accordance with the general rules for head and neck cancer. However, detailed rules based on the specific characteristics of oral cancer are essential. The objective of this article was to contribute to the development of the diagnosis, treatment, and research of oral cancer, based on the correct and useful medical information of clinical, surgical, pathological, and imaging findings accumulated from individual patients at various institutions. Our general rules were revised as the UICC was revised for the 8th edition and were published as the Japanese second edition in 2019. In this paper, the English edition of the "Rules" section is primarily presented.

Effect of Patient Clinical Variables in Osteoporosis Classification Using Hip X-rays in Deep Learning Analysis.

Background and Objectives: A few deep learning studies have reported that combining image features with patient variables enhanced identification accuracy compared with image-only models. However, previous studies have not statistically reported the additional effect of patient variables on the image-only models. This study aimed to statistically evaluate the osteoporosis identification ability of deep learning by combining hip radiographs with patient variables. Materials andMethods: We collected a dataset containing 1699 images from patients who underwent skeletal-bone-mineral density measurements and hip radiography at a general hospital from 2014 to 2021. Osteoporosis was assessed from hip radiographs using convolutional neural network (CNN) models (ResNet18, 34, 50, 101, and 152). We also investigated ensemble models with patient clinical variables added to each CNN. Accuracy, precision, recall, specificity, F1 score, and area under the curve (AUC) were calculated as performance metrics. Furthermore, we statistically compared the accuracy of the image-only model with that of an ensemble model that included images plus patient factors, including effect size for each performance metric. Results: All metrics were improved in the ResNet34 ensemble model compared with the image-only model. The AUC score in the ensemble model was significantly improved compared with the image-only model (difference 0.004; 95% CI 0.002-0.0007; p = 0.0004, effect size: 0.871). Conclusions: This study revealed the additional effect of patient variables in identification of osteoporosis using deep CNNs with hip radiographs. Our results provided evidence that the patient variables had additive synergistic effects on the image in osteoporosis identification.

Risk Factors for Mandibular Fracture After Marginal Mandibular Resection.

PURPOSE: This study aimed to examine the relationship between post-operative mandibular fractures and its predictable risk factors in patients with marginal mandibular resection. Additionally, the timing of post-operative mandibular fractures was assessed. PATIENTS AND METHODS: Records of 37 patients with mandibular gingival carcinoma who underwent marginal mandibular resection at the Department of Oral and Maxillofacial Surgery, Kagawa Prefectural Central Hospital, from April 2011 to March 2019 were retrospectively analyzed. The following variables were investigated: age, sex, location of carcinoma, tumor size, mandibular height on the surgical and healthy sides, surgical approach, number of residual teeth, post-operative radiotherapy, chemotherapy, and the presence or absence of diabetes and osteoporosis. Various risk factors for post-operative mandibular fractures were statistically investigated. RESULTS: Post-operative mandibular fracture was observed in 5 (13.5%) of the 37 mandibular marginal resection cases. The average residual mandibular height in patients with post-operative mandibular fracture was 8.5 mm. A significant difference in residual mandibular height (P = 0.013) was observed between patients with post-operative mandibular fracture and those with no fracture. The average time to post-operative fracture of the mandible was 305.4 days, and it was found to be correlated to the remaining height of the mandibular body. CONCLUSIONS: A decrease in mandibular height below 9 mm results in post-operative mandibular fracture. Furthermore, a correlation between the height of the mandibular bone and the period until the post-operative mandibular fracture was noted in this study. These findings contribute to the prediction and management of mandibular fractures after mandibular margin resection.

Maxillofacial Trauma Surgery Patients With Titanium Osteosynthesis Miniplates: Remove or Not?

The authors examined the timing and causes of titanium miniplate removal after maxillofacial trauma surgery. The authors performed a retrospective study of maxillofacial fracture patients in whom maxillofacial osteosynthesis miniplates were inserted or removed at the Kagawa Prefectural Central Hospital, between 2008 and 2017. Predictive variables were age, sex, fracture site distribution, and time to miniplate removal with or without complications in relation to primary outcome variables. Among 185 patients, 440 miniplates were inserted and 272 miniplates were removed. In total, 116 patients (73.4%) had 282 miniplates (64.1%) removed, of which 4.8% fracture sites and 5.7% miniplates were removed because of complications. The mean time to miniplate removal was 630.9 and 258.0 days in patients with and without complications, respectively. There was a statistically significant difference in miniplate removal and miniplate retention relative to age and sex. This difference was not related to the presence or absence of sex- or age-related complications. The miniplates as osteosynthesis material were safe and useful for a long period of time with relatively few complications. Because complications requiring miniplate removal occurred within 1 or after 5 years postoperatively, osteosynthesis miniplate treatments should be decided while considering the patient's age and sex. Long-term follow-up is recommended for miniplates that remain implanted for >1 year.

Impact of the Stroma on the Biological Characteristics of the Parenchyma in Oral Squamous Cell Carcinoma.

Solid tumors consist of the tumor parenchyma and stroma. The standard concept of oncology is that the tumor parenchyma regulates the tumor stroma and promotes tumor progression, and that the tumor parenchyma represents the tumor itself and defines the biological characteristics of the tumor tissue. Thus, the tumor stroma plays a pivotal role in assisting tumor parenchymal growth and invasiveness and is regarded as a supporter of the tumor parenchyma. The tumor parenchyma and stroma interact with each other. However, the influence of the stroma on the parenchyma is not clear. Therefore, in this study, we investigated the effect of the stroma on the parenchyma in oral squamous cell carcinoma (OSCC). We isolated tumor stroma from two types of OSCCs with different invasiveness (endophytic type OSCC (ED-st) and exophytic type OSCC (EX-st)) and examined the effect of the stroma on the parenchyma in terms of proliferation, invasion, and morphology by co-culturing and co-transplanting the OSCC cell line (HSC-2) with the two types of stroma. Both types of stroma were partially positive for alpha-smooth muscle actin. The tumor stroma increased the proliferation and invasion of tumor cells and altered the morphology of tumor cells in vitro and in vivo. ED-st exerted a greater effect on the tumor parenchyma in proliferation and invasion than EX-st. Morphological analysis showed that ED-st changed the morphology of HSC-2 cells to the invasive type of OSCC, and EX-st altered the morphology of HSC-2 cells to verrucous OSCC. This study suggests that the tumor stroma influences the biological characteristics of the parenchyma and that the origin of the stroma is strongly associated with the biological characteristics of the tumor.

MCSF orchestrates branching morphogenesis in developing submandibular gland tissue.

The importance of macrophages in tissue development and regeneration has been strongly emphasized. However, the specific roles of macrophage colony-stimulating factor (MCSF), the key regulator of macrophage differentiation, in glandular tissue development have been unexplored. Here, we disclose new macrophage-independent roles of MCSF in tissue development. We initially found that MCSF is markedly upregulated at embryonic day (E)13.5, at a stage preceding the colonization of macrophages (at E15.5), in mouse submandibular gland (SMG) tissue. Surprisingly, MCSF-induced branching morphogenesis was based on a direct effect on epithelial cells, as well as indirectly, by modulating the expression of major growth factors of SMG growth, FGF7 and FGF10, via the phosphoinositide 3-kinase (PI3K) pathway. Additionally, given the importance of neurons in SMG organogenesis, we found that MCSF-induced SMG growth was associated with regulation of neurturin expression and neuronal network development during early SMG development in an in vitro organogenesis model as well as in vivo These results indicate that MCSF plays pleiotropic roles and is an important regulator of early SMG morphogenesis.

Involvement of miR-140-3p in Wnt3a and TGFß3 signaling pathways during osteoblast differentiation in MC3T3-E1 cells.

The Wnt/ß-catenin signaling and TGFß signaling pathways play a key role in osteoblast differentiation. The miRNAs play important roles in regulating gene expression at the post-transcriptional level through fine-tuning of protein-encoding gene expression. However, involvement of miRNAs is not established for Wnt3a and TGFß signaling pathways in osteoblast differentiation. Here, we examined the role of miRNAs expressed differentially after Wnt3a expression during osteoblast differentiation. Over-expression of the Wnt3a gene increased ALP transcription, but decreased Col1, Runx2, and OCN transcription in osteoblastic MC3T3-E1 cells. Expression profiling and quantitative PCR for miRNAs showed that miR-140-3p decreased in Wnt3a-over-expressing osteoblastic cells. Wnt3a over-expression increased TGFß3 expression, whereas transfection of the miR-140-3p mimic into MC3T3-E1 cells significantly inhibited TGFß3 expression. Luciferase assay for the TGFß3 transcript showed that TGFß3 was a direct target of miR-140-3p. miR-140-3p mimic transfection resulted in significantly increased OCN transcription, but did not affect ALP, Col1, and Runx2 transcription in MC3T3-E1 cells. rTGFß3 treatment decreased OCN transcription in MC3T3-E1 cells. These results suggest that the miR-140-3p is involved in osteoblast differentiation as a critical regulatory factor between Wnt3a and TGFß3 signaling pathways.

Immunohistochemistry of YAP and dNp63 and survival analysis of patients bearing precancerous lesion and oral squamous cell carcinoma.

Background: Yes-associated protein (YAP) is a candidate oncogene in various human cancers, and recently, it has been reported that YAP expression and its activity was enhanced by ΔNp63. However, the role of YAP and ΔNp63 expression in carcinogenesis and progression of oral squamous cell carcinoma (OSCC) has been unknown. Therefore, we investigated how YAP and ΔNp63 influence carcinogenesis and progression of OSCC. Methods: We performed immunohistochemical analyses in whole tissue samples to investigate YAP and ΔNp63 expression in normal oral mucosa, epithelial hyperplasia, oral epithelial dysplasia (OED; low/high grade), carcinoma in situ (CIS), and OSCC. Furthermore, in OSCC, we analyzed clinical significance by using Kaplan-Meier survival analysis. Results: In normal oral mucosa and epithelial hyperplasia, YAP expression was primarily confined to the basal and parabasal layers, but YAP expression was elevated in OED, CIS, and OSCC. In OED, YAP and ΔNp63 expression levels were markedly higher in high grade than in low grade. In OSCC groups, YAP and ΔNp63 expression patterns tended to differ according to histopathological differentiation of OSCC. Furthermore, the YAP high expression group, which showed YAP staining in >50% positive cells with strong cytoplasmic staining or >10% positive cells with nuclear reactivity, showed a tendency to have a poor survival rate. Conclusion: YAP and ΔNp63 expression levels correlated with grade of oral OED. Additionally, YAP expression was associated with OSCC survival rate. Our results suggested that YAP and ΔNp63 expression might serve as predictive markers to distinguish OSCC development and progression.

Feasible Advantage of Bioactive/Bioresorbable Devices Made of Forged Composites of Hydroxyapatite Particles and Poly-L-lactide in Alveolar Bone Augmentation: A Preliminary Study.

Purpose: We aimed to document the clinical usefulness of uncalcined and unsintered hydroxyapatite (u-HA) particles and poly-L-lactide (PLLA) composite materials and their advantageous properties. Methods: Between April 2016 and March 2018, five patients required anterior maxillary alveolar ridge augmentation using fixation with u-HA/PLLA screws for an onlay block bone graft harvested from the mandibular ramus at our institute. Bone biopsies were obtained from the dental implantation site following bone healing for histomorphometric and immunohistochemical (IHC) measurements. Results: Many stromal cells were positive for Osterix, RUNX2, and SOX9 but were negative for CD68. On cell counting, based on IHC staining for Osterix, RUNX2, SOX9 and CD68 from peripheral u-HA/PLLA screw or bone areas, both areas consistently showed no significant difference in terms of Osterix, RUNX2, and SOX9. Hematoxylin-eosin staining revealed direct bone connection to the biomaterials, and no inflammatory cells infiltrated the areas surrounding the bone or artificial material. Area between the bone and u-HA/PLLA screw was seamless with no boundary. Round small cells and immature fibroblasts were noted. The new bone showed the presence of bone lamellae, normal osteocytes, and osteoblasts. Conclusion: The u-HA/PLLA materials showed excellent biodegradability and bioactive osteoconductivity. In addition, this material induced no apparent inflammatory or foreign body reactions following implantation, and it directly bonded to the human bone. Therefore, this u-HA/PLLA material seems ideal and most suitable for use as a substitute for osteosynthesis.

Notch Signaling Affects Oral Neoplasm Cell Differentiation and Acquisition of Tumor-Specific Characteristics.

Histopathological findings of oral neoplasm cell differentiation and metaplasia suggest that tumor cells induce their own dedifferentiation and re-differentiation and may lead to the formation of tumor-specific histological features. Notch signaling is involved in the maintenance of tissue stem cell nature and regulation of differentiation and is responsible for the cytological regulation of cell fate, morphogenesis, and/or development. In our previous study, immunohistochemistry was used to examine Notch expression using cases of odontogenic tumors and pleomorphic adenoma as oral neoplasms. According to our results, Notch signaling was specifically associated with tumor cell differentiation and metaplastic cells of developmental tissues. Notch signaling was involved in the differentiation of the ductal epithelial cells of salivary gland tumors and ameloblast-like cells of odontogenic tumors. However, Notch signaling was also involved in squamous metaplasia, irrespective of the type of developmental tissue. In odontogenic tumors, Notch signaling was involved in epithelial-mesenchymal interactions and may be related to tumor development and tumorigenesis. This signaling may also be associated with the malignant transformation of ameloblastomas. Overall, Notch signaling appears to play a major role in the formation of the characteristic cellular composition and histological features of oral neoplasms, and this involvement has been reviewed here.

The Role of Sonic Hedgehog Signaling in the Tumor Microenvironment of Oral Squamous Cell Carcinoma.

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of SHH expression appear to correlate with cancer progression. However, the role of SHH in the tumor microenvironment (TME) of oral squamous cell carcinoma (OSCC) is still unclear. No studies have compared the expression of SHH in different subtypes of OSCC and focused on the relationship between the tumor parenchyma and stroma. In this study, we analyzed SHH and expression of its receptor, Patched-1 (PTCH), in the TME of different subtypes of OSCC. Fifteen endophytic-type cases (ED type) and 15 exophytic-type cases (EX type) of OSCC were used. H&E staining, immunohistochemistry (IHC), double IHC, and double-fluorescent IHC were performed on these samples. ED-type parenchyma more strongly expressed both SHH and PTCH than EX-type parenchyma. In OSCC stroma, CD31-positive cancer blood vessels, CD68- and CD11b-positive macrophages, and α-smooth muscle actin-positive cancer-associated fibroblasts partially expressed PTCH. On the other hand, in EX-type stroma, almost no double-positive cells were observed. These results suggest that autocrine effects of SHH induce cancer invasion, and paracrine effects of SHH govern parenchyma-stromal interactions of OSCC. The role of the SHH pathway is to promote growth and invasion.

Significance of PD-L1 Expression in Tongue Cancer Development.

Aims: Immunohistochemistry of PD-L1 has been recently established as a surrogate method to predict if immunotherapy targeting PD-L1/PD-1 has a significant effect on suppression of cancers such as lung non-small cell carcinoma, melanoma, and renal cell carcinoma. Here we performed immunohistochemistry for PD-L1 expression in squamous cell carcinoma (SCC) of the tongue to investigate the potential correlation between PD-L1 expression and clinicopathological factors and whether PD-L1 expression would be associated with prognosis. Methods: Tissue microarray cores of paraffin-embedded blocks from 135 cases with surgically resected tongue SCC were immunohistochemically analysed for PD-L1 expression. Results: We observed a positive correlation between PD-L1 expression and tongue SCC pT1 and pT2 tumours, but a negative correlation with pT2, pT3 and pT4 tumours. We also observed a positive correlation with lymph node metastasis. However, no positive correlation was demonstrated between PD-L1 expression and overall survival. Conclusions: PD-L1 tends to be overexpressed at the early stage of tongue SCC, showing a close correlation with initial development of tongue. However, PD-L1 expression may not affect prognosis.

The Role of Bone Marrow-Derived Cells during Ectopic Bone Formation of Mouse Femoral Muscle in GFP Mouse Bone Marrow Transplantation Model.

Multipotential ability of bone marrow-derived cells has been clarified, and their involvement in repair and maintenance of various tissues has been reported. However, the role of bone marrow-derived cells in osteogenesis remains unknown. In the present study, bone marrow-derived cells during ectopic bone formation of mouse femoral muscle were traced using a GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) mice were transplanted into C57BL/6 J wild type mice. After transplantation, insoluble bone matrix (IBM) was implanted into mouse muscle. Ectopic bone formation was histologically assessed at postoperative days 7, 14, and 28. Immunohistochemistry for GFP single staining and GFP-osteocalcin double staining was then performed. Bone marrow transplantation successfully replaced hematopoietic cells with GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts and osteocytes involved in ectopic bone formation were GFP-negative, whereas osteoclasts and hematopoietic cells involved in bone formation were GFP-positive. These results indicate that bone marrow-derived cells might not differentiate into osteoblasts. Thus, the main role of bone marrow-derived cells in ectopic osteogenesis may not be to induce bone regeneration by differentiation into osteoblasts, but rather to contribute to microenvironment formation for bone formation by differentiating tissue stem cells into osteoblasts.

Characterization and potential roles of bone marrow-derived stromal cells in cancer development and metastasis.

Background: The tumor microenvironment and its stromal cells play an important role in cancer development and metastasis. Bone marrow-derived cells (BMDCs), a rich source of hematopoietic and mesenchymal stem cells, putatively contribute to this tumoral stroma. However their characteristics and roles within the tumor microenvironment are unclear. In the present study, BMDCs in the tumor microenvironment were traced using the green fluorescent protein (GFP) bone marrow transplantation model. Methods: C57BL/6 mice were irradiated and rescued by bone marrow transplantation from GFP-transgenic mice. Lewis lung cancer cells were inoculated into the mice to generate subcutaneous allograft tumors or lung metastases. Confocal microscopy, immunohistochemistry for GFP, α-SMA, CD11b, CD31, CD34 and CD105, and double-fluorescent immunohistochemistry for GFP-CD11b, GFP-CD105 and GFP-CD31 were performed. Results: Round and dendritic-shaped GFP-positive mononuclear cells constituted a significant stromal subpopulation in primary tumor peripheral area (PA) and metastatic tumor area (MA) microenvironment, thus implicating an invasive and metastatic role for these cells. CD11b co-expression in GFP-positive cells suggests that round/dendritic cell subpopulations are possibly BM-derived macrophages. Identification of GFP-positive mononuclear infiltrates co-expressing CD31 suggests that these cells might be BM-derived angioblasts, whereas their non-reactivity for CD34, CD105 and α-SMA implies an altered vascular phenotype distinct from endothelial cells. Significant upregulation of GFP-positive, CD31-positive and GFP/CD31 double-positive cell densities positively correlated with PA and MA (P<0.05). Conclusion: Taken together, in vivo evidence of traceable GFP-positive BMDCs in primary and metastatic tumor microenvironment suggests that recruited BMDCs might partake in cancer invasion and metastasis, possess multilineage potency and promote angiogenesis.