Role of Stromal Fibroblast-Induced WNT7A Associated with Cancer Cell Migration Through the AKT/CLDN1 Signaling Axis in Oral Squamous Cell Carcinoma.

Wnt signaling plays a crucial role in the progression of various cancers, including oral squamous cell carcinoma (OSCC). However, the tumor microenvironment (TME) regulating Wnt signaling has not yet been fully elucidated. In this study, we investigated whether cancer-associated fibroblasts (CAFs), the primary components of the TME, activate Wnt signaling and promote tumor progression in OSCC. We conducted a Transwell coculture assay using human OSCC cell lines and normal human dermal fibroblasts (NHDFs). NHDFs stimulated WNT7A expression in several OSCC cell lines, especially HO-1-N-1 and HSC-5. An immunohistochemical study using 122 human OSCC samples indicated that high WNT7A expression in tumor cells was significantly associated with invasion depth and poor prognosis. Moreover, WNT7A expression in OSCC cells was positively correlated with α-smooth muscle actin expression in CAFs. WNT7A knockdown in OSCC cells demonstrated that OSCC cells cocultured with NHDFs significantly promoted tumor cell migration and invasion, which was dependent on WNT7A expression in OSCC cells. We also isolated HSC-5 cells from the coculture and conducted microarray analysis to investigate the factors that promote tumor progression induced by WNT7A. Among the various differentially expressed genes, we identified a downregulated gene encoding CLDN1 and confirmed that WNT7A negatively regulated CLDN1 expression in OSCC cells and CLDN1 knockdown in OSCC cells promoted their migration. Phosphokinase array analysis showed that WNT7A activates protein kinase B (AKT) phosphorylation. Activating AKT signaling using the SC79 agonist induced CLDN1 downregulation in OSCC cells. In the coculture assay, the AKT inhibitor MK2206 significantly recovered CLDN1 expression downregulated by WNT7A, resulting in OSCC cell migration suppression. These results suggest that CAFs stimulate OSCC cells to produce WNT7A, following CLDN1 expression downregulation by activating AKT signaling, promoting cancer cell migration. These findings highlight the importance of molecular therapies targeting the TME in OSCC.

Comparison of Osteoconductive Ability of Two Types of Cholesterol-Bearing Pullulan (CHP) Nanogel-Hydrogels Impregnated with BMP-2 and RANKL-Binding Peptide: Bone Histomorphometric Study in a Murine Calvarial Defect Model.

The receptor activator of NF-κB ligand (RANKL)-binding peptide is known to accelerate bone morphogenetic protein (BMP)-2-induced bone formation. Cholesterol-bearing pullulan (CHP)-OA nanogel-crosslinked PEG gel (CHP-OA nanogel-hydrogel) was shown to release the RANKL-binding peptide sustainably; however, an appropriate scaffold for peptide-accelerated bone formation is not determined yet. This study compares the osteoconductivity of CHP-OA hydrogel and another CHP nanogel, CHP-A nanogel-crosslinked PEG gel (CHP-A nanogel-hydrogel), in the bone formation induced by BMP-2 and the peptide. A calvarial defect model was performed in 5-week-old male mice, and scaffolds were placed in the defect. In vivo µCT was performed every week. Radiological and histological analyses after 4 weeks of scaffold placement revealed that the calcified bone area and the bone formation activity at the defect site in the CHP-OA hydrogel were significantly lower than those in the CHP-A hydrogel when the scaffolds were impregnated with both BMP-2 and the RANKL-binding peptide. The amount of induced bone was similar in both CHP-A and CHP-OA hydrogels when impregnated with BMP-2 alone. In conclusion, CHP-A hydrogel could be an appropriate scaffold compared to the CHP-OA hydrogel when the local bone formation was induced by the combination of RANKL-binding peptide and BMP-2, but not by BMP-2 alone.

IL-1 Generated by Oral Squamous Cell Carcinoma Stimulates Tumor-Induced and RANKL-Induced Osteoclastogenesis: A Possible Mechanism of Bone Resorption Induced by the Infiltration of Oral Squamous Cell Carcinoma.

We previously observed a novel osteoclastogenesis system that is induced by oral squamous cell carcinoma (OSCC) cells, which target osteoclast precursor cells (OPC) without upregulation of the master transcriptional factor of osteoclastogenesis, NFATc1. Here, we analyzed inflammatory cytokines that were preferentially expressed in one of the osteoclastogenic OSCC cell lines, namely NEM, compared with the subclone that had lost its osteoclastogenic properties. Based on a gene expression microarray and a protein array analyses, IL-1, IL-6, IL-8, and CXCL1 were chosen as candidates responsible for tumor-induced osteoclastogenesis. From the results of the in vitro osteoclastogenesis assay using OPCs cultured with OSCC cells or their culture supernatants, IL-1 was selected as a stimulator of both OSCC-induced and RANKL-induced osteoclastogenesis. The IL-1 receptor antagonist significantly attenuated osteoclastogenesis induced by NEM cells. The stimulatory effects of IL-1 for OSCC-induced and RANKL-induced osteoclastogenesis were effectively attenuated with cannabidiol and denosumab, respectively. These results suggest that IL-1 secreted from OSCC cells stimulates not only tumor-induced osteoclastogenesis targeting OPCs but also physiological RANKL-induced osteoclastogenesis, and this may be the biological mechanism of bone resorption induced by the infiltration of OSCC. These results also suggest that IL-1 inhibitors are candidates for therapeutic agents against bone resorption induced by OSCC.

Perforated Hydrogels Consisting of Cholesterol-Bearing Pullulan (CHP) Nanogels: A Newly Designed Scaffold for Bone Regeneration Induced by RANKL-Binding Peptides and BMP-2.

The receptor activator of NF-κB ligand (RANKL)-binding peptide, OP3-4, is known to stimulate bone morphogenetic protein (BMP)-2-induced bone formation, but peptides tend to aggregate and lose their bioactivity. Cholesterol-bearing pullulan (CHP) nanogel scaffold has been shown to prevent aggregation of peptides and to allow their sustained release and activity; however, the appropriate design of CHP nanogels to conduct local bone formation needs to be developed. In the present study, we investigated the osteoconductive capacity of a newly synthesized CHP nanogel, CHPA using OP3-4 and BMP-2. We also clarified the difference between perforated and nonperforated CHPA impregnated with the two signaling molecules. Thirty-six, five-week-old male BALB/c mice were used for the calvarial defect model. The mice were euthanized at 6 weeks postoperatively. A higher cortical bone mineral content and bone formation rate were observed in the perforated scaffold in comparison to the nonperforated scaffold, especially in the OP3-4/BMP-2 combination group. The degradation rate of scaffold material in the perforated OP3-4/BMP-2 combination group was lower than that in the nonperforated group. These data suggest that perforated CHPA nanogel could lead to local bone formation induced by OP3-4 and BMP-2 and clarified the appropriate degradation rate for inducing local bone formation when CHPA nanogels are designed to be perforated.

Primordial odontogenic tumor occurred in the maxilla with unique calcifications and its crucial points for differential diagnosis.

Primordial odontogenic tumor (POT) is a newly classified, mixed epithelial and mesenchymal odontogenic tumor, with only 17 reported cases to date. Herein, we report a case of POT that occurred in the right maxilla of a 10-year-old boy and reveal unique features in comparison with those previously reported. Radiologically, the lesion presented as a well-defined, unilocular radiolucency with notable radiopaque foci on the periphery. Microscopically, the tumor was mainly composed of dental papilla-like myxoid fibrous connective tissue, largely surrounded by non-keratinized squamous epithelium with numerous calcified particles, and partly enclosed by inner enamel epithelium-like columnar cells and enamel organ-like structures accompanied with cuboidal and/or stellate reticulum-like cells. Immunohistochemically, the epithelium tested positive for cytokeratin 14 and 19. Moreover, amelogenin and ameloblastin, matrix proteins relating to enamel formation, were positive in the covering epithelium. The tumor was enucleated as a whole, and no recurrence was recorded thereafter. Although the presence of numerous calcified particles was unique, we diagnosed this lesion as POT based on the above-described features. Furthermore, we emphasize the importance of the differential diagnosis of POT and other odontogenic tumors that resemble corresponding tooth germ components.

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.

Intracellular claudin-1 at the invasive front of tongue squamous cell carcinoma is associated with lymph node metastasis.

Claudins are the major component of tight junctions, which form a primary barrier to paracellular diffusion and maintain cell polarity in normal epithelia and endothelia. In cancer cells, claudins play additional roles besides serving as components of the tight junctions, and participate in anoikis or invasion. Among the claudin family proteins, claudin-1 has the most promising potential, both diagnostically and prognostically, in many types of cancers, including oral, gastric, liver, and colon cancers. However, conflicting results have been reported in relation to the degree of claudin-1 expression and the prognosis, suggesting that the expression level of claudin-1 alone is not sufficient to analyze the relationship between claudin-1 and cancer progression. As endocytic trafficking of claudin-1 has been reported in several epithelial cell types in vitro, we aimed to determine whether intracellular localization of claudin-1 is the missing aspect between claudin-1 and cancer. We investigated the expression of claudin-1 in 83 tongue squamous cell carcinoma (TSCC) pathological specimens. Although the expression level of claudin-1 based on immunohistochemistry was not associated with TSCC progression, within the high claudin-1 expression group, the incidence of intracellular localization of claudin-1 was correlated with cervical lymph node metastasis. In an in vitro experiment, claudin-1 was constitutively internalized in TSCC-derived cells. Motility of TSCC-derived cells was increased by deficiency of claudin-1, suggesting that the decrease in cell-surface claudin-1 promoted the cell migration. Therefore, intracellular localization of claudin-1 at the invasion front may represent a promising diagnostic marker of TSCC.

Co-expression of EGFR and MET has a synergistic effect on the prognosis of patients with oral squamous cell carcinoma.

BACKGROUND: This study aimed to elucidate the correlation between gene amplification, protein expression of receptor tyrosine kinase, and prognosis of patients with oral squamous cell carcinoma (OSCC) using immunohistochemistry (IHC) and next-generation sequencing data. METHODS: We evaluated data pertaining to 208 patients with OSCC using IHC for epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET). RESULTS: High expressions of EGFR and MET were detected in 60 and 41 patients, respectively. We evaluated the association of clinicopathological variables with high expressions of EGFR and/or MET. Distant metastasis was found in 9 of 41 patients (22.0%) and 6 of 15 patients (40.0%) with high expression of MET and high co-expressions of EGFR and MET, respectively; statistically significant differences were detected in both high expression of MET (P = .003) and high co-expressions of EGFR and MET (P = 3.41 × 10-5 ). The cumulative 5-year survival rate of patients with high and low expressions of EGFR or MET was approximately 65% and 85%, respectively. Conversely, among cases with high expressions of EGFR or MET, there was no additional decrease in the survival rate of patients harboring TP53 mutations. Moreover, the survival rate of patients with high co-expression of both EGFR and MET was very poor (22.0%) (P < 1.0 × 10-9 ). CONCLUSION: Our findings suggest that evaluation of protein expressions of EGFR and MET may facilitate prognostic assessment of patients with OSCC; in addition, patients with OSCC should be screened for enrollment in clinical trials of combination therapy with EGFR and MET inhibitors.

Invasive raccoon management systems and challenges in regions with active control.

BACKGROUND: The raccoon (Procyon lotor) is an invasive, non-native species in Japan. Throughout the country, it causes significant agricultural damage and negatively affects native biodiversity. Most of the responsibility for raccoon management lies with local government, and there are still many challenges to be overcome. Although raccoon populations have not been eradicated, intensive control campaigns such as focus on the early stages of invasion have controlled raccoons in some regions. To improve the national management of raccoons, we conducted a survey on raccoon management systems in local government departments considered to solve the challenges recognized in many areas. During 2014 and 2015, we surveyed three different municipal departments about raccoon management measures. The semi-structured interview survey covered two topics: (1) the situation leading up to the current management system; (2) the current management system. RESULTS: Our results describe the scope and methods used in raccoon management. The government staff managed raccoons using monitoring, employing a variety of methods, a range of budgets, and various role divisions. The management practices are similar in that they share a sense of taking precautions, collaborating with stakeholders, understanding that adequate methods must be used, and obtaining support from experts. CONCLUSIONS: Our case studies reveal the challenges in raccoon management faced by local government officers in regions with active control. The management systems and methods that we surveyed seemed to be effective in solving problems in both developed and undeveloped areas.

Genetic and histopathological analysis of a case of primary intraosseous carcinoma, NOS with features of both ameloblastic carcinoma and squamous cell carcinoma.

BACKGROUND: Primary intraosseous carcinoma (PIOC), NOS is an odontogenic carcinoma with unknown etiology. Its diagnosis may be used when central jaw carcinoma cannot be categorized as any other type of carcinoma. Further information on this extremely rare tumor is needed to improve our understanding and evaluate the classification of odontogenic carcinomas. CASE PRESENTATION: We herein presented two patients with PIOC, NOS with different clinical and histopathological features and analyzed gene mutations in these patients using next-generation sequencing (NGS). The typical PIOC, NOS case had many histopathological similarities to oral squamous cell carcinoma (OSCC), including the missense point mutations of TP53 Glu285Val, KDR Gln472His, and APC Pro1433Leu, which are similar to those in other cancers; however, no mutations were detected in the other patient with an atypical presentation of PIOC, NOS, which was derived from a precursor cystic lesion with similarities to both ameloblastic carcinoma and OSCC. CONCLUSIONS: Genetic analysis suggested that these two PIOC, NOS cases have different features and can be subcategorized.

A new osteoclastogenesis pathway induced by cancer cells targeting osteoclast precursor cells.

The precise mechanism of osteolysis induced by tumors infiltrating into the bone remains unclear. The main hypothesis is that tumor cells generate receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor-alpha (TNF-α), or other molecules that activate the expression of RANKL in osteoblasts, osteocytes, or bone marrow stromal cells. Administration of bisphosphonates or anti-RANKL antibody drugs, which suppress systemic bone resorption, prevents osteolysis induced by tumors infiltrating into the bone. However, these therapeutic agents may cause medication-related osteonecrosis of the jaw. In this study, we found a novel tumor-associated osteoclastogenesis pathway in osteoclast precursor cells. Co-culture with human oral squamous cell carcinoma cells, 3A or NEM, or culture with each of their conditioned medium induced many osteoclasts from osteoclast precursor cells, which were generated by a 24-h pretreatment of RANKL or TNF-α. Osteoprotegerin, a decoy RANKL receptor, denosumab, an anti-RANKL antibody drug, and infliximab, an anti-TNF-α antibody drug, did not prevent this tumor-associated osteoclastogenesis. Quantitative RT-PCR analysis showed that the expression of NFATc1 was decreased in this tumor-associated osteoclastogenesis, which was suggested to be independent of NFATc1. These results revealed a novel pathway for tumor-associated osteoclastogenesis, which may be a new therapeutic target for osteolysis induced by tumors infiltrating into the bone without affecting systemic bone metabolism.

Quantitation and distribution of metallic elements in sequestra of medication-related osteonecrosis of jaw (MRONJ) using inductively coupled plasma atomic emission spectroscopy and synchrotron radiation X-ray fluorescence analysis.

Medication-related osteonecrosis of the jaw (MRONJ) is a serious adverse effect of antiresorptive agents like bisphosphonates. Abnormal concentrations of various trace metallic elements contained in bone minerals have been associated with MRONJ. In this study, we focused on trace metallic elements contained in the MRONJ sequestrum; their content and distribution were compared to those in osteomyelitis and non-inflammatory bones using inductively coupled plasma atomic emission spectroscopy (ICP-AES) and synchrotron radiation X-ray fluorescence analysis (SR-XRF). On ICP-AES analyses, various trace elements (Co, Cr, Cu, Fe, K, Mg, Ni, Sb, Ti, V, Pb) were significantly more in MRONJ sequestra than non-inflammatory bones. The Cu content was significantly higher in MRONJ sequestra than osteomyelitis and non-inflammatory bones. The Cu content in MRONJ sequestra was high even after decalcification. Additionally, Cu was distributed along the trabecular structures in decalcified MRONJ specimens, as observed using SR-XRF analysis. Therefore, this study was indicative of the characteristic behavior of Cu in MRONJ.

Loss of Notch1 predisposes oro-esophageal epithelium to tumorigenesis.

Notch signaling functions in diverse developmental and homeostatic processes, including stem cell self-renewal and cell fate determination. Notch1-inactivating mutations are frequently detected in skin and oro-esophageal cancers, suggesting a role for Notch1 as a tumor suppressor. Here, we clarify the contribution of Notch1 deficiency to oro-esophageal tumorigenesis using a physiological experimental model. Tongue and esophageal tumors induced in mice by 4-nitroquinoline-1-oxide (4-NQO) showed pathophysiological similarities to human tumors, including decreased Notch1 expression in the basal cells. We created mutant mice (N1cKO), in which the Notch1 gene was disrupted specifically in the squamous epithelium. The epithelium formed normally in N1cKO mice, and although multiple skin tumors were detected at 65 weeks, no tumors developed in the tongue and esophagus. However, 4-NQO-induced tumorigenesis assays revealed that tumor onset occurred earlier in N1cKO mice than in wild-type littermates, and the tumors arose preferentially from the Notch1-negative epithelium, indicating the tumor susceptibility of Notch1-deficient epithelium. Notch1 regulates the expression of TERT, and age-related telomere erosion was more rapid in Notch1-deficient basal cells. Our results indicated that although Notch1 deficiency had little effect on squamous epithelium formation, it predisposed the affected epithelium to tumor development, at least in part through accelerated telomere erosion.

Alpha-L-fucosidase-1 is a diagnostic marker that distinguishes mucoepidermoid carcinoma from squamous cell carcinoma.

Alpha-L-fucose is a component of glycans on the cell surface. Alpha-L-fucose is correlated with tumorigenesis and malignancy, and alpha-L-fucosidase-1 (FUCA1), the enzyme that removes terminal α-L-fucose residues from glycoproteins, is downregulated in some high malignancy cancers. The expression profile of FUCA1 in head and neck tumors remains unknown, and we analyzed the expression profiles of FUCA1 and an upstream molecule p53 in mucoepidermoid carcinoma (MEC) and oral squamous cell carcinoma (OSCC). FUCA1 was expressed in most MECs irrespective of histopathological grading, whereas expression in OSCCs was low. High immunohistochemical intensity of p53 was detected in OSCCs at high frequency, but rarely detected in MECs. Genetic mutation analysis using next-generation sequencing revealed no significant mutation of TP53 in MECs. We further analyzed the expression profiles of FUCA1 in normal major and minor salivary glands and found strong expression in the intercalated duct, moderate expression in mucous acinar cells and no expression in serous acinar cells. These contrasting immunohistochemical profiles and anatomical distribution in normal salivary glands suggest that FUCA1 is a useful marker to distinguish MEC from OSCC, and many MECs have similar immunohistochemical phenotypes to intercalated duct and mucous acinar cells.

Metastasis of lower gingival squamous cell carcinoma to buccinator lymph node: case report and review of the literature.

BACKGROUND: Metastasis of oral cancer to the buccinator lymph nodes (BN) is uncommon. The antegrade lymphatic flow in patients with normal anatomy and physiology makes metastasis of lower gingival cancer to BN unlikely. CASE PRESENTATION: A 67-year-old woman presented with a 46 × 25-mm tumor on her lower gingiva, along with metastatic foci in BN and cervical lymph nodes. After neoadjuvant chemotherapy, she underwent radical resection of the primary tumor and BN, along with neck dissection. Following surgery, she received adjuvant chemoradiotherapy. Two years after treatment, there has been no evidence of tumor recurrence or metastasis. CONCLUSION: This is the first report of lower gingival squamous cell carcinoma with metastasis to BN. Metastasis to BN from lower gingival cancer is very rare but should be considered in patients with locally advanced tumors or tumors that metastasize to the submandibular node.

A Novel, Tumor-Induced Osteoclastogenesis Pathway Insensitive to Denosumab but Interfered by Cannabidiol.

Bone metabolism is strictly regulated, and impaired regulation caused by hormonal imbalances induces systemic bone loss. Local bone loss caused by tumor invasion into bone is suggested to be induced by the generation of cytokines, which affect bone metabolism, by tumor cells. The major cause of systemic and local bone losses is excess bone resorption by osteoclasts, which differentiate from macrophages by receptor activator of nuclear factor kappa-B ligand (RANKL) or tumor necrosis factor-alpha (TNF-α). We previously found a novel pathway for tumor-induced osteoclastogenesis targeting osteoclast precursor cells (OPCs). Tumor-induced osteoclastogenesis was resistant to RANKL and TNF-α inhibitors. In the present study, we confirmed that exosomes derived from oral squamous cell carcinoma (OSCC) cells induced osteoclasts from OPCs. We also showed that the depletion of exosomes from culture supernatants of OSCC cells partially interfered with osteoclastogenesis, and cannabidiol, an innoxious cannabinoid without psychotropic effects, almost completely suppressed tumor-induced osteoclastogenesis. Osteoclastogenesis and its interference by cannabidiol were independent of the expression of nuclear factor of T cell c1 (NFATc1). These results show that osteoclastogenesis induced by OSCC cells targeting OPCs is a novel osteoclastogenic pathway independent of NFATc1 expression that is partially caused by tumor-derived exosomes and suppressed by cannabidiol.

A lesion categorized between ghost cell odontogenic carcinoma and dentinogenic ghost cell tumor with CTNNB1 mutation.

Ghost cell odontogenic carcinoma (GCOC) is a rare malignant neoplasm characterized by the presence of ghost cells. It is considered to arise either de novo or from a preexisting benign precursor, calcifying odontogenic cyst (COC), or dentinogenic ghost cell tumor (DGCT). We report a case of a 44-year-old Japanese male with a left maxillary tumor. The patient received treatment to resect the left maxillary cyst 25 years prior; however, the details were uncertain. The tumor was resected with clear margins. Taken together with the results of histological and immunohistochemical examinations, the tumor was categorized between GCOC and DGCT, and we diagnosed the tumor as GCOC suggesting similarity to DGCT. Further, we focused on CTNNB1, which encodes ß-catenin and is frequently mutated in COCs. In this tumor, we identified CTNNB1 Ser33Cys, one of the mutations typically found in COCs. This finding suggests that CTNNB1 is a common target for the pathogenesis of tumors accompanied by ghost cells.

Eos Is Redundant for Regulatory T Cell Function but Plays an Important Role in IL-2 and Th17 Production by CD4+ Conventional T Cells.

Eos belongs to the Ikaros family of transcription factors. It was reported to be a regulatory T cell (Treg) signature gene, to play a critical role in Treg suppressor functions, and to maintain Treg stability. We used mice with a global deficiency in Eos to re-examine the role of Eos expression in both Tregs and conventional T cells (Tconvs). Tregs from Eos-deficient (Eos(-/-)) mice developed normally, displayed a normal Treg phenotype, and exhibited normal suppressor function in vitro. Eos(-/-) Tregs were as effective as Tregs from wild-type (WT) mice in suppressing inflammation in a model of inflammatory bowel disease. Bone marrow (BM) from Eos(-/-) mice was as effective as that from WT mice in controlling T cell activation when used to reconstitute immunodeficient mice in the presence of scurfy fetal liver cells. Surprisingly, Eos was expressed in activated Tconvs and was required for IL-2 production, CD25 expression, and proliferation in vitro by CD4(+) Tconvs. Eos(-/-) mice developed more severe experimental autoimmune encephalomyelitis than WT mice, displayed increased numbers of effector T cells in the periphery and CNS, and amplified IL-17 production. In conclusion, our studies are not consistent with a role for Eos in Treg development and function but demonstrate that Eos plays an important role in the activation and differentiation of Tconvs.

The CCL2-CCR2 Axis in Lymph Node Metastasis From Oral Squamous Cell Carcinoma: An Immunohistochemical Study.

PURPOSE: Cytokine or chemokine networks involve lymphatic and distant metastasis of various malignancies, including oral squamous cell carcinoma (OSCC). Immunohistochemical analysis was used to investigate the contribution of the axis of the CC chemokine receptor-2 (CCR2) and the CC chemokine receptor-2 ligand (CCL2) to lymphatic metastasis, particularly the relation between primary OSCC and marginal sinus histiocytosis in regional lymph nodes. MATERIALS AND METHODS: Thirteen metastasis-free cases, 15 metastatic cases at resection of primary tumor resection, and 13 postresection metastasis cases were examined. No patient was treated with radiotherapy or chemotherapy before neck dissection. Samples were fixed in formalin, embedded in paraffin, and subjected to immunohistochemical analysis using antibodies against CCL2, CCR2, podoplanin, and α-smooth muscle actin (α-SMA). RESULTS: Marginal sinus histiocytosis was frequently observed in metastatic cases. CCL2 was expressed in tumor-associated neutrophils (TANs) and moderately or poorly differentiated SCC was detected at primary tumor sites. TANs expressing CCL2 flowed into the marginal sinus in the lymph nodes. CCR2-positive macrophages and mesenchymal cells infiltrated the tumor stroma and were seen within the carcinoma nests. They were predominantly present in the marginal sinus of metastatic cases. In small metastatic foci, α-SMA-positive spindle cells resembling carcinoma-associated fibroblasts (CAFs) were observed adjacent to the macrophages. CONCLUSION: The CCL2-CCR2 axis is associated with lymphatic metastasis. To clarify the mechanism of lymphatic metastasis from OSCC, further functional analyses of the CCL2-positive TANs, CCR2-positive macrophages, and CAF-like cells detected in this study are recommended.

CD163-Positive Macrophages Within the Tumor Stroma Are Associated With Lymphangiogenesis and Lymph Node Metastasis in Oral Squamous Cell Carcinoma.

PURPOSE: Increasing evidence shows that tumor stromal components, particularly tumor-associated macrophages (TAMs), play an important role in the tumor progression of various solid malignant tumor types. However, their roles in oral squamous cell carcinoma (OSCC) have not been fully elucidated. MATERIALS AND METHODS: Seventy human tongue OSCC samples were analyzed in the present study. Immunohistochemistry was used to investigate the correlations between the densities of CD68-, CD163-, and CD204-positive TAMs and clinicopathologic parameters. Lymphatic vessel density (LVD) was estimated using the D2-40 antibody. In vitro studies also were conducted to investigate the effect of conditioned medium (CM) derived from OSCC cell lines on cytokine and chemokine expression in RAW264.7 mouse monocytic leukemia cells. RESULTS: Increased densities of CD68-, CD163-, and CD204-positive TAMs were significantly correlated with lymph node metastasis (P = .035, .0082, and .038, respectively). Higher LVD occurred considerably more frequently in patients with nodal metastasis than in those without such metastasis. Moreover, LVD was considerably increased in patients with higher CD163-positive TAM densities. Studies using immunofluorescence showed that vascular endothelial growth factor (VEGF)-C was expressed in 52 of 70 patients with CD163-positive TAMs (74.2%). Moreover, CM derived from OSCC cell lines stimulated the expression of Il-10, Ccl22, Vegf-a, and Vegf-c in RAW264.7 cells; however, Il-12p35 expression levels were not changed. CONCLUSION: CD163-positive TAMs promote lymphangiogenesis through VEGF-C expression, which contributes to regional lymph node metastasis in OSCC.