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The objective of the current study was to elucidate the clinicopathological significance and appearance of in vitro three-dimension (3D) spheroid models of oral malignant tumors that were prepared from four pathologically different squamous cell carcinoma (OSCC; low-grade; SSYP and MO-1000, intermediate-grade; LEM2) and oral adenosquamous carcinoma (OASC; high-grade; Mesimo) obtained from patients with different malignant stages. To characterize the biological significance of these cell lines themselves, two-dimensional (2D) cultured cells were subjected to cellular metabolic analysis by a Seahorse bioanalyzer alongside the measurement of the cytotoxicity of cisplatin (CDDP). The appearance of their 3D spheroids was then observed by phase contrast microscopy, and both 2D and 3D cultured cells were subject to trypsin digestion and qPCR analysis of factors related to oncogenic signaling and other related analyses. ATP-linked respiration and proton leaking were significantly different among the four cell lines, and the malignant stages of these cultures were significantly associated with increased ATP-linked respiration and decreased proton leakage. Alternatively, the appearances of these 3D spheroids were also significantly diverse among them, and their differences increased in the order of LEM2, MO-1000, SSYP, and Mesimo. Interestingly, these orders were exactly the same in that the efficacies of CDDP-induced cytotoxicity increased in the same order. qPCR analysis indicated that the levels of expression of oncogenic signaling-related factors varied among these four cell lines, and the values for fibronectin and a key regulator of mitochondrial biogenesis, PGC-1α, were prominently elevated in cultures of the worst malignant Mesimo cells. In addition, although 0.25% trypsin-induced destruction was comparable among all four 2D cultured cells, the values for the 3D spheroids were also substantially varied among these cultures. The findings reported herein indicate that cellular metabolic functions and 3D spheroid architectures may be valuable and useful indicators for estimating the pathological and drug-sensitive aspects of OSCC and OASC malignancies.

Long noncoding RNAs (lncRNAs) are deeply involved in cancer development. We previously reported that DLEU1 (deleted in lymphocytic leukemia 1) is one of the lncRNAs overexpressed in oral squamous cell carcinoma (OSCC) cells, where it exhibits oncogenic activity. In the present study, we further clarified the molecular function of DLEU1 in the pathogenesis of OSCC. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that DLEU1 knockdown induced significant changes in the levels of histone H3 lysine 4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27ac) in OSCC cells. Notably, DLEU1 knockdown suppressed levels of H3K4me3/ H3K27ac and expression of a number of interferon-stimulated genes (ISGs), including IFIT1, IFI6 and OAS1, while ectopic DLEU1 expression activated these genes. Western blot analysis and reporter assays suggested that DLEU1 upregulates ISGs through activation of JAK-STAT signaling in OSCC cells. Moreover, IFITM1, one of the ISGs induced by DLUE1, was frequently overexpressed in primary OSCC tumors, and its knockdown inhibited OSCC cell proliferation, migration and invasion. These findings suggest that DLEU1 exerts its oncogenic effects, at least in part, through activation of a series ISGs in OSCC cells.

Background/aim The role of tumour-infiltrating CD45Ro+ T-cells in oral squamous cell carcinoma (OSCC) is unclear. This study aimed to evaluate prognostic biomarkers for OSCC. Patients and methods We determined the density of tumour-infiltrating CD45Ro+ T cells in the parenchyma and stroma at the tumour centre (TCe) and invasive front (IF) and examined the association between the density of these cells and histopathological status in 142 patients. Results Five-year overall survival (OS) and recurrence-free survival were favourable in patients with high CD45Ro+ T-cell density in the TCe stroma. OS was favourable in patients with high CD45Ro+ T-cell density in the IF stroma. Stepwise Cox regression model analysis indicated that CD45Ro+ T-cells in the stroma of the IF and TCe were an independent prognostic factor for OS. Conclusion CD45Ro+ T-cells in the stroma of the IF and TCe play a role in cancer immune surveillance and may be a useful prognostic factor.

The CXCL12/CXCR4 axis plays a pivotal role in the progression of various malignancies, including oral squamous cell carcinoma (OSCC). In this study, we aimed to clarify the biological and clinical significance of CXCL12 in the tumor microenvironment of OSCCs.Publicly available single-cell RNA-sequencing (RNA-seq) datasets were used to analyze CXCL12 expression in head and neck squamous cell carcinomas (HNSCC). Immunohistochemical analysis of CXCL12, α-smooth muscle antigen (α-SMA), fibroblast activation protein (FAP) and CD8 was performed in a series of 47 surgically resected primary tongue OSCCs. Human skeletal muscle cells were co-cultured with or without OSCC cells, after which CXCL12 expression was analyzed using quantitative reverse-transcription PCR.Analysis of the RNA-seq data suggested CXCL12 is abundantly expressed in stromal cells within HNSCC tissue. Immunohistochemical analysis showed that in grade 1 primary OSCCs, CXCL12 is expressed in both tumor cells and muscle cells. By contrast, grade 3 tumors were characterized by disruption of muscle structure and reduced CXCL12 expression. Quantitative analysis of CXCL12-positive areas within tumors revealed that reduced CXCL12 expression correlated with poorer overall survival. Levels of CXCL12 expression tended to inversely correlate α-SMA expression and positively correlate with infiltration by CD8+ lymphocytes, though these relations did not reach statistical significance. CXCL12 was significantly upregulated in muscle cells co-cultured with OSCC cells.Our results suggest that tongue OSCC cells activate CXCL12 expression in muscle cells, which may contribute to tumor progression. However, CXCL12 is reduced in advanced OSCCs due to muscle tissue destruction.

Immune checkpoint inhibitor-based cancer immunotherapy has provided an additional therapeutic option for oral squamous cell carcinoma (OSCC) with recurrence or distant metastases. However, further improvement of OSCC treatment is required to develop the optimal combination or order for chemoradiotherapy and immunotherapy. Along with the accumulation of clinical knowledge and evidence, it is also essential to clarify the biological impact of chemo-radiotherapeutic agents on the cancer immune microenvironment. In this study, we investigated the effects of cisplatin (CDDP), a key therapeutic agent for OSCC, on programmed death-ligand 1 (PD-L1) expression in OSCC lines. Although CDDP treatment increased the surface levels of PD-L1 on OSCC cell lines, the gene and total protein expression levels of PD-L1 were not altered. We also demonstrated that the phosphorylation of heat shock factor 1 and heat shock protein 90 was involved in this process. In addition, CDDP-induced PD-L1 attenuated the target-specific cytotoxic T lymphocyte reaction to OSCC. These results provide an immunobiological basis for the response of OSCC to CDDP and will contribute to our biological understanding of the action of novel combination therapy including immunotherapy together with platinum-based chemotherapy for OSCC.

Background/aim The tumor microenvironment (TME) balances tumor growth and suppression through humoral factors and cell-cell interactions. In oral squamous cell carcinoma (OSCC), TMEs have been associated with prognosis of cancer patients and are evaluated by microscopy; however, these methods of evaluation vary among studies. Materials and methods To evaluate the TME, borderline microenvironment fibrosis (bMF) was evaluated histologically in 236 OSCC cases and used to determine the clinicopathological status. Results bMF was observed in 47% (110 in 236 cases) of OSCC cases and associated with higher T category, N category, stage, histological grade and mode of invasion. bMF-positive was related to overall survival (OS) and progression-free survival (PFS). Multivariate analysis revealed that bMF-positive was an independent factor for OS in all cases [n=226; HR=1.683 (1.018-2.781); p=0.042], especially in T1+T2 cases [n=186; HR=1.926 (1.079-3.440); p=0.024], and PFS in all cases [n=226; HR=2.254 (1.397-3.637); p=0.001]. Conclusion bMF may act as a novel biomarker for OSCC.

Human leukocyte antigen (HLA) class Ⅰ molecules play a central role in anticancer immunity, but their prognostic value in oral squamous cell carcinoma (OSCC) remains unclear. We examined HLA class I expression in 2 distinct tumor compartments, namely, the tumor center and invasive front, and evaluated the association between its expression pattern and histopathological status in 137 cases with OSCC. Human leukocyte antigen class Ⅰ expression was graded semiquantitatively as high, low, and negative. At the invasive front of the tumor, HLA class I expression was high in 72 cases (52.6%), low in 44 cases (32.1%), and negative in 21 cases (15.3%). The HLA class I expression in the tumor center was high in 48 cases (35.0%), low in 58 cases (42.4%), and negative in 31 cases (22.6%). The 5‐year overall survival and disease‐specific survival rates were good in cases with high HLA class I expression at the invasive front; however, there was no significant difference in survival based on HLA class I expression in the tumor center. In addition, high HLA class I expression was correlated with high CD8+ T cell density, whereas negative HLA class I expression was correlated with low CD8+ T cell density at the invasive front. These results suggest that it is easier for CD8+ T cells to recognize presented peptides in the case of high HLA class Ⅰ expression at the tumor invasive front and could be a prognostic factor for OSCC., Human leukocyte antigen class Ⅰ expression at the tumor invasive front could be a useful prognostic factor for oral squamous cell carcinoma.

BACKGROUND The presence of tumor-infiltrating lymphocytes (TILs) is associated with improved survival in head and neck squamous cell carcinoma. However, the prognostic value of TILs remains unclear in oral squamous cell carcinoma (OSCC). METHODS We evaluated the associations between tumor-infiltrating CD8+ T-cell density and survival in five distinct compartments in 139 OSCC cases. RESULTS There was a significant association between increased tumor-infiltrating CD8+ T cells and their distribution. High parenchymal CD8+ T-cell density at the invading tumor edge was associated with improved overall survival (OS) and disease-specific survival (DSS; P

Objective Epithelial–mesenchymal transition (EMT) plays an important role in cancer invasion and metastasis induced by hypoxia. Here, we examined whether phosphorylation of GSK3-β via phosphoinositide 3-kinase (PI3 K)/Akt signaling is involved in enhancing the hypoxia-induced EMT in oral squamous cell carcinoma (OSCC). Study Design Experiments were performed in OSCC cell lines (HSC-2, HSC-3, HSC-4, SAS, and HO-1-U-1) under normoxic or hypoxic conditions. The EMT was assessed by Matrigel invasion assays and wound healing assays. OSCC cell lines (HSC-2 and HSC-4) overexpressing hypoxia-inducible factor (HIF)-1α were established to examine the effects of HIF-1α on EMT-related factors. Immunohistochemical staining was performed to examine phosphorylation of GSK3-β in 33 cases of tongue squamous cell carcinoma. Results Under hypoxic conditions, OSCC cell lines exhibited HIF-1α expression and showed evidence of the EMT. In cells overexpressing HIF-1α, the levels of phospho-Akt and phospho-GSK3-β were increased, resulting in induction of the EMT. Inhibition of GSK3-β phosphorylation suppressed these effects. Moreover, the intensity of pGSK3-β staining was significantly increased with cN stage and cTNM stage in patients with tongue squamous cell carcinoma. Conclusions Our data showed that the hypoxia-induced EMT in OSCC was enhanced by GSK3-β phosphorylation, suggesting that GSK3-β may be important in the invasion and metastasis of OSCC.

Background Tumor-infiltrating lymphocytes include tumor-reactive lymphocytes and regulatory T-cells. However, the prognostic value of tumor-infiltrating lymphocytes in oral squamous cell carcinoma (OSCC) remains unclear. Methods We used immunohistochemistry to evaluate the presence of tumor-infiltrating FoxP3⁺ T-cells and CTLA-4⁺ cells in four distinct histological compartments (tumor parenchyma and stroma at the tumor center, and parenchyma and stroma at the invasive front) and assessed the association between the prevalence of these cells and the histopathological status of 137 patients with OSCC. Results Five-year overall survival, disease-specific survival, and recurrence-free survival were favorable in patients with high numbers of FoxP3⁺ T-cells in the parenchyma of the invasive front. Recurrence-free survival and metastasis-free survival were decreased in patients with high numbers of CTLA-4⁺ cells in the parenchyma of the invasive front. Conclusions The presence of FoxP3⁺ T-cells in the parenchyma of the invasive front may be a useful prognostic factor. Our results indicate that FoxP3⁺ T-cells may exert site-specific anti-tumor effects but may not play an immunosuppressive role in OSCC. In addition, our results suggest that CTLA-4+ cells suppress the function of FoxP3+ T-cells and promote anti-tumor immunity in OSCC.

Recent studies have shown that long noncoding RNAs (lncRNAs) have pivotal roles in human malignancies, although their significance in oral squamous cell carcinoma (OSCC) is not fully understood. In the present study, we identified lncRNAs functionally associated with OSCC. By analyzing RNA-seq datasets obtained from primary head and neck squamous cell carcinoma (HNSCC), we identified 15 lncRNAs aberrantly expressed in cancer tissues. We then validated their expression in 18 OSCC cell lines using qRT-PCR and identified 6 lncRNAs frequently overexpressed in OSCC. Among those, we found that knocking down DLEU1 (deleted in lymphocytic leukemia 1) strongly suppressed OSCC cell proliferation. DLEU1 knockdown also suppressed migration, invasion, and xenograft formation by OSCC cells, which is suggestive of its oncogenic functionality. Microarray analysis revealed that DLEU1 knockdown significantly affects expression of a number of cancer-related genes in OSCC cells, including HAS3, CD44, and TP63, suggesting that DLEU1 regulates HA-CD44 signaling. Expression of DLEU1 was elevated in 71% of primary OSCC tissues, and high DLEU1 expression was associated with shorter overall survival of HNSCC patients. These data suggest that elevated DLEU1 expression contributes to OSCC development, and that DLEU1 may be a useful therapeutic target in OSCC.

Colorectal cancer consists of a small number of cancer stem cells (CSC) and many non-CSCs. Although rare in number, CSCs are a target for cancer therapy, because they survive conventional chemo- and radiotherapies and perpetuate tumor formation in vivo. In this study, we conducted an HLA ligandome analysis to survey HLA-A24 peptides displayed by CSCs and non-CSCs of colorectal cancer. The analysis identified an antigen, ASB4, which was processed and presented by a CSC subset but not by non-CSCs. The ASB4 gene was expressed in CSCs of colorectal cancer, but not in cells that had differentiated into non-CSCs. Because ASB4 was not expressed by normal tissues, its peptide epitope elicited CD8+ cytotoxic T-cell (CTL) responses, which lysed CSCs of colorectal cancer and left non-CSCs intact. Therefore, ASB4 is a tumor-associated antigen that can elicit CTL responses specific to CSCs and can discriminate between two cellular subsets of colorectal cancer. Adoptively transferred CTLs specific for the CSC antigen ASB4 could infiltrate implanted colorectal cancer cell tumors and effectively prevented tumor growth in a mouse model. As the cancer cells implanted in these mice contained very few CSCs, the elimination of a CSC subset could be the condition necessary and sufficient to control tumor formation in vivo. These results suggest that CTL-based immunotherapies against colorectal CSCs might be useful for preventing relapses. Cancer Immunol Res; 6(3); 358–69. ©2018 AACR.

Background It has well known that, compared to normal cells, tumor cells have a different manner of energy metabolism, which influences the sensitivity of radiotherapy (RT). However, whether inhibition of glycolysis enhances the efficacy of radiotherapy is a matter of debate in oral squamous cell carcinoma (OSCC). The aim of this study was to characterize whether the combination of radiotherapy with the glucose inhibitor 2-deoxy-D-glucose (2-DG) affected DNA repair kinetics. Methods To compare the synergistic effect of 2-DG, we examined the cell survival after treatment with radiation, 2-DG and a combination of the two in 5 OSCC cell lines and one lip fibroblast cell line, determined using clonogenic survival assay. Changes in the protein levels of DNA repair kinetics such as PARP, Rad51 and Ku-70 were analyzed by western blotting. Then, using one of the 5 OSCC cell lines, we assessed the inhibition of xenograft tumor growth in vivo. Results We found that 2-DG with radiation induced significant inhibition of cell proliferation in cell line SAS (p

Background It is essential that cytotoxic T-lymphocytes (CTLs) recognize tumor-associated antigens (TAAs) and exhibit antitumor effects in immunological responses for tumor rejection. However, only a few cases with oral squamous cell carcinoma (OSCC) have been studied for these antitumor mechanisms. We established a cancer cell line and autologous CTL pair for identifying TAAs in OSCC, despite difficulties in establishing such a pair. Materials and methods A cell line (OTM) from a primary lesion and a CTL line (TcOTM) were derived from OSCC in a 64-year-old female patient. CTL clones were generated by repetitive limiting dilutions. Accurate characterization was performed by in vitro analysis. Results The TcOTM clone showed specific cytotoxic activity against OTM cells in an human leukocyte antigen (HLA)-A24-restricted manner. Furthermore, it exerted cytotoxicity against the allogenic HLA-A24 cell lines. Conclusion These data indicated that the TcOTM clone recognized a TAA presented by HLA-A24 cells. This unique method will allow for identification of unknown TAAs for OSCC in the future.

The most important prognostic factor in oral squamous cell carcinoma (OSCC) is neck metastasis, which is treated by neck dissection. Although selective neck dissection (SND) is a useful tool for clinically node-negative OSCC, its efficacy for neck node-positive OSCC has not been established. Sixty-eight OSCC patients with pN1&ndash, 3 disease who were treated with curative surgery using SND and/or modified-radical/radical neck dissection (MRND/RND) were retrospectively reviewed. The neck control rate was 94% for pN1&ndash, 3 patients who underwent SND. The five-year overall survival (OS) and disease-specific survival (DSS) in pN1-3 OSCC patients were 62% and 71%, respectively. The multivariate analysis of clinical and pathological variables identified the number of positive nodes as an independent predictor of SND outcome (OS, hazard ratio (HR) = 4.98, 95% confidence interval (CI): 1.48&ndash, 16.72, p &lt, 0.01, DSS, HR = 6.44, 95% CI: 1.76&ndash, 23.50, p &lt, 0.01). The results of this retrospective study showed that only SND for neck node-positive OSCC was appropriate for those with up to 2 lymph nodes that had a largest diameter &le, 3 cm without extranodal extension (ENE) of the neck and adjuvant radiotherapy. However, the availability of postoperative therapeutic options for high-risk OSCC, including ENE and/or multiple positive lymph nodes, needs to be further investigated.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are considered to be essential for tumor maintenance, recurrence and metastasis. Therefore, eradication of CSCs/CICs is essential to cure cancers. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we investigated the molecular mechanism of the cell growth of oral CSCs/CICs. Oral CSCs/CICs were isolated as aldehyde dehydrogenase 1 bright (ALDH1(br)) cells by the ALDEFLUOR assay. Small proline-rich protein-1B (SPRR1B) gene was shown to be overexpressed in ALDH1(br) cells by a cDNA microarray and RT-PCR. SPRR1B was shown to have a role in cell growth and maintenance of ALDH1(br) cells by SPRR1B overexpression and knockdown experiments. To elucidate the molecular mechanism by which SPRR1B regulates cell growth, further cDNA microarray analysis was performed using SPRR1B-overexpressed cells and cells with SPRR1B knocked down by siRNA. Expression of the tumor suppressor gene Ras association domain family member 4 (RASSF4) was found to be suppressed in SPRR1B-overexpressed cells. On the other hand, the expression of RASSF4 was enhanced in cells in which SPRR1B expression was knocked down by SPRR1B-specific siRNA. RASSF4 has an RA (Ras association) domain, and we thus hypothesized that RASSF4 modulates the MAP kinase signal downstream of the Ras signal. MAP kinase signal was activated in SPRR1B-overexpressed cells, whereas the signal was suppressed in SPRR1B knocked down cells. Taken together, the results indicate that the expression of SPRR1B is upregulated in oral CSCs/CICs and that SPRR1B has a role in cell growth by suppression of RASSF4.

Background: Antitumor effects of chemotherapeutic agents are commonly associated with the induction of apoptosis. Bax belongs to the Bcl-2 family and induces apoptosis. The present study was conducted to investigate the relationship between enhanced Bax expression in oral squamous cell carcinoma (SCC; cell lines and clinical cases) and the antitumor effects of chemotherapy. Methods: In three oral SCC cell lines, Bax expression before and after treatment with chemotherapeutic agents [docetaxel (TXT), cisplatin and 5-fluorouracil] was examined by reverse transcriptase-polymerase chain reaction and immunoblotting. The effects of treatment were assessed by counting the number of viable cells and determining sub-G1 cells. Tissue samples (both biopsy specimens before chemotherapy and surgically excised specimens after chemotherapy) from nine patients with oral SCC who underwent neoadjuvant chemotherapy were immunostained for Bax. The relationship between enhancement of Bax expression and chemotherapeutic effects was established. Results: Two of three cell lines did not express Bax mRNA or protein before treatment. After treatment, Bax expression was enhanced only by TXT in one cell line, but by all chemotherapeutic agents in the other two cell lines. In three of nine patients, Bax expression was not found before chemotherapy. Two of these three patients showed enhanced Bax expression after chemotherapy including TXT, but one still failed to express Bax. Both in cell lines and clinical cases, enhancement of Bax after chemotherapy was associated with antitumor effects. Conclusion: Certain chemotherapeutic agents enhance Bax expression in oral SCC, and it is suggested that this contributes to the antitumor effects of chemotherapy.

A large number of human tumor antigens recognized by CD8+ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4+ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC-20, was effectively lysed by HLA-DRB1*08032 (HLA-DR8)-restricted autologous CD4+ T cell line, TcOSC-20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse-phase high-performance liquid chromatography (RP-HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano-liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA-DR8 binding motif, and TcOSC-20 showed a rather strong cytotoxic response to one of the synthetic peptides, namely, amino acid residues 321-336 of human alpha-enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4+ T cells.

口腔扁平上皮癌における核DNA量の予後因子としての意義を検討した。イメージサイトメトリーを用いて, 197例の口腔扁平上皮癌の生検組織のパラフィン包埋切片における腫瘍発育先進部の癌細胞の核DNA量を測定した。得られたDNAヒストグラムの評価として, 超4c率 (4cを越える癌細胞の比率) を用いた。197例の超4c率は最低値0から最高値51%にわたり, 中央値は9.7%であった。各種臨床病理組織所見との関連をみると, 癌の進展と悪性度との関連がみられた。全症例の5年累積生存率は75%で, 超4c率9.7%未満群: 90%, 超4c率9.7%以上群: 60%であった。癌浸潤様式 (山本・小浜分類) の各モードごとに超4c率と生存率との関係をみると, モード1および2を除き, 超4c率が低値の群で良好な傾向がみられたが, 推計学的に有意差がみられたのはモード3のみであった。そこで, モード3のみを対象に予後因子の多変量解析を行ったところ, 超4c率が最も独立性の高い因子であった。以上より, 超4c率は有用な口腔癌の予後因子となり得ることが示唆された。

Although CD8+ killer T cells reacting against human autologous tumor cells have recently been studied in detail, little is known about the cytotoxic mechanism of CD4+ T cells against such tumor cells. In order to investigate this, we have established CD4+ cytotoxic T lymphocyte TcOSC-20 lines. TcOSC-20 showed selective cytotoxic activity against autologous OSC-20 cells, derived from a cancer of the tongue, in an HLA-DR-restricted fashion. HLA-DR8 (DRB1*08032) is the only DR molecule expressed on OSC-20 cells, and anti-DR8 monoclonal antibody could inhibit the cytotoxicity, suggesting that HLA-DRB1*08032 is the tumor rejection antigen-presenting molecule to TcOSC-20. The Fas ligand was expressed on TcOSC-20 lines, and its expression was induced upon mixed lymphocyte-tumor cell culture of autologous peripheral blood lymphocytes. Furthermore, the cytotoxicity of TcOSC-20 was inhibited by anti-Fas ligand antibody. These data imply that TcOSC-20 lines recognize the tumor antigenic peptide presented by HLA-DR8, and exert cytotoxicity against autologous tumor cells via a Fas-mediated cytotoxic pathway.

Background During tumor development, cells are exposed to a hypoxic microenvironment. Tumor hypoxia also has a profound influence on the sensitivity of cancer chemotherapy. The objective of this study was to investigate the mechanism of cisplatin (CDDP) resistance of oral squamous cell carcinoma (OSCC) cells under hypoxia by analyzing gene expression profiles to identify key genes and factors involved. Methods Cell viability was measured following culture of the cells in the presence or absence of CDDP, under normoxic or hypoxic conditions, using a CCK-8 assay. Analysis of the expression of HIF target genes in hypoxia-treated cells was performed using an HIF-regulated cDNA plate array. Changes in the mRNA expression of selected HIF target genes were analyzed using RT-PCR, and changes in the protein levels of these genes were analyzed by Western blotting. Tumor cell apoptosis was assessed by flow cytometry. Results The OSCC cell lines responded differently to CDDP under normoxic and hypoxic conditions. The expression of glucose transporter protein-1 (GLUT-1) was up-regulated in human squamous cell carcinoma of mouth (HSC-2) cells under hypoxia. Furthermore, there was little correlation between the cisplatin sensitivity of human squamous cell carcinoma of tongue (SAS) in normoxia and hypoxia. After GLUT-1 knockdown, CDDP treatment resulted in increased rates of apoptosis under hypoxia as compared with normoxia in cell lines HSC-2, Ca9-22, and SAS (P = 0.025). Conclusion The results of this study suggest that knockdown of GLUT-1 inhibits sensitization of oral squamous cells to CDDP during hypoxia in HSC-2, Ca9-22, and SAS cells.

For the doctors and other medical staff treating oral cancers, it is necessary to standardize basic concepts and rules on oral cancers to progress in the treatment, research and diagnosis. Oral cancers are integrated in head and neck cancers and are applied to the general rules on head and neck cancer, but it is considered that more detailed rules based on the characteristics of oral cancers are essential. The objectives of this 'General Rules for Clinical and Pathological Studies on Oral Cancer' are to contribute to the development of the diagnosis, treatment and research of oral cancers based on the correct and useful medical information of clinical, surgical, pathological and image findings accumulated from individual patients at various institutions.

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is abundantly expressed in most malignancies, but is hardly detectable in normal adult tissues. Previously we have identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8(+) cytotoxic T lymphocytes (CTL). Survivin-2B80-88-specific CTL were induced efficiently from peripheral blood mononuclear cells (PBMC) of oral cancer patients after stimulation with the peptide in vitro. We conducted a phase I clinical study to evaluate the safety and the efficacy of survivin-2B80-88 peptide vaccination in HLA-A24-positive patients with advanced or recurrent oral cancer. The vaccines were given subcutaneously or intratumorally six times at 14-day intervals. Eleven patients were enrolled and 10 patients completed the vaccination protocol. No adverse events were observed in any patients. In two patients, the levels of serum squamous cell carcinoma (SCC) antigen decreased transiently during the period of vaccination. Tumor regression that was compatible with a partial response (PR) was noted in one patient. The remaining nine patients experienced progressive disease (PD). Immunologically, an increase of the peptide-specific CTL frequency was detected in six of the eight patients evaluated by HLA-A24/peptide tetramer analysis. The present clinical trial revealed that survivin-2B peptide vaccination was safe and had therapeutic potential for oral cancer patients. However, subsequent clinical trials in combination with various adjuvant drugs will be required to improve the immunological and therapeutic efficacy. This trial was registered with University Hospital Medical Information Network (UMIN) number UMIN000000976.

Summary The purpose of this clinical trial was to assess the toxicity and efficacy of docetaxel plus nedaplatin induction chemotherapy in patients with locally advanced oral squamous cell carcinoma (OSCC). Twenty-one patients were enrolled in this phase I/II clinical study. The toxicities, response rates, and the maximum tolerated dose of nedaplatin that could be safely given preoperatively were assessed. Patients received escalating doses of nedaplatin (60, 70, 80, 90 mg/m 2 ) combined with a fixed dose of docetaxel (60 mg/m 2 ) on day one. Dose-limiting toxicity (DLT), grade 4 leukopenia lasting for two days or more, was seen in one patient at dose level 3; no other DLT was observed at any dose level. The overall response rate was 66.7%. The response rate was 100% at nedaplatin dose level 4, while that at dose level 1 was low (33.3%). Given these results, the recommended dose of nedaplatin in this regimen combined with fixed dose docetaxel (60 mg/m 2 ) was determined to be 90 mg/m 2 . Docetaxel 60 mg/m 2 plus nedaplatin 90 mg/m 2 induction chemotherapy can be recommended for patients with locally advanced oral squamous cell carcinoma. Based on these results, an early phase II clinical study using this dose level was conducted; docetaxel plus nedaplatin induction chemotherapy appears to be a useful regimen for the treatment of OSCC. A late phase II clinical study is warranted.