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Immunotherapy with immune‐checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor‐β (TGF‐β) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T‐cells (Tregs) and cancer‐associated fibroblasts and inhibiting the function of cytotoxic T‐lymphocytes (CTLs) and natural killer cells. TGF‐β may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF‐β on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T‐cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. Additionally, TGF‐β inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T‐cell/Treg ratio and between TGFB1 mRNA expression and the Ki‐67 expression in CD8+ T‐cells, indicating that TGF‐β also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF‐β function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune‐checkpoint inhibitors and TGF‐β inhibitors, for OSCCs. We found that, TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. And, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T‐cell/Treg ratio. Inhibition of TGF‐β restores the immunosuppressive TME to active status by a mechanism different from that of immune‐checkpoint inhibitors, suggesting that the combination of both may lead to a new therapeutic strategy for OSCCs.

Thin film compound semiconductor solar cells, including copper indium sulfide (CIS), have potential as next-generation power supply sources. However, recent fabrication processes include gas phase reactions, which consume much energy and generate abundant loss of material resources. Therefore, the development of alternative eco-friendly methods without a gas phase reaction has been actively researched. Herein, we synthesize CIS nanoparticles via a liquid phase reaction using iminodiacetic acid (IDA) and glycine (Gly) as the complex reagent to homogenize copper and indium complexes. The X-ray diffraction (XRD) profiles of the as-synthesized nanoparticles in a buffer solution exhibit broad peaks, which nearly correspond to CIS. These peaks grow sharply after annealing. Selected area electron diffraction and high-resolution transmission electron microscopies indicate the presence of the (112) phase of CIS but the crystallinity of our CIS with IDA and Gly differs. The results indicate that maintaining a homogenized condition of metal complexes by pH stabilization using a buffer solution might be maybe important for aqueous synthesis of CIS.

Regulatory T-cells (Tregs) play a major role in suppressing anti-tumor immune responses. Mogamulizumab, an anti-CC chemokine receptor type 4 (CCR4) monoclonal antibody, depletes effector Tregs (eTregs). However, the clinical efficacy of mogamulizumab was limited in phase Ia/Ib studies for solid tumors (NCT01929486); the finding suggests that mogamulizumab may also deplete beneficial CCR4 + CD8 + T-cells in patients. Therefore, we focused on CTLs and aimed to identify a way to protect CCR4 + CTLs. Here, we evaluated the association of CCR4 expression in cytotoxic T-lymphocytes (CTLs) with antigen and cytokine stimulations and kinase inhibition using cytomegalovirus antigen instead of tumor antigen. CCR4 expression in CTLs was induced by antigen stimulation (mean 3.14–29.0%), enhanced by transforming growth factor-β1 (TGF-β1) (mean 29.0–51.2%), and downregulated by trametinib with (mean 51.2–11.4%) or without TGF-β1 treatment (mean 29.0–6.98%). Phosphorylation of ERK in CD8 + T-cells was suppressed by trametinib. Regarding the effect on immunological function of CTL, trametinib reduced cytokine production but not affected cytotoxicity. Importantly, trametinib alleviated CTL reduction by anti-CCR4 antibody without affecting eTreg depletion because CCR4 expression in eTregs was not downregulated. In conclusion, combination therapy with trametinib may improve the clinical efficacy of mogamulizumab.

The expression of programmed death ligand-1 (PD-L1) is controlled by complex mechanisms. The elucidation of the molecular mechanisms of PD-L1 expression is important for the exploration of new insights into PD-1 blockade therapy. Detailed mechanisms of the in situ expression of PD-L1 in tissues of oral squamous cell carcinomas (OSCCs) have not yet been clarified. We examined the mechanisms of PD-L1 expression focusing on the phosphorylation of downstream molecules of epidermal growth factor (EGF) and interferon gamma (IFN-γ) signaling in vitro and in vivo by immunoblotting and multi-fluorescence immunohistochemistry (MF-IHC), respectively. The in vitro experiments demonstrated that PD-L1 expression in OSCC cell lines is upregulated by EGF via the EGF receptor (EGFR)/PI3K/AKT pathway, the EGFR/STAT1 pathway, and the EGFR/MEK/ERK pathway, and by IFN-γ via the JAK2/STAT1 pathway. MF-IHC demonstrated that STAT1 and EGFR phosphorylation was frequently shown in PD-L1-positive cases and STAT1 phosphorylation was correlated with lymphocyte infiltration and EGFR phosphorylation. Moreover, the phosphorylation pattern of the related molecules in PD-L1-positive cells differed among the cases investigated. These findings indicate that PD-L1 expression mechanisms differ depending on the tissue environment and suggest that the examination of the tissue environment and molecular alterations of cancer cells affecting PD-L1 expression make it necessary for each patient to choose the appropriate combination drugs for PD-1 blockade cancer treatment.

Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucial role in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here, we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)–labeled anti-podoplanin antibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma–xenografted tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin expression–dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using a handheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative, and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection and subsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.

This research explores strategies to support first-year STEM Brazilian undergraduates in two programs, computer science and mathematics, through partnerships among faculty and student members and the implementation of an active methodology known as “Method 300” within an elementary mathematics course. The primary goal of these initiatives was to enhance the academic performance of freshmen. In Method 300, for students to benefit from grade improvements, they must assist each other, regularly attend team meetings, and commit to the activities that constitute the method. The method was implemented only during the first half of the course, fortunately it also implicated into the second part of the course, showing that a tenth of the students who performed poorly at the beginning achieved satisfactory and excellent performance in the last assessment, even though the content was more complex. According to the assessment system of the whole course, by monitoring students who initially had poor academic performance, it was found that half of them achieved satisfactory performance by the end of the course, which contributed to a final approval rate above 73% of all students. The condition of student heterogeneity is crucial for the effective application of Method 300; without it, this could become a potential limitation to its successful implementation. In addition to this strategy, the course was supported by voluntary extracurricular tutors, although these were underutilized by the freshmen, prompting us to consider new ways of providing support. These results suggest that future research in this field should focus on improving students’ academic performance by systematically replicating Method 300 in other courses for freshmen students.

In recent years, intraoperative surgical guides have been widely used in oral and maxillofacial surgery to navigate the resection sites. However, most of them are designed for segmental mandibulectomy and determine only the anterior-posterior cutting sites. In the case of marginal mandibulectomy, the depth and angle of the resection need to be considered in addition to the anterior-posterior cutting site. This report describes a method for creating a translucent mandible model with a colored tumor that enables visualization of the tumor depth and a surgical guide for marginal mandibulectomy with a planned resection angle. If accurate surgical planning and intraoperative navigation are established using this method, personalized surgery is realized according to tumor features and hence avoids over- or under-resection.

Background/Objectives: Oral squamous cell carcinoma (OSCC) often presents at an advanced stage; therefore, the early detection of precursor lesions is crucial. However, the risk assessment of precursor lesions such as oral epithelial dysplasia (OED) remains challenging because of the subjectivity of histopathological grading. We aimed to identify molecular markers that enhance the diagnostic accuracy and prognostic stratification of OSCC and explore the differences in the molecular characterization of OED and OSCC using a few selected markers. Methods: A two-step diagnostic workflow was applied: (1) FISH evaluation of EGFR amplification and CDKN2A deletion to distinguish OED from OSCC and identify EGFR-dependent tumors, and (2) HRAS immunohistochemistry performed exclusively in EGFR-negative OSCCs to stratify EGFR-independent cases. Fluorescence in situ hybridization (FISH) was used to assess seven EGFR/cell cycle-related genes (CCND1, CDKN2A, EGFR, PIK3CA, PTEN, TP53, and 1p36 locus) in 117 formalin-fixed paraffin-embedded samples (66 OED and 51 OSCC) and 10 normal mucosa samples. HRAS expression was evaluated using immunohistochemistry (IHC) in 36 EGFR amplification-negative OSCCs samples. Results:EGFR amplification was frequent in OSCC, whereas CDKN2A deletion was common in OED. The EGFR-amplified/ CDKN2A-intact profile showed high specificity for OSCC and improved diagnostic performance (area under the curve = 0.77) when combined with the Ki-67 labeling index. It also predicted poor disease-free survival (hazard ratio [HR] = 5.08, p = 0.016) and overall survival (HR = 6.10, p = 0.047). Among EGFR-negative OSCCs, HRAS overexpression was associated with advanced-stage disease and a poor prognosis (HR = 6.15, p = 0.043). Conclusions:EGFR amplification was frequent in OSCC, and CDKN2A deletion was prevalent in OED, supporting their use as molecular markers for differential diagnoses. FISH for EGFR/CDKN2A and HRAS IHC can stratify OSCC by diagnosis and prognosis, enabling practical molecular subclassification, including EGFR-negative cases.

Oral cancer may become advanced because of delay in diagnosis. In order to promote oral cancer screening, simple and highly reliable screening methods that can be implemented at general dental clinics are required. Herein we investigated differential salivary gene expression between oral squamous cell carcinoma (OSCC) patients and healthy volunteers (HV) to identify new biomarkers for OSCC detection. Candidate genes were selected by microarrays, nuclear undecaprenyl pyrophosphate synthase 1 (NUS1) and reticulocalbin 1 (RCN1) were selected for further investigation. We used real-time quantitative reverse transcription PCR (qRT-PCR) to determine NUS1 and RCN1 expression levels in saliva and tissues. qRT-PCR analysis of clinical samples revealed that OSCC patients had significantly higher expression of salivary NUS1 and RCN1 than HV. A combination of NUS1 and RCN1 accurately distinguished patients from controls, and this combination can be implemented as a screening test for OSCC.

Heated tobacco products (HTPs) appear to be less harmful to health than conventional cigarettes (CCs). However, limited analytical data are available to support this claim. This study aimed to compare the cytotoxic, genotoxic, and toxicogenomic effects of HTPs and CCs in carcinogenesis via multistep gene mutations in the oral mucosal cells.INTRODUCTIONHeated tobacco products (HTPs) appear to be less harmful to health than conventional cigarettes (CCs). However, limited analytical data are available to support this claim. This study aimed to compare the cytotoxic, genotoxic, and toxicogenomic effects of HTPs and CCs in carcinogenesis via multistep gene mutations in the oral mucosal cells.Cigarette smoke extract (CSE) was obtained from HTPs and CCs. Primary human oral keratinocytes (HOKs) were treated with 5% and 20% CSE from HTPs and CCs. Cell survival rate assays were performed after 6, 12, and 24 h. After 6 h, DNA double-strand breaks (DSBs) were evaluated using anti-γH2AX antibodies with immunohistochemistry. mRNAs expressions of mediator of DNA damage checkpoint 1 (MDC1) and ataxia telangiectasia and Rad3-related protein (ATR), were analyzed. Expressions of miR-22 and miR-185 were analyzed because miR-22 targets MDC1 and miR-185, ATR.METHODSCigarette smoke extract (CSE) was obtained from HTPs and CCs. Primary human oral keratinocytes (HOKs) were treated with 5% and 20% CSE from HTPs and CCs. Cell survival rate assays were performed after 6, 12, and 24 h. After 6 h, DNA double-strand breaks (DSBs) were evaluated using anti-γH2AX antibodies with immunohistochemistry. mRNAs expressions of mediator of DNA damage checkpoint 1 (MDC1) and ataxia telangiectasia and Rad3-related protein (ATR), were analyzed. Expressions of miR-22 and miR-185 were analyzed because miR-22 targets MDC1 and miR-185, ATR.The HOKs had equivalent survival rates after exposure to the same concentrations of CSE from CCs and HTPs. HTPs increased foci formation of γH2AX in HOKs, as did CCs (without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of MDC1 and ATR decreased in cells exposed to CSE from CCs and HTPs (MDC1: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05; ATR: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of miR-22 and miR-185 were not significantly increased when exposed to CSE from CCs or HTPs.RESULTSThe HOKs had equivalent survival rates after exposure to the same concentrations of CSE from CCs and HTPs. HTPs increased foci formation of γH2AX in HOKs, as did CCs (without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of MDC1 and ATR decreased in cells exposed to CSE from CCs and HTPs (MDC1: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05; ATR: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of miR-22 and miR-185 were not significantly increased when exposed to CSE from CCs or HTPs.HTPs and CCs had similar cytotoxic effects. HTPs are genotoxic, can cause DSBs, and have toxicogenomic damage because they inhibit the MDC1 and ATR-CHK1 DNA repair pathways in the oral mucosa. The miRNA-mRNA axis was not related to these inhibitions.CONCLUSIONSHTPs and CCs had similar cytotoxic effects. HTPs are genotoxic, can cause DSBs, and have toxicogenomic damage because they inhibit the MDC1 and ATR-CHK1 DNA repair pathways in the oral mucosa. The miRNA-mRNA axis was not related to these inhibitions.