Oral Squamous Cell Carcinoma Xenograft Research Articles

Porphyromonas gingivalis Promotes Oral Squamous Cell Carcinoma Progression by Modulating Autophagy.

Porphyromonas gingivalis (P. gingivalis) is a keystone periodontal pathogen associated with various gastro-intestinal tract cancers. However, whether P. gingivalis can promote oral squamous cell carcinoma (OSCC) and the underlying mechanism associated with such promotion remain unclear. In this study, OSCC xenograft models were used to evaluate the effects of P. gingivalis on tumor progression. The functional studies were done on several OSCC cell lines invitro. P. gingivalis-specific 16S rRNA fluorescent insitu hybridization (FISH) was used to test its prevalence in clinical samples. We found that P. gingivalis increased tumor volume and tumor growth in OSCC nude models. Functional studies demonstrated that P. gingivalis inhibited the apoptosis of OSCC cells by promoting cellular autophagy. P. gingivalis was more prevalent in FISH samples from patients with OSCC than from patients with leukoplakia or healthy subjects (70% vs. 47.2% vs. 33.3%, p = 0.045 and p < 0.001, respectively). These data suggest that P. gingivalis plays an accelerating role in OSCC progression and contributes to OSCC by enhancing the autophagy pathway to reduce carcinoma apoptosis.

The critical role of Gαi3 in oral squamous cell carcinoma cell growth.

The identification of novel and effective therapeutic targets for oral squamous cell carcinoma (OSCC) is of paramount importance. This study investigates the expression, potential functions, and mechanistic insights of G protein inhibitory subunit 3 (Gαi3) in OSCC. Gαi3 is found to be upregulated in human OSCC tissues as well as in various primary and established OSCC cells. In different OSCC cells, silencing of Gαi3 through shRNA resulted in inhibited cell proliferation and migration, while also inducing apoptosis. Knockout (KO) of Gαi3 via the CRISPR/Cas9 method produced significant anti-cancer effects in OSCC cells. Conversely, ectopic overexpression of Gαi3 enhanced OSCC cell growth, promoting cell proliferation and migration. Gαi3 plays a crucial role in activating the Akt-mTOR signaling pathway in OSCC cells. Silencing or KO of Gαi3 led to decreased phosphorylation levels of Akt and S6K, whereas overexpression of Gαi3 increased their phosphorylation. Restoration of Akt-mTOR activation through a constitutively active mutant Akt1 mitigated the anti-OSCC effects induced by Gαi3 shRNA. In vivo, Gαi3 silencing significantly suppressed the growth of subcutaneous OSCC xenografts in nude mice, concomitant with inactivation of the Akt-mTOR pathway and induction of apoptosis. Collectively, these findings underscore the critical role of Gαi3 in OSCC cell growth both in vitro and in vivo.

The efficacy and potential mechanisms of pyrotinib in targeting EGFR and HER2 in advanced oral squamous cell carcinoma

BackgroundHuman epidermal growth factor receptor 2 (HER2) plays an important role in the progression of multiple solid tumors and induces resistance to epidermal growth factor receptor (EGFR) target treatment. However, the expression status and the clinical significance of HER2 in oral squamous cell carcinoma (OSCC) is still controversial. Pyrotinib (PYR) is a promising novel EGFR/HER2 dual inhibitor, whose efficacy in OSCC has not been determined.Methods57 locally advanced de novo OSCC patients were included in this study to investigate the relationship between the HER2 expression levels and the prognosis by the tissue microarray analysis (TMA). In vitro and in vivo experiments were performed to retrieve the efficacy of PYR in OSCC. The main downstream of HER2 was evaluated by western blotting in OSCC cell lines and xenograft tumors to explore the potential mechanism of PYR.ResultsThis study revealed the primary tumor of OSCC had higher HER2 expression levels. Patients with HER2 overexpression had poor overall survival (P < 0.014) and poor disease free survival (P < 0.042). In vitro, PYR suppressed the proliferation, colony formation and migration of OSCC cells. It also promoted apoptosis of OSCC cells and induced cell cycle arrest. Furthermore, PYR was able to inhibit the occurrence and development of OSCC effectively in vivo. Western blotting revealed that PYR suppressed OSCC by inhibiting the phosphorylation of HER2, AKT and ERK.ConclusionsThis study exhibited the anti-OSCC effects of PYR in vitro and in vivo, and demonstrated PYR inhibited OSCC cells by inducing apoptosis via the HER2/ AKT and ERK pathway. The result of this study also indicated locally advanced OSCC patients might benefit from HER2 assay and EGFR/HER2 dual inhibit treatment.

TCF12 induces ferroptosis by suppressing OTUB1-mediated SLC7A11 deubiquitination to promote cisplatin sensitivity in oral squamous cell carcinoma.

Chemotherapy resistance is a major obstacle to effective cancer treatment, and promotion of ferroptosis can suppress cisplatin resistance in tumor cells. TCF12 plays a suppressive role in oral squamous cell carcinoma (OSCC), but whether it participates in the regulation of cisplatin resistance by modulating ferroptosis remains unclear. Here, we found that TCF12 expression was decreased in OSCC cells compared with normal oral cells, and it was reduced in cisplatin (DDP)-resistant OSCC cells compared with parental cells. Moreover, overexpression of TCF12 sensitized DDP-resistant cells to DDP by promoting ferroptosis. Intriguingly, silencing TCF12 reversed the promotion effect of the ferroptosis activator RSL3 on ferroptosis and DDP sensitivity, and overexpressing TCF12 antagonized the effect of the ferroptosis inhibitor liproxstatin-1 on ferroptosis and DDP resistance. Mechanically, TCF12 promoted ubiquitination of SLC7A11 and decreased SLC7A11 protein stability through transcriptional repression of OTUB1, thereby facilitating ferroptosis. Consistently, SLC7A11 overexpression neutralized the promotion effect of TCF12 on ferroptosis and DDP sensitivity. Additionally, upregulation of TCF12 hindered the growth of mouse OSCC xenografts and enhanced the DDP sensitivity of xenografts by inducing ferroptosis. In conclusion, TCF12 enhanced DDP sensitivity in OSCC cells by promoting ferroptosis, which was achieved through modulating SLC7A11 expression via transcriptional regulation of OTUB1.

Combretastatin A4 phosphate encapsulated in hyaluronic acid nanoparticles is highly cytotoxic to oral squamous cell carcinoma.

To investigate the toxicity of combretastatin A4 phosphate (CA4P) hyaluronic acid (HA) gel nanoparticles (HA-CA4P-NPs) in OSCC (oral squamous cell carcinoma). Toxicity was investigated using fluorescence microscopy, MTT assay, flow cytometry, and OSCC xenograft mouse models. Compared with CA4P, HA-CA4P-NPs generated nearly 10 times more fluorescence in OSCC cells. Cytotoxicity assays showed that HACA4P-NPs were more toxic to SCC-4 cells but not to HNECs. Remarkable necrosis was induced in SCC-4 cells after exposure to HA-CA4P-NPs, and related proteins were upregulated. Furthermore, HA-CA4P-NPs significantly reduced the tumour size. HA-CA4P-NPs improved drug release and delivery, and increased cytotoxicity to cancer cells.

Efficacy of Cisplatin-CXCR4 Antagonist Combination Therapy in Oral Cancer.

Cisplatin is a platinum-based compound that is widely used for treating inoperable oral squamous cell carcinoma (OSCC) in Japan; however, resistance to cisplatin presents a challenge and innovative approaches are required. We aimed to investigate the therapeutic potential of targeting the chemokine receptor CXCR4, which is involved in angiogenesis and tumor progression, using the CXCR4 inhibitor AMD3100, in combination with cisplatin. AMD3100 induced necrosis and bleeding in OSCC xenografts by inhibiting angiogenesis. We investigated the combined ability of AMD3100 plus cisplatin to enhance the antitumor effect in cisplatin-resistant OSCC. An MTS assay identified HSC-2 cells as cisplatin-resistant cells in vitro. Mice treated with the cisplatin-AMD combination exhibited the most significant reduction in tumor volume, accompanied by extensive hemorrhage and necrosis. Histological examination indicated thin and short tumor vessels in the AMD and cisplatin-AMD groups. These results indicated that cisplatin and AMD3100 had synergistic antitumor effects, highlighting their potential for vascular therapy of refractory OSCC. Antitumor vascular therapy using cisplatin combined with a CXCR4 inhibitor provides a novel strategy for addressing cisplatin-resistant OSCC.

Nociceptive adenosine A2A receptor on trigeminal nerves orchestrates CGRP release to regulate the progression of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) associated pain commonly predicts adverse events among patients. This clinical feature indicates the engagement of nociceptors on sensory neurons during the development of malignancy. However, it is yet to be determined if targeting oncometabolite-associated nociception processes can hinder OSCC progression. In this study, we reported that nociceptive endings infiltrating both clinical samples and mouse tumor xenografts were associated with poorer clinical outcomes and drove tumor progression in vivo, as evidenced by clinical tissue microarray analysis and murine lingual denervation. We observed that the OSCC microenvironment was characteristic of excessive adenosine due to CD73 upregulation which negatively predicted clinical outcomes in the TCGA-HNSC patient cohort. Notably, such adenosine concentrative OSCC niche was associated with the stimulation of adenosine A2A receptor (A2AR) on trigeminal ganglia. Antagonism of trigeminal A2AR with a selective A2AR inhibitor SCH58261 resulted in impeded OSCC growth in vivo. We showed that trigeminal A2AR overstimulation in OSCC xenograft did not entail any changes in the transcription level of CGRP in trigeminal ganglia but significantly triggered the release of CGRP, an effect counteracted by SCH58261. We further demonstrated the pro-tumor effect of CGRP by feeding mice with the clinically approved CGRP receptor antagonist rimegepant which inhibited the activation of ERK and YAP. Finally, we diminished the impact of CGRP on OSCC with istradefylline, a clinically available drug that targets neuronal A2AR. Therefore, we established trigeminal A2AR-mediated CGRP release as a promising druggable circuit in OSCC treatment.

Dehydroepiandrosterone attenuated the immune escape of oral squamous cell carcinoma through NF-κB p65/miR-15b-5p/B7-H4 axis

ObjectivesWe aimed to explore the effects and mechanisms of action of dehydroepiandrosterone (DHEA) on immune evasion of oral squamous cell carcinoma (OSCC) to provide evidence for enhancing the effect of immunotherapy. Materials and MethodsA xenograft mouse model and immunohistochemistry were used to reveal the patterns of tumor-infiltrating lymphocytes (TILs). The CAL27 and SCC VII cell lines were used for the in vitro study. Western blotting, qPCR, immunofluorescence, and flow cytometry were used to evaluate the expression of B7-H4. Recombinant mouse B7-H4 protein (rmB7-H4) and PG490, an inhibitor of NF-κB p65 were used for the “rescue study.” Gain- and loss-of-function, luciferase reporter, and chromatin immunoprecipitation assays were performed to verify this mechanism. ResultsDHEA inhibited tumor growth in an OSCC xenograft mouse model, increased CD8 + cells, and decreased FOXP3 + cells in TILs. DHEA reduced the expression of B7-H4 in CAL27 and SCC VII cells RmB7-H4 reverses the effect of DHEA on tumor growth and TIL patterns. DHEA increased the expression of miR-15b-5p and activated its transcriptional factor NF-κB p65. Further experiments demonstrated that miR-15b-5p inhibited B7-H4 expression by binding to its 3′-UTR regions, and NF-κB p65 activated miR-15b transcription. PG490 reversed the effects of DHEA on tumor growth, antitumor immunity in the OSCC xenograft model, and the expression/phosphorylation of NF-κB p65, miR-15b-5p, and B7-H4. ConclusionsThis study indicates that DHEA attenuates the immune escape of OSCC cells by inhibiting B7-H4 expression, providing new insights for cancer immunotherapy.

MiR-617 interacts with the promoter of DDX27 and positively regulates its expression: implications for cancer therapeutics

BackgroundPervasive transcription of the eukaryotic genome generates noncoding RNAs (ncRNAs), which regulate messenger RNA (mRNA) stability and translation. MicroRNAs (miRNAs/miRs) represent a group of well-studied ncRNAs that maintain cellular homeostasis. Thus, any aberration in miRNA expression can cause diseases, including carcinogenesis. According to microRNA microarray analyses, intronic miR-617 is significantly downregulated in oral squamous cell carcinoma (OSCC) tissues compared to normal oral tissues.MethodsThe miR-617-mediated regulation of DDX27 is established by performing experiments on OSCC cell lines, patient samples, and xenograft nude mice model. Overexpression plasmid constructs, bisulphite sequencing PCR, bioinformatics analyses, RT-qPCR, Western blotting, dual-luciferase reporter assay, and cell-based assays are utilized to delineate the role of miR-617 in OSCC.ResultsThe present study shows that miR-617 has an anti-proliferative role in OSCC cells and is partly downregulated in OSCC cells due to the hypermethylation of its independent promoter. Further, we demonstrate that miR-617 upregulates DDX27 gene by interacting with its promoter in a dose-dependent and sequence-specific manner, and this interaction is found to be biologically relevant in OSCC patient samples. Subsequently, we show that miR-617 regulates cell proliferation, apoptosis, and anchorage-independent growth of OSCC cells by modulating DDX27 levels. Besides, our study shows that miR-617 exerts its effects through the PI3K/AKT/MTOR pathway via regulating DDX27 levels. Furthermore, the OSCC xenograft study in nude mice shows the anti-tumorigenic potential of miR-617.ConclusionmiR-617-mediated upregulation of DDX27 is a novel mechanism in OSCC and underscores the therapeutic potential of synthetic miR-617 mimics in cancer therapeutics. To the best of our knowledge, miR-617 is the 15th example of a miRNA that upregulates the expression of a protein-coding gene by interacting with its promoter.

Abstract 6992: Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma

Abstract Background:Oral squamous cell carcinoma (OSCC) stands as a prevalent malignancy worldwide. Profilin 2 (PFN2), an actin-binding protein regulating actin polymerization dynamics and associated with cell motility, has recently gained attention as a significant regulator in various cancers. However, the clinical significance and biological function of PFN2 in OSCC remain debatable. This study aims to elucidate the role of PFN2 in OSCC. Methods:We assessed PFN2 expression in OSCC and adjacent normal oral mucosa tissues through Q-RT-PCR and immunohistochemistry analysis. Associations between PFN2 expression, clinicopathological features, and OSCC prognosis were analyzed. Cell proliferation, invasion, and migration effects were examined via MTT and Transwell assays. Western blotting assessed the protein expression related to epithelial–mesenchymal transition (EMT). Additionally, we established OSCC xenografts to determine tumorigenicity in vivo using female Nude mice. Results:PFN2 RNA and protein expression were significantly increased in OSCC compared to normal oral mucosa. PFN2 expression positively correlated with lymphovascular invasion and cervical lymph node metastasis. MTT and Transwell assays demonstrated that PFN2 promoted OSCC cell proliferation, migration, invasion, and endothelial invasion. Downregulation of PFN2 induced an EMT phenotype, including an increase in the expression of the epithelial marker E-cadherin and a decrease in the expression of mesenchymal markers Vimentin, Snail, and Slug in OSCC cells in vitro. Similarly, OSCC cells with reduced PFN2 expression exhibited weaker tumorigenicity in vivo. Conclusions:Our findings unveil a novel role for PFN2 in promoting OSCC progression and metastasis, suggesting its potential as an early biomarker for high-risk populations. Targeting PFN2 may offer a promising therapeutic strategy for OSCC treatment. Citation Format: Chung Ji LIu, Li-Han Lin, Kuo-Wei Chang, Hui-Wen Cheng. Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6992.

Network pharmacology and experiment validation investigate the potential mechanism of triptolide in oral squamous cell carcinoma.

Objective: This study aimed to investigate the molecular mechanism of triptolide in the treatment of oral squamous cell carcinoma (OSCC) via network pharmacology and experimental validation. Methods: The network pharmacological method was used to predict the key targets, detect the signal pathways for the treatment of OSCC, and screen the critical components and targets for molecular docking. Predicted targets were validated in cellular and xenograft mouse model. Results: In this study, we predicted action on 17 relevant targets of OSCC by network pharmacology. PPI network demonstrated that Jun, MAPK8, TP53, STAT3, VEGFA, IL2, CXCR4, PTGS2, IL4 might be the critical targets of triptolide in the treatment of OSCC. These potential targets are mainly closely related to JAK-STAT and MAPK signaling pathways. The analysis of molecular docking showed that triptolide has high affinity with Jun, MAPK8 and TP53. Triptolide can suppress the growth of OSCC cells and xenograft mice tumor, and downregulate the expression of Jun, MAPK8, TP53, STAT3, VEGFA, IL2, CXCR4, PTGS2 to achieve the therapeutic effect of OSCC. Conclusion: Through network pharmacological methods and experimental studies, we predicted and validated the potential targets and related pathways of triptolide for OSCC treatment. The results suggest that triptolide can inhibit the growth of OSCC via several key targets.

GAD1-mediated GABA elicits aggressive characteristics of human oral cancer cells

Studies suggest that the expression of glutamate decarboxylase 1 (GAD1), γ-aminobutyric acid (GABA), and GABA receptors are involved in tumor progression. However, the underlying mechanisms of high expression and potential functions of GAD1 and GABA in oral squamous cell carcinoma (OSCC) are not known. In this study, we found that the expressions of GAD1 and GABA were considerably increased in OSCC samples, which were closely associated with clinical stage and lymph node metastasis. The knockdown of GAD1 expression significantly inhibited the proliferation, migration and invasion abilities of OSCC cells by reducing the expression of GABA-mediated GABAB receptors, which could be reversed by exogenous GABA, but did not cause excessive OSCC cell proliferation. And GABA secreted by OSCC cells promoted M2 macrophage polarization for inhibiting anti-tumor immunity by activating GABBR1/ERK/Ca2+. In addition, GABA/GABABR promoted the proliferation and progression of OSCC xenograft tumor. Altogether, our results showed that GAD1 synthetized GABA to promote the malignant progression of OSCC and limits the anti-tumor immunity of macrophages, thereby targeting GABA can be a novel strategy for treating OSCC.

Transcutaneous carbon dioxide application suppresses the expression of cancer-associated fibroblasts markers in oral squamous cell carcinoma xenograft mouse model.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. Cancer-associated fibroblasts (CAFs) are the main stromal cells in the tumor microenvironment (TME). As CAFs promote tumor progression and hypoxia in the TME, regulating the conversion of normal fibroblasts (NFs) into CAFs is essential for improving the prognosis of patients with OSCC. We have previously reported the antitumor effects of transcutaneous carbon dioxide (CO2) application in OSCC. However, the effects of reducing hypoxia in the TME remain unclear. In this study, we investigated whether CO2 administration improves the TME by evaluating CAFs marker expression. Human OSCC cells (HSC-3) and normal human dermal fibroblasts (NHDF) were coinjected subcutaneously into the dorsal region of mice. CO2 gas was applied twice a week for 3 weeks. The tumors were harvested six times after transcutaneous CO2 application. The expression of CAFs markers (α-SMA, FAP, PDPN, and TGF-β) were evaluated by using real-time polymerase chain reaction and immunohistochemical staining. The expression of α-SMA, FAP, PDPN, and TGF-β was significantly increased over time after co-injection. In the CO2-treated group, tumor growth was significantly suppressed after treatment initiation. In addition, the mRNA expression of these markers was significantly inhibited. Furthermore, immunohistochemical staining revealed a significant decrease in the protein expression of all CAFs markers in the CO2-treated group. We confirmed that transcutaneous CO2 application suppressed CAFs marker expression and tumor growth in OSCC xenograft mouse model.

Yin Yang 1-Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1-Mediated EZH2 Degradation.

LncRNAs play a critical role in oral squamous cell carcinoma (OSCC) progression. However, the function and detailed molecular mechanism of most lncRNAs in OSCC are not fully understood. Here, a novel nuclear-localized lncRNA, DUXAP9 (DUXAP9), that is highly expressed in OSCC is identified. A high level of DUXAP9 is positively associated with lymph node metastasis, poor pathological differentiation, advanced clinical stage, worse overall survival, and worse disease-specific survival in OSCC patients. Overexpression of DUXAP9 significantly promotes OSCC cell proliferation, migration, invasion, and xenograft tumor growth and metastasis, and upregulates N-cadherin, Vimentin, Ki67, PCNA, and EZH2 expression and downregulates E-cadherin in vitro and in vivo, whereas knockdown of DUXAP9 remarkably suppresses OSCC cell proliferation, migration, invasion, and xenograft tumor growth in vitro and in vivo in an EZH2-dependent manner. Yin Yang 1 (YY1) is found to activate the transcriptional expression of DUXAP9 in OSCC. Furthermore, DUXAP9 physically interacts with EZH2 and inhibits EZH2 degradation via the suppression of EZH2 phosphorylation, thereby blocking EZH2 translocation from the nucleus to the cytoplasm. Thus, DUXAP9 can serve as a promising target for OSCC therapy.

FBXW7 promotes autophagy and inhibits proliferation of oral squamous cell carcinoma.

F-box and WD repeat domain containing 7 (FBXW7) is a critical tumor suppressor. The expression of FBXW7 is decreased in oral squamous cell carcinoma (OSCC) tissues and shows diagnosis value. We aimed to investigate the influence of FBXW7 overexpression on OSCC cell proliferation and autophagy. In Balb/c nude mice, CAL27 xenograft tumor model was established. Western blot was employed to evaluate protein level. Messenger RNA level was analyzed by quantitative reverse transcription-polymerase chain reaction. Colony formation assay and MTT assay were employed to evaluate cell proliferation. FBXW7 expression was decreased in OSCC cell lines. FBXW7 inhibited cell proliferation of SCC9 and CAL27. FBXW7 increased Autophagy related 7 (Atg7), Beclin1 (BECN1), B-cell lymphoma 2 (BCL2) -associated X (BAX), BCL2 antagonist killer (BAK), and microtubule-associated protein 1 light chain 3 (LC3) levels and decreased MCL1 and BCL2 levels in CAL27 cells. FBXW7 decreased tumor volume and weight in CAL27 xenograft tumor model. FBXW7 increased BECN1, Atg7, and LC3 levels in CAL27 xenograft tumor model. In conclusion, decreased expression of FBXW7 is confirmed in diverse OSCC cell lines. The enhanced FBXW7 expression inhibits cancer cell proliferation and promotes autophagy in both OSCC cells and xenograft tumor model.

CircZNF236 facilitates malignant progression in oral squamous cell carcinoma by sequestering miR-145-5p.

A number of non-coding circular RNAs (circRNAs) have recently been implicated in the modulation of gene expression in cancer models. We therefore sought to explore if circZNF236 has a role in oral squamous cell carcinoma (OSCC). We first examined circZNF236 expression in 32 pairs of OSCC and noncancerous tissues. We then investigated a functional role for circZNF236 using knockdown and overexpression approaches in OSCC cancer cell lines. Cell counting kit-8, wound healing, Transwell, and flow cytometry were employed to assess circZNF236 function in vitro. The association between circZNF236 and miR-145-5p, or that between miR-145-5p and malignant brain tumor domain containing 1 (MBTD1) was predicted by bioinformatics and demonstrated by dual-luciferase reporter assays, RNA pull-down assays as well as RNA immunoprecipitation (RIP) assays. A mouse OSCC xenograft model was employed to demonstrate the impacts of circZNF236 inhibition on tumor development in vivo. OSCC tissues and cells had higher levels of circZNF236 expression compared with normal controls. Furthermore, high circZNF236 levels in patients with OSCC correlated with a poor prognosis. CircZNF236 silencing decreased the malignant properties of OSCC cells and suppressed OSCC tumor formation in the mouse model. We then noticed that miR-145-5p can be regulated by circZNF236, and that circZNF2361 promoted OSCC development by absorbing miR-145-5p and consequently upregulating MBTD1 expression. CircZNF236 modulates OSCC via the miR-145-5p/MBTD1 axis. These results support the potential of circZNF236 as a treatment target for OSCC.

Artemisinin suppressed tumour growth and induced vascular normalisation in oral squamous cell carcinoma via inhibition of macrophage migration inhibitory factor.

Tumour vascular normalisation therapy advocates a balance between pro-angiogenic factors and anti-angiogenic factors in tumours. Artemisinin (ART), which is derived from traditional Chinese medicine, has been shown to inhibit tumour growth; however, the relationship between ART and tumour vascular normalisation in oral squamous cell carcinoma (OSCC) has not been previously reported. Different concentrations(0mg/kg, 25 mg/kg, 50 mg/kg, 100 mg/kg)of ART were used to treat the xenograft nude mice model of OSCC. The effects of ART on migration and proliferation of OSCC and human umbilical vein endothelial cells (HUVEC) cells were detected by scratch assay and CCK-8 assay. OSCC cells with macrophage migration inhibitory factor (MIF) silenced were constructed to explore the effect of MIF. Treatment with ART inhibited the growth and angiogenesis of OSCC xenografts in nude mice and downregulated vascular endothelial growth factor (VEGF), IL-8, and MIF expression levels. ART reduced the proliferation, migration, and tube formation of HUVEC, as well as the expression of VEGFR1 and VEGFR2. When the dose of ART was 50 mg/kg, vascular normalisation of OSCC xenografts was induced. Moreover, VEGF and IL-8 were needed in rhMIF restoring tumour growth and inhibit vascular normalisation after the addition of rhMIF to ART-treated cells. Artemisinin might induce vascular normalisation and inhibit tumour growth in OSCC through the MIF-signalling pathway.

Oral Cancer Stem Cell-Derived Small Extracellular Vesicles Promote M2 Macrophage Polarization and Suppress CD4+ T-Cell Activity by Transferring UCA1 and Targeting LAMC2.

Cancer-derived small extracellular vesicles (sEVs) are emerging as crucial mediators of intercellular communication between cancer cells and M2-tumor-associated macrophages (M2-TAMs) via transferring lncRNAs. We previously reported that miR-134 blocks the expression of its targeting protein LAMC2 via the PI3K/AKT pathway and inhibits cancer stem cell (CSC) migration and invasion in oral squamous cell carcinoma (OSCC). This study hypothesize that OSCC-CSC-derived small extracellular vesicles (OSCC-CSC-sEVs) transfer a ceRNA of miR-134 and consequently promote M2 macrophage polarization by targeting LAMC2 via the PI3K/AKT pathway through in vitro and in vivo experiment methods. The results showed that sEVs derived from CD133+CD44+ OSCC cells promoted M2 polarization of macrophages by detecting several M2 macrophage markers (CD163, IL-10, Arg-1, and CD206+CD11b+). Mechanistically, we revealed that the lncRNA UCA1, by binding to miR-134, modulated the PI3K/AKT pathway in macrophages via targeting LAMC2. Importantly, OSCC-CSC-sEV transfer of UCA1, by targeting LAMC2, promoted M2 macrophage polarization and inhibited CD4+ T-cell proliferation and IFN-γ production in vitro and in vivo. Functionally, we demonstrated that M2-TAMs, by transferring exosomal UCA and consequently targeting LAMC2, enhanced cell migration and invasion of OSCC in vitro and the tumorigenicity of OSCC xenograft in nude mice. In conclusion, our results indicated that OSCC-CSC-sEV transfer of UCA1 promotes M2 macrophage polarization via a LAMC2-mediated PI3K/AKT axis, thus facilitating tumor progression and immunosuppression. Our findings provide a new understanding of OSCC-CSC molecular mechanisms and suggest a potential therapeutic strategy for OSCC through targeting CSC-sEVs and M2-TAMs.

Rapid and Quantitative Intraoperative Pathology-Assisted Surgery by Paired-Agent Imaging-Derived Confidence Map.

In nonmetastatic head and neck cancer treatment, surgical margin status is the most important prognosticator of recurrence and patient survival. Fresh frozen sectioning (FFS) of tissue margins is the standard of care for intraoperative margin assessment. However, FFS is time intensive, and its accuracy is not consistent among institutes. Mapping the epidermal growth factor receptor (EGFR) using paired-agent imaging (PAI) has the potential to provide more consistent intraoperative margin assessment in a fraction of the time as FFS. PAI was carried out through IV injection of an anti-epidermal growth factor receptor (EGFR) affibody molecule (ABY-029, eIND 122,681) and an untargeted IRDye680LT carboxylate. Imaging was performed on 4 µm frozen sections from three oral squamous cell carcinoma xenograft mouse models (n = 24, 8 samples per cell line). The diagnostic ability and tumor contrast were compared between binding potential, targeted, and untargeted images. Confidence maps were constructed based on group histogram-derived tumor probability curves. Tumor differentiability and contrast by confidence maps were evaluated. PAI outperformed ABY-029 and IRDye 680LT alone, demonstrating the highest individual receiver operating characteristic (ROC) curve area under the curve (PAI AUC: 0.91, 0.90, and 0.79) and contrast-to-noise ratio (PAI CNR: 1, 1.1, and 0.6) for FaDu, Det 562, and A253. PAI confidence maps (PAI CM) maintain high tumor diagnostic ability (PAI CMAUC: 0.91, 0.90, and 0.79) while significantly enhancing tumor contrast (PAI CMCNR: 1.5, 1.3, and 0.8) in FaDu, Det 562, and A253. Additionally, the PAI confidence map allows avascular A253 to be differentiated from a healthy tissue with significantly higher contrast than PAI. Notably, PAI does not require additional staining and therefore significantly reduces the tumor delineation time in a 5 [Formula: see text] 5mm slice from ~ 35min to under a minute. This study demonstrated that PAI improved tumor detection in frozen sections with high diagnostic accuracy and rapid analysis times. The novel PAI confidence map improved the contrast in vascular tumors and differentiability in avascular tumors. With a larger database, the PAI confidence map promises to standardize fluorescence imaging in intraoperative pathology-assisted surgery (IPAS).

Inhibitor of Growth 4 (ING4) Plays a Tumor-repressing Role in Oral Squamous Cell Carcinoma via Nuclear Factor Kappa-B (NF-kB)/DNA Methyltransferase 1 (DNMT1) Axis-mediated Regulation of Aldehyde Dehydrogenase 1A2 (ALDH1A2).

Inhibitor of growth 4 (ING4) level was reported to be decreased in head and neck squamous cell carcinoma (HNSC) tissue, however, it is unknown whether and how ING4 participates in regulating the development of oral squamous cell carcinoma (OSCC). This study aimed to investigate the role and mechanism of ING4 in OSCC. ING4 was forced to up- or down-regulated in two OSCC cell lines, and its effects on the malignant behavior of OSCC cells were investigated in vitro. The ubiquitination level of NF-kB p65 in ING4 upregulated cells was measured by co-immunoprecipitation. Moreover, the effects of ING4 on the methylation level of ALDH1A2 were evaluated by methylation-specific polymerase chain reaction (MSP) assay. The role of ING4 in OSCC growth in vivo was observed in nude mice. Our results showed that the expression of ING4 in OSCC cell lines was lower than that in normal oral keratinocyte cells. In vitro, ING4 overexpression inhibited the proliferation, migration, and invasion of OSCC cell lines and ING4 silencing exhibited opposite results. We also demonstrated that ING4 overexpression promoted the ubiquitination and degradation of P65 and reduced DNA methyltransferase 1 (DNMT1) expression and Aldehyde dehydrogenase 1A2 (ALDH1A2) methylation. Moreover, overexpression of p65 rescued the suppression of malignant behavior, induced by ING4 overexpression. In addition, ING4 negatively regulated the growth of OSCC xenograft tumors in vivo. Our data evidenced that ING4 played a tumor-repressing role in OSCC in vivo and in vitro via NF-κB/DNMT1/ALDH1A2 axis.