Mouse Oral Squamous Cell Carcinoma Research Articles

Black raspberry modulates cecal and oral microbiome at the early stage of a dibenzo[def,p]chrysene-induced murine oral cancer model.

While tobacco smoking is a risk factor in the development of oral squamous cell carcinoma (OSCC), only a fraction of smokers develop the disease. Compelling evidence shows that microbial community composition is associated with carcinogenesis, suggesting that the microbiome may play a role in cancer development of smokers. We previously showed that black raspberry (BRB) protects against OSCC induced by the tobacco constituent dibenzo[def,p]chrysene (DBP) altering genetic and epigenetic markers in a manner consistent with its cancer preventive activity. In the present study, we conducted a mouse experiment to investigate the effects of BRB and DBP individually and in combination on the oral and gut microbiota. DBP-induced DNA damage in the mouse oral cavity which is an essential step for the development of OSCC in mice. 16S rRNA gene sequencing revealed that BRB significantly increased microbial diversity and shifted microbiome composition in the gut and oral cavity, whereas DBP had no significant effect. In both gut and oral microbiota, Akkermansia muciniphila was significantly reduced after BRB treatment; however, this was not consistent with pure culture in vitro assays suggesting that the impact of BRB on A. muciniphila may be mediated through indirect mechanisms including the host or other microbes. Indeed BRB, but not DBP, was found to modulate the growth kinetics of human gut microbes in vitro including lactic acid bacteria and Bacteroides spp. The results of the current study further emphasize the interplay of microbiome and environmental factors in the development and prevention of OSCC.

Integration of host gene regulation and oral microbiome reveals the influences of smoking during the development of oral squamous cell carcinoma.

This study aims to investigate the regulation of host gene transcription and microbial changes during the development of oral squamous cell carcinoma (OSCC) associated with smoking. The OSCC mouse model and smoking mouse model were established using 200 μg/mL 4-nitroquinoline-1-oxide (4NQO) in drinking water and exposure to cigarette smoke (four cigarettes per session, once a day, 5 days a week). Tongue tissues were harvested at 4 weeks and 16 weeks. Histopathological changes were evaluated using hematoxylin and eosin staining and Ki67 staining. RNA sequencing was performed on the mouse tongue tissues to identify differentially expressed genes (DEGs), and the results were validated by RT-PCR and immunohistochemistry. 16S rDNA sequencing was used to analyze changes in the oral microbiota during the early development of OSCC, identifying differentially abundant taxa associated with smoking. Finally, associations between the relative abundances of the oral microbiome and host gene expression were modeled using the Origin software. DEGs associated with smoking during the development of OSCC were identified. There were 12 upregulated genes, including NR4A3 and PPP1R3C, and 23 downregulated genes, including CD74 and ANKRD1. These genes were enriched in functions related to the signal transduction of cellular processes such as inflammation, differentiation, immunity, and PI3K/AKT, NF-κB signaling pathways. 4NQO and smoking treatment decreased oral microbial diversity and reduced the abundance of Bacteroidetes, Proteobacteria, and Lactobacillus but increased the abundance of Staphylococcus. Integrative analysis showed that the expression of CD74 was positively correlated with the relative abundance of Lactobacillus, while PPP1R3C was negatively correlated with Bacteroidota. In addition to characterizing host gene expression and the oral microbiome, our study explored the potential role of host-microbiome interactions in the development of OSCC. These findings enhance our understanding of smoking-related OSCC occurrence and development, providing new insights for its prevention.

Iontophoresis-Enhanced Buccal Delivery of Cisplatin-Encapsulated Chitosan Nanoparticles for Treating Oral Cancer in a Mouse Model.

Cisplatin is one of the most effective chemotherapeutic drugs used in oral cancer treatment, but systemic administration has side effects. The purpose of this study was to evaluate the effect of iontophoresis on the enhancement of cisplatin release from cisplatin-encapsulated chitosan nanoparticles. The effect of different mass ratios of chitosan to tripolyphosphate (TPP) (5:1, 10:1, 15:1, 20:1) on the encapsulation efficiency of cisplatin was investigated. Uptake of cisplatin-encapsulated chitosan by cells was observed using a confocal laser scanning microscope. The cell viability at different cisplatin concentrations was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Three iontophoresis methods, namely constant-current chronopotentiometry (CCCP), cyclic chronopotentiometry (CCP), and differential pulse voltammetry (DPV), were used to enhance cisplatin release from cisplatin-encapsulated chitosan nanoparticles. In addition, mouse oral squamous cell carcinoma cell lines were implanted into the mouse oral mucosa to induce oral cancer. The effects of enhanced cisplatin release by CCCP, CCP, and DPV on tumor suppression in mice were evaluated. Tumors and lymph nodes were isolated for hematoxylin-eosin staining and immunohistochemistry staining including Ki-67 and pan CK after sacrifice. Inductively coupled plasma mass spectrometry was conducted to quantify the platinum content within the tumors. The results showed that nanoparticles with a mass ratio of 15:1 exhibited the highest cisplatin encapsulation efficiency (approximately 15.6%) and longest continued release (up to 35 days) in phosphate buffered saline with a release rate of 100%. Cellular uptake results suggested that chitosan nanoparticles were delivered to the cytoplasm via endocytosis. The results of the MTT assay revealed that the survival rate of cells decreased as the cisplatin concentration increased. The CCP (1 mA, on:off = 1 s: 1 s) and DPV (0-0.06 V) groups were the most effective in inhibiting tumor growth, and both groups exhibited the lowest percentage of Ki-67 positive and pan CK positive. This study is the first to investigate and determine the efficacy of DPV in enhancing in vivo drug release from nanoparticles for the treatment of cancer in animals. The results suggest that the CCP and DPV methods have the potential to be combined with surgery for oral cancer treatment.

Multispectral fluorescence imaging of EGFR and PD-L1 for precision detection of oral squamous cell carcinoma: a preclinical and clinical study

BackgroundEarly detection and treatment are effective methods for the management of oral squamous cell carcinoma (OSCC), which can be facilitated by the detection of tumor-specific OSCC biomarkers. The epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) are important therapeutic targets for OSCC. Multispectral fluorescence molecular imaging (FMI) can facilitate the detection of tumor multitarget expression with high sensitivity and safety. Hence, we developed Nimotuzumab-ICG and Atezolizumab-Cy5.5 imaging probes, in combination with multispectral FMI, to sensitively and noninvasively identify EGFR and PD-L1 expression for the detection and comprehensive treatment of OSCC.MethodsThe expression of EGFR and PD-L1 was analyzed using bioinformatics data sources and specimens. Nimotuzumab-ICG and Atezolizumab-Cy5.5 imaging probes were developed and tested on preclinical OSCC cell line and orthotopic OSCC mouse model, fresh OSCC patients’ biopsied samples, and further clinical mouthwash trials were conducted in OSCC patients.ResultsEGFR and PD-L1 were specifically expressed in human OSCC cell lines and tumor xenografts. Nimotuzumab-ICG and Atezolizumab-Cy5.5 imaging probes can specifically target to the tumor sites in an in situ human OSCC mouse model with good safety. The detection sensitivity and specificity of Nimotuzumab-ICG in patients were 96.4% and 100%, and 95.2% and 88.9% for Atezolizumab-Cy5.5.ConclusionsEGFR and PD-L1 are highly expressed in OSCC, the combination of which is important for a precise prognosis of OSCC. EGFR and PD-L1 expression can be sensitively detected using the newly synthesized multispectral fluorescence imaging probes Nimotuzumab-ICG and Atezolizumab-Cy5.5, which can facilitate the sensitive and specific detection of OSCC and improve treatment outcomes.Trial registrationChinese Clinical Trial Registry, ChiCTR2100045738. Registered 23 April 2021, https://www.chictr.org.cn/bin/project/edit?pid=125220

DAZAP1 Phase Separation Regulates Mitochondrial Metabolism to Facilitate Invasion and Metastasis of Oral Squamous Cell Carcinoma.

Tumor invasion and metastasis are the underlying causes of the high mortality rate of oral squamous cell carcinoma (OSCC). Energy metabolism reprogramming has been identified as a crucial process mediating tumor metastasis, thus indicating an urgent need for in-depth investigation of the specific mechanisms of tumor energy metabolism. Here, we identified an RNA-binding protein, DAZ associated protein 1 (DAZAP1), as a tumor-promoting factor with an important role in OSCC progression. DAZAP1 was significantly upregulated in OSCC, which enhanced the migration and invasion of OSCC cells and induced the epithelial-mesenchymal transition (EMT). RNA-seq analysis and experimental validation demonstrated that DAZAP1 regulates mitochondrial energy metabolism in OSCC. Mechanistically, DAZAP1 underwent liquid-liquid phase separation (LLPS) to accumulate in the nucleus where it enhanced cytochrome-c oxidase 16 (COX16) expression by regulating pre-mRNA alternative splicing, thereby promoting OSCC invasion and mitochondrial respiration. In mouse OSCC models, loss of DAZAP1 suppressed EMT, downregulated COX16, and reduced tumor growth and metastasis. In OSCC patient samples, expression of DAZAP1 positively correlated with COX16, and high expression of both proteins was associated with poor patient prognosis. Together, these findings revealed a mechanism by which DAZAP1 supports mitochondrial metabolism and tumor development of OSCC, suggesting the potential of therapeutic strategies targeting DAZAP1 to block OSCC invasion and metastasis.

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.

Double-faced CX3CL1 enhances lymphangiogenesis-dependent metastasis in an aggressive subclone of oral squamous cell carcinoma.

Because cancer cells have a genetically unstable nature, they give rise to genetically different variant subclones inside a single tumor. Understanding cancer heterogeneity and subclone characteristics is crucial for developing more efficacious therapies. Oral squamous cell carcinoma (OSCC) is characterized by high heterogeneity and plasticity. On the other hand, CX3C motif ligand 1 (CX3CL1) is a double-faced chemokine with anti- and pro-tumor functions. Our study reported that CX3CL1 functioned differently in tumors with different cancer phenotypes, both in vivo and in vitro. Mouse OSCC 1 (MOC1) and MOC2 cells responded similarly to CX3CL1 in vitro. However, in vivo, CX3CL1 increased keratinization in indolent MOC1 cancer, while CX3CL1 promoted cervical lymphatic metastasis in aggressive MOC2 cancer. These outcomes were due to double-faced CX3CL1 effects on different immune microenvironments indolent and aggressive cancer created. Furthermore, we established that CX3CL1 promoted cancer metastasis via the lymphatic pathway by stimulating lymphangiogenesis and transendothelial migration of lymph-circulating tumor cells. CX3CL1 enrichment in lymphatic metastasis tissues was observed in aggressive murine and human cell lines. OSCC patient samples with CX3CL1 enrichment exhibited a strong correlation with lower overall survival rates and higher recurrence and distant metastasis rates. In conclusion, CX3CL1 is a pivotal factor that stimulates the metastasis of aggressive cancer subclones within the heterogeneous tumors to metastasize, and our study demonstrates the prognostic value of CX3CL1 enrichment in long-term monitoring in OSCC.

DNMT1-targeting remodeling global DNA hypomethylation for enhanced tumor suppression and circumvented toxicity in oral squamous cell carcinoma

BackgroundThe faithful maintenance of DNA methylation homeostasis indispensably requires DNA methyltransferase 1 (DNMT1) in cancer progression. We previously identified DNMT1 as a potential candidate target for oral squamous cell carcinoma (OSCC). However, how the DNMT1- associated global DNA methylation is exploited to regulate OSCC remains unclear.MethodsThe shRNA-specific DNMT1 knockdown was employed to target DNMT1 on oral cancer cells in vitro, as was the use of DNMT1 inhibitors. A xenografted OSCC mouse model was established to determine the effect on tumor suppression. High-throughput microarrays of DNA methylation, bulk and single-cell RNA sequencing analysis, multiplex immunohistochemistry, functional sphere formation and protein immunoblotting were utilized to explore the molecular mechanism involved. Analysis of human samples revealed associations between DNMT1 expression, global DNA methylation and collaborative molecular signaling with oral malignant transformation.ResultsWe investigated DNMT1 expression boosted steadily during oral malignant transformation in human samples, and its inhibition considerably minimized the tumorigenicity in vitro and in a xenografted OSCC model. DNMT1 overexpression was accompanied by the accumulation of cancer-specific DNA hypomethylation during oral carcinogenesis; conversely, DNMT1 knockdown caused atypically extensive genome-wide DNA hypomethylation in cancer cells and xenografted tumors. This novel DNMT1-remodeled DNA hypomethylation pattern hampered the dual activation of PI3K-AKT and CDK2-Rb and inactivated GSK3β collaboratively. When treating OSCC mice, targeting DNMT1 achieved greater anticancer efficacy than the PI3K inhibitor, and reduced the toxicity of blood glucose changes caused by the PI3K inhibitor or combination of PI3K and CDK inhibitors as well as adverse insulin feedback.ConclusionsTargeting DNMT1 remodels a novel global DNA hypomethylation pattern to facilitate anticancer efficacy and minimize potential toxic effects via balanced signaling synergia. Our study suggests DNMT1 is a crucial gatekeeper regarding OSCC destiny and treatment outcome.

Spatial Analysis of NETotic Neutrophil Crosstalk with Monocytes/Mφ in Oral Squamous Cell Carcinoma

Abstract Neutrophils are specialized immune cells that exhibit dual roles in cancer immunity; both pro and anti-tumor functions have been characterized thus far. It is now recognized that previously identified Myeloid-Derived Suppressor Cells (MDSCs) that impede anti-tumor immunity in various cancers are comprised of pathologic monocyte and neutrophil types. However, the interplay between tumor-associated neutrophils, monocytes, and their impact on tumor immunity remains unclear. Neutrophils execute their immune functions via NETosis, an exceptional mechanism that extrudes neutrophil extracellular traps (NETs), which play a dual role in cancer. In some cancers, NETs recruit macrophages toward an anti-tumor response, yet prolonged interaction induces apoptosis, contributing to immune suppression in the tumor microenvironment (TME). In oral squamous cell carcinoma (OSCC), linked to poor prognosis and high mortality, NETotic neutrophil infiltration at the tumor site correlates with improved survival. Conversely, heightened neutrophil presence in late-stage metastatic lymph nodes reduces survival. Consistently, elevated NET concentration in patient serum was identified. In this study, we use CosMx Spatial Molecular Imager (CosMx SMI) to investigate in-depth transcriptome changes of monocytes/Mφs induced by crosstalk with NETotic neutrophils in the tumor site from MOC2 tumors (OSCC mouse model) to demonstrate the contribution of NETotic neutrophil-monocyte/Mφ crosstalk to OSCC initiation.

Harnessing tetrahedral framework nucleic acids for enhanced delivery of microRNA-149-3p: A new frontier in oral squamous cell carcinoma therapy.

Oral squamous cell carcinoma (OSCC), a type of malignant tumour that primarily occurs in the oral mucosa, has drawn considerable attention owing to its aggressive growth and potentially high metastatic rate. Surgical resection is the primary treatment method for OSCC and is typically combined with radiation therapy and chemotherapy. microRNA-149-3p (miR-149) is a negative regulator of the Pi3k/Akt pathway and can effectively inhibit the proliferation of tumour cells. However, the application of miR-149 is limited owing to its relatively low efficiency of cellular uptake and poor stability when used alone. To overcome these challenges, this study adopted a novel nucleic acid nanostructured material, tetrahedral framework nucleic acids (tFNAs). The use of tFNAs as carriers to assemble the T-miR-149 complex reduced the expression of Pi3k and Akt involved in tumorigenesis and alterations in proteins related to cell apoptosis. The results indicated that the bionic drug delivery system has an effective tumour suppressive effect on OSCC in mice, revealing its potential clinical value in the treatment of OSCC.

Analysis of the effect of CCR7 on the microenvironment of mouse oral squamous cell carcinoma by single-cell RNA sequencing technology

BackgroundStudies have shown that CCR7, an important inflammatory factor, can promote the proliferation and metastasis of oral squamous cell carcinoma (OSCC), but its role in the tumor microenvironment (TME) remains unclear. This paper explores the role of CCR7 in the TME of OSCC.MethodsIn this work, we constructed CCR7 gene knockout mice and OSCC mouse models. Single-cell RNA sequencing (scRNA-seq) and bioinformatics were used to analyze the differences in the OSCC microenvironment between three CCR7 gene knockout mice (KO) and three wild-type mice (WT). Immunohistochemistry, immunofluorescence staining, and flow cytometry were used to analyze the expression of key genes in significantly different cell types between the KO and WT groups. An in vitro experiment was used to verify the effect of CCR7 on M2 macrophage polarization.ResultsIn the mouse OSCC models, the tumor growth rate in the KO group was significantly lower than that in the WT group. Eight main cell types (including tumor cells, fibroblasts, macrophages, granulocytes, T cells, endothelial cells, monocytes, and B cells) were identified by Seurat analysis. The scRNA-seq results showed that the proportion of tumor cells was lower, but the proportion of inflammatory cells was significantly higher in the KO group than in the WT group. CellPhoneDB analysis results indicated a strong interaction relationship between tumor cells and macrophages, T cells, fibroblasts, and endothelial cells. Functional enrichment results indicated that the expression level of the Dusp1 gene in the KO group was generally higher than that in the WT group in various cell types. Macrophage subclustering results indicated that the proportion of M2 macrophages in the KO group was lower than that in the WT group. In vitro experimental results showed that CCR7 can promote M2 macrophage polarization, thus promoting the proliferation, invasion and migration of OSCC cells.ConclusionsCCR7 gene knockout can significantly inhibit the growth of mouse oral squamous cell carcinoma by promoting the polarization of M2 macrophages.

Pharmacological blockade of HDAC6 attenuates cancer progression by inhibiting IL-1β and modulating immunosuppressive response in OSCC

Interleukin-1-beta (IL-1β) one of the biomarkers for oral squamous cell carcinoma (OSCC), is upregulated in tumor-microenvironment (TME) and associated with poor patient survival. Thus, a novel modulator of IL-1β would be of great therapeutic value for OSCC treatment. Here we report regulation of IL-1β and TME by histone deacetylase-6 (HDAC6)-inhibitor in OSCC. We observed significant upregulation of HDAC6 in 4-nitroquniline (4-NQO)-induced OSCC in mice and 4-NQO & Lipopolysaccharide (LPS) stimulated OSCC and fibroblast cells. Tubastatin A (TSA)-attenuated the OSCC progression in mice as observed improvement in the histology over tongue and esophagus, with reduced tumor burden. TSA treatment to 4-NQO mice attenuated protein expression of HDAC6, pro-and-mature-IL-1β and pro-and-cleaved-caspase-1 and ameliorated acetylated-tubulin. In support of our experimental work, human TCGA analysis revealed HDAC6 and IL-1β were upregulated in the primary tumor, with different tumor stages and grades. We found TSA modulate TME, indicated by downregulation of CD11b+Gr1+-Myeloid-derived suppressor cells, CD11b+F4/80+CD206+ M2-macrophages and increase in CD11b+F4/80+MHCII+ M1-macrophages. TSA significantly reduced the gene expression of HDAC6, IL-1β, Arginase-1 and iNOS in isolated splenic-MDSCs. FaDu-HTB-43 and NIH3T3 cells stimulated with LPS and 4-NQO exhibit higher IL-1β levels in the supernatant. Interestingly, immunoblot analysis of the cell lysate, we observed that TSA does not alter the expression as well as activation of IL-1β and caspase-1 but the acetylated-tubulin was found to be increased. Nocodazole pre-treatment proved that TSA inhibited the lysosomal exocytosis of IL-1β through tubulin acetylation. In conclusion, HDAC6 inhibitors attenuated TME and cancer progression through the regulation of IL-1β in OSCC.

Abstract 3783: Oral squamous carcinoma, LN metastasis

Abstract Due to genetically unstable nature of cancer cells, they give rise to genetically different variant subclones inside a single tumor. To enable the development of more efficacious therapies, it is crucial to understand cancer heterogeneity and subclone characteristics. Oral squamous cell carcinoma (OSCC) has high heterogeneity and plasticity. C-X3-C motif ligand 1 (CX3CL1) is the chemokine with anti- and pro-tumor functions. Our study reported CX3CL1 functions on cancer subclones with different phenotypes, both in vivo and in vitro. We found that Mouse OSCC (MOC) 1 and MOC2 cells responded in similar manners to CX3CL1 in vitro. However, in vivo, CX3CL1 increases cellular differentiation in indolent cancer (MOC1) while increasing metastasis in aggressive cancer (MOC2). These phenomena are influenced by different phenotypic tumor microenvironments created by indolent and aggressive cancer. From there, we established that when aggressive cancer cells gain CX3CL1 expression, they stimulate protumor immune system, promoting cancer metastasis via lymphatic vessels. A similar phenomenon was observed in metastatic OSCC patient samples, where CX3CL1 expression became intense in metastasis regions, consistent with aggressive subclone (MOC2) character; we correctly predicted patient outcomes by using CX3CL1 as prognostic indicator for long-term follow-up in metastatic OSCC patients. In conclusion, CX3CL1 is the key factor for stimulating aggressive cancer subclones to create new lymphatic networks for lymph node metastasis. Citation Format: Hotaka Kawai, Htoo Shwe Eain, Toshiaki Ohara, May Wathone Oo, Yamin So, Hitoshi Nagatsuka. Oral squamous carcinoma, LN metastasis [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 3783.

Heat-killed Prevotella intermedia promotes the progression of oral squamous cell carcinoma by inhibiting the expression of tumor suppressors and affecting the tumor microenvironment

BackgroundOral microbial dysbiosis contributes to the development of oral squamous cell carcinoma (OSCC). Our previous study showed that Prevotella intermedia (P. intermedia) were enriched in the oral mucosal surface, plaque, and saliva of patients with OSCC. Intratumoral microbiome could reshape the immune system and influence the development of various tumors. However, the invasion status of human OSCC tissues by P. intermedia and the pathway through which intratumoral P. intermedia potentiates tumor progression remain unexplored.MethodsP. intermedia in human OSCC or normal tissues was detected by FISH. A mouse OSCC cell line SCC7 was adopted to investigate the effects of heat-killed P. intermedia treatment on cell proliferation, invasion, and cytokine release by using CCK-8 assay, transwell invasion assay and ELISA. Moreover, we established a mouse transplanted tumor model by using SCC7 cells, injected heat-killed P. intermedia into tumor tissues, and investigated the effects of heat-killed P. intermedia on tumor growth, invasion, cytokine levels, immune cell infiltrations, and expression levels by using gross observation, H&E staining, ELISA, immunohistochemistry, mRNA sequencing, and transcriptomic analysis.ResultsOur results indicated that P. intermedia were abundant in OSCC and surrounding muscle tissues. Heat-killed P. intermedia promoted SCC7 cell proliferation, invasion and proinflammatory cytokine secretions, accelerated transplanted tumor growth in mice, exacerbate muscle and perineural invasion of OSCC, elevated the serum levels of IL-17A, IL-6, TNF-α, IFN-γ, and PD-L1, induced Treg cells M2 type macrophages in mouse transplanted tumors. The data of transcriptomic analysis revealed that heat-killed P. intermedia increased the expression levels of inflammatory cytokines and chemokines while reduced the expression levels of some tumor suppressor genes in mouse transplanted tumors. Additionally, IL-17 signaling pathway was upregulated whereas GABAergic system was downregulated by heat-killed P. intermedia treatment.ConclusionsTaken together, our results suggest that P. intermedia could inhibit the expression of tumor suppressors, alter the tumor microenvironment, and promote the progression of OSCC.

Lingual Denervation Improves the Efficacy of Anti-PD-1 Immunotherapy in Oral Squamous Cell Carcinomas by Downregulating TGFβ Signaling.

Intratumoral nerve infiltration relates to tumor progression and poor survival in oral squamous cell carcinoma (OSCC). How neural involvement regulates antitumor immunity has not been well characterized. This study aims to investigate molecular mechanisms of regulating tumor aggressiveness and impairing antitumor immunity by nerve-derived factors. We performed the surgical lingual denervation in an immunocompetent mouse OSCC model to investigate its effect on tumor growth and the efficacy of anti-PD-1 immunotherapy. A trigeminal ganglion neuron and OSCC cell coculture system was established to investigate the proliferation, migration, and invasion of tumor cells and the PD-L1 expression. Both the neuron-tumor cell coculture in vitro model and the OSCC animal model were explored. Lingual denervation slowed down tumor growth and improved the efficacy of anti-PD-1 treatment in the OSCC model. Coculturing with neurons not only enhanced the proliferation, migration, and invasion but also upregulated TGFβ-SMAD2 signaling and PD-L1 expression of tumor cells. Treatment with the TGFβ signaling inhibitor galunisertib reversed nerve-derived tumor aggressiveness and downregulated PD-L1 on tumor cells. Similarly, lingual denervation in vivo decreased TGFβ and PD-L1 expression and increased CD8+ T-cell infiltration and the expression of IFNγ and TNFα within tumor. Neural involvement enhanced tumor aggressiveness through upregulating TGFβ signaling and PD-L1 expression in OSCC, while denervation of OSCC inhibited tumor growth, downregulated TGFβ signaling, enhanced activities of CD8+ T cells, and improved the efficacy of anti-PD-1 immunotherapy. This study will encourage further research focusing on denervation as a potential adjuvant therapeutic approach in OSCC. This study revealed the specific mechanisms for nerve-derived cancer progression and impaired antitumor immunity in OSCC, providing a novel insight into the cancer-neuron-immune network as well as pointing the way for new strategies targeting nerve-cancer cross-talk as a potential adjuvant therapeutic approach for OSCC.

Novel vaccination strategies based on optimal stimulation of CD4+ T helper cells for the treatment of oral squamous cell carcinoma.

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant tumor of the oral cavity. Despite recent advances in the field of oral cancer therapy, including the introduction of immunotherapeutic approaches, the 5-year survival rate remains steadily assessed around 50%. Thus, there is an urgent need for new therapeutic strategies. After the characterization of the immune phenotype of three human OSCC cell lines (CAL-27, SCC-25, and SCC-4) and one mouse OSCC cell line (MOC2) showing their similarities to resected patient tumors, we explored for the first time an experimental preclinical model of therapeutic vaccination with mouse OSCC MOC2 cell line stably expressing MHC class II antigens after CIITA gene transfection (MOC2-CIITA). Mice injected with MOC2-CIITA reject or strongly retard tumor growth; more importantly, vaccinated animals that fully reject MOC2-CIITA tumors display anti-tumor immunological memory protective against challenge with parental MOC2 tumor cells. Further experiments of adoptive cell transfer or in vivo cell depletion show that both CD4+ and CD8+ T lymphocytes prove fundamental in tumor rejection. This unprecedented approach for oral cancer opens the way for possible future translation of novel immunotherapeutic strategies to the human setting for the treatment of this tumor.

Near-infrared-II Ag2S quantum dot probes targeting podoplanin enhance immunotherapy in oral squamous cell carcinoma

Partial epithelial-mesenchymal transition (pEMT) plays a vital role in oral squamous cell carcinoma (OSCC) cervical lymph node metastasis and tumor immune escape as an immune barrier. However, targeted interventions for pEMT have yet to be established. In this study, we generated an αPDPN-Ag2S probe by modifying a Podoplanin(PDPN) monoclonal antibody on the surface of near infrared (NIR)-II Ag2S quantum dots (QDs) with carboxyl groups through an amide reaction. Then, we evaluated its in vivo targeting ability, therapeutic efficacy of eliminating pEMT using αPDPN-Ag2S-mediated NIR-II photoimmunotherapy (PIT) and biological safety. Here, we found that pEMT is related to CD8 + T-cell infiltration in our human OSCC tissue microarray. Compared with PdpnWT SCC7, the slower growth rate of subcutaneous graft tumors implanted with PdpnKD SCC7 was associated with a change in the tumor immune microenvironment (TIM) in an immunocompetent C3H/HeJ mouse model. In vitro, αPDPN-Ag2S plus NIR 808 nm laser irradiation induced SCC7 cell death. In vivo, NIR-II imaging results show that the αPDPN-Ag2S probe has a good active-targeting ability in a 4-nitroquinoline 1-oxide (4NQO)-induced C57 mouse OSCC model and C3H/HeJ SCC7 subcutaneous graft tumor model. Elimination of pEMT cells by NIR-II αPDPN-Ag2S probe-mediated PIT significantly reversed the local immunosuppressive tumor microenvironment and enhanced PD-1 immunotherapy efficacy. The safety profiles of αPDPN-Ag2S in BALB/c mice were also acceptable. Thus, αPDPN-Ag2S has important clinical translational value in predicting the risk of cervical lymph node metastasis. Importantly, our study proposed a new way to improve the efficacy of tumor immunotherapy.

VISTA blockade alleviates immunosuppression of MDSCs in oral squamous cell carcinoma

V-domain Ig suppressor of T-cell activation (VISTA) is a novel immune checkpoint regulator that can inhibit T cell-mediated antitumor immunity. Although the use of anti-VISTA monoclonal antibody has demonstrated encouraging outcomes in the therapy of various malignancies, its specific impact and underlying mechanisms in oral squamous cell carcinoma (OSCC) remain to be explored. In this work, we analyzed human OSCC tissue microarrays, human peripheral blood mononuclear cells, and immunocompetent transgenic mouse models to investigate the relationship between high VISTA expression and markers of myeloid-derived immunosuppressive cells (MDSCs; CD11b, CD33, Arginase-1), tumor-associated macrophages (CD68, CD163, CD206), and T cell function (CD8, PD-L1, Granzyme B). In OSCC, we discovered that VISTA was highly expressed and stably expressed in MDSCs. Furthermore, we established a mouse OSCC orthotopic xenograft tumor model to investigate the impact of VISTA blockade on the tumor microenvironment. We found that VISTA blockade reduces the immunosuppressive microenvironment and delays tumor growth. This is achieved by suppressing the quantity and function of MDSCs while boosting the function of tumor-infiltrating T cells. Our research indicated that VISTA expressed by MDSCs has a crucial function in the progression of OSCC and that VISTA blockade therapy is a promising immune checkpoint blockade therapy.

Redox-Activatable Magnetic Nanoarchitectonics for Self-Enhanced Tumor Imaging and Synergistic Photothermal-Chemodynamic Therapy.

Oral squamous cell carcinoma (OSCC) is a prevalent malignancy of the head and neck region associated with high recurrence rates and poor prognosis under current diagnostic and treatment methods. The development of nanomaterials that can improve diagnostic accuracy and therapeutic efficacy is of great importance for OSCC. In this study, a redox-activatable nanoarchitectonics is designed via the construction of dual-valence cobalt oxide (DV-CO) nanospheres, which can serve as a contrast agent for magnetic resonance (MR) imaging, and exhibit enhanced transverse and longitudinal relaxivities through the release and redox of Co3+ /Co2+ in an acidic condition with glutathione (GSH), resulting in self-enhanced T1 /T2 -weighted MR contrast. Moreover, DV-CO demonstrates properties of intracellular GSH-depletion and hydroxyl radicals (•OH) generation through a Fenton-like reaction, enabling strengthened chemodynamic (CD) effect. Additionally, DV-CO displays efficient near-infrared laser-induced photothermal (PT) effect, thereby exhibiting synergistic PT-CD therapy for suppressing OSCC tumor cells. It further investigates the tumor-specific self-enhanced MR imaging of DV-CO both in subcutaneous and orthotopic OSCC mouse models, and demonstrate the therapeutic effects of DV-CO in orthotopic OSCC mouse models. Overall, the in vitro and in vivo findings highlight the excellent theranositc potentials of DV-CO for OSCC and offer new prospects for future advancement of nanomaterials.

A novel near-infrared EGFR targeting probe for metastatic lymph node imaging in preclinical mouse models

For the treatment of patients with oral squamous cell carcinoma (OSCC), the imaging of cervical lymph nodes and the evaluation of metastastic progression are of great significance. In recent years, the development of new non-radioactive lymph node tracers has been an area of intense research. Here, we report the synthesis, good biocompatibility, and in vivo evaluation of a new small molecule near-infrared (NIR) fluorescence probe by the conjugation of Lapatinib to S0456 (LP-S). We show that like Lapatinib, LP-S binds to the epidermal growth factor receptor (EGFR) resulting in high quality fluorescence imaging of metastatic lymph nodes in OSCC mouse models. After local injection of LP-S into the tumor, the lymphatic drainage pathway and lymph nodes can be clearly identified by NIR fluorescence imaging. Further, the LP-S probe shows higher contrast and longer retention in metastatic lymph nodes, allowing them to be differentiated from normal lymph nodes, and affording a new choice for fluorescence-guided surgery.Graphical abstractScheme. Chemical synthesis and application of EGFR targeting probe LP-S for imaging of metastatic lymph nodes (mLNs) in OSCC