Oral Carcinogenesis Model Research Articles

Tumor-colonized Streptococcus mutans metabolically reprograms tumor microenvironment and promotes oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) remains a major death cause in head and neck cancers, but the exact pathogenesis mechanisms of OSCC are largely unclear.ResultsSaliva derived from OSCC patients but not healthy controls (HCs) significantly promotes OSCC development and progression in rat models, and metabolomic analyses reveal saliva of OSCC patients but not HCs and OSCC tissues but not adjacent non-tumor tissues contain higher levels of kynurenic acid (KYNA). Furthermore, large amounts of Streptococcus mutans (S. mutans) colonize in OSCC tumor tissues, and such intratumoral S. mutans mediates KYNA overproductions via utilizing its protein antigen c (PAc). KYNA shifts the cellular types in the tumor microenvironment (TME) of OSCC and predominantly expedites the expansions of S100a8highS100a9high neutrophils to produce more interleukin 1β (IL-1β), which further expands neutrophils and induces CD8 + T cell exhaustion in TME and therefore promotes OSCC. Also, KYNA compromises the therapeutic effects of programmed cell death ligand 1 (PD-L1) and IL-1β blockades in oral carcinogenesis model. Moreover, KYNA-mediated immunosuppressive program and aryl hydrocarbon receptor (AHR) expression correlate with impaired anti-tumor immunity and poorer survival of OSCC patients.ConclusionsThus, aberration of oral microbiota and intratumoral colonization of specific oral bacterium such as S. mutans may increase the production of onco-metabolites, exacerbate the oral mucosal carcinogenesis, reprogram a highly immunosuppressive TME, and promote OSCC, highlighting the potential of interfering with oral microbiota and microbial metabolism for OSCC preventions and therapeutics.5ViVJ5sCbraGF_6Zi1UXojVideo

Single-cell transcriptome dissecting the microenvironment remodeled by PD1 blockade combined with photodynamic therapy in a mouse model of oral carcinogenesis.

Oral squamous cell carcinoma (OSCC) stands as a predominant and perilous malignant neoplasm globally, with the majority of cases originating from oral potential malignant disorders (OPMDs). Despite this, effective strategies to impede the progression of OPMDs to OSCC remain elusive. In this study, we established mouse models of oral carcinogenesis via 4-nitroquinoline 1-oxide induction, mirroring the sequential transformation from normal oral mucosa to OPMDs, culminating in OSCC development. By intervening during the OPMDs stage, we observed that combining PD1 blockade with photodynamic therapy (PDT) significantly mitigated oral carcinogenesis progression. Single-cell transcriptomic sequencing unveiled microenvironmental dysregulation occurring predominantly from OPMDs to OSCC stages, fostering a tumor-promoting milieu characterized by increased Treg proportion, heightened S100A8 expression, and decreased Fib_Igfbp5 (a specific fibroblast subtype) proportion, among others. Notably, intervening with PD1 blockade and PDT during the OPMDs stage hindered the formation of the tumor-promoting microenvironment, resulting in decreased Treg proportion, reduced S100A8 expression, and increased Fib_Igfbp5 proportion. Moreover, combination therapy elicited a more robust treatment-associated immune response compared with monotherapy. In essence, our findings present a novel strategy for curtailing the progression of oral carcinogenesis.

4-nitroquinoline 1-oxide-induced oral epithelial lesions exhibit time- and stage-dependent changes in the tumor immune microenvironment.

Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity and is associated with high morbidity due to local invasion and lymph node metastasis. Tumor infiltrating lymphocytes (TILs) are associated with good prognosis in oral cancer patients and dictate response to treatment. Ectopic sites for immune activation in tumors, known as tertiary lymphoid structures (TLS), and tumor-associated high-endothelial venules (TA-HEVs), which are specialized lymphocyte recruiting vessels, are associated with a favorable prognosis in OSCC. Why only some tumors support the development of TLS and HEVs is poorly understood. In the current study we explored the infiltration of lymphocyte subsets and the development of TLS and HEVs in oral epithelial lesions using the 4-nitroquinoline 1-oxide (4NQO)-induced mouse model of oral carcinogenesis. We found that the immune response to 4NQO-induced oral epithelial lesions was dominated by T cell subsets. The number of T cells (CD4+, FoxP3+, and CD8+), B cells (B220+) and PNAd+ HEVs increased from the earliest to the latest endpoints. All the immune markers increased with the severity of the dysplasia, while the number of HEVs and B cells further increased in SCCs. HEVs were present already in early-stage lesions, while TLS did not develop at any timepoint. This suggests that the 4NQO model is applicable to study the dynamics of the tumor immune microenvironment at early phases of oral cancer development, including the regulation of TA-HEVs in OTSCC.

Analysis of Co-expression of HSP 70 and BCL 2 in Oral Precancer and Cancer Patients Using Immunohistochemistry.

Understanding of molecular model of oral carcinogenesis has carried cancer chemotherapy far forward from conventional drug therapies. Small molecule inhibitors have gained acceptance as it has fewer adverse effects and also provide targeted drug therapy. The association of HSP 70 (Heat Shock Protein 70) and BCL 2 (B-cell lymphoma 2) proteins with oral precancer and cancer is already established. However, the complex interaction between these two proteins and how they affect each other's expression is still not understood completely. In our study, we aimed to correlate the expression of HSP 70 and BCL 2 with different histopathological grades of oral precancer and cancer tissue samples using tissue immunohistochemistry. Tissue samples were taken from a total of 250 patients (100 OPMDs and 150 OSCCs) and subjected to immunohistochemistry using anti-human mouse monoclonal antibodies to HSP70 and BCL2. Immunostaining was done, and the immunostaining intensity distribution (IID) index was calculated. Immunoreactivity scores for both HSP 70 and BCL 2 correlated with different grades of dysplasia. However, only HSP 70 had a statistically significant association (p=0.066). We also found that HSP 70 showed an inverse correlation, with higher expression majorly seen in well-differentiated OSCCs. Our study unveiled the HSP 70-BCL 2 interaction and provides insights about how this might affect drug designing and help overcome therapeutic lags. However, further studies are needed to provide a comprehensive review of such interactions among various small molecules.

Abstract LB156: Targeting epigenetic activity of beta-catenin/CBP impedes oral cancer progression

Abstract Head and neck cancer is a complex malignancy with its major anatomical subsite, cancer of the oral cavity, ranking among the most deadly and disfiguring cancers. Oral cancer presents primarily as HPV-negative oral squamous cell carcinoma (OSCC) whose etiology is associated with tobacco and alcohol use and for which there are limited treatments. Whereas immunotherapies, such as anti-PD-1 antibodies, have become a promising therapeutic option, only ~20% of head neck cancer patients benefit from anti-PD-1 immunotherapy. We and others have shown that non-mutational epigenetic reprogramming mediated by the β-catenin/CBP complex promotes aggressive cell phenotypes in OSCC by establishing open chromatin at the transcription start sites of genes associated with cancer stemness. Accordingly, pharmacological inhibition of this complex dramatically inhibits OSCC evolution in a syngeneic mouse model of OSCC. Here, we evaluated whether the inhibition of β-catenin/CBP epigenetic activity would enhance the effects of anti-PD-1 immunotherapy. We adapted a syngeneic mouse model of tobacco-associated oral carcinogenesis that utilizes a mouse cell line, 4MOSC1, derived from 4-nitroquinoline-1 oxide- (4NQO)-induced tongue tumors of C57BL/6J mice. After tumors developed for 7 days, mice were blindly separated into treatment cohorts (n=13, each) to receive vehicle control (PBS), monotherapy (either 10 mg/kg anti-PD-1 EOD IP, or 100 mg/kg E7386, a Wnt signaling pathway modulator, QD oral gavage) or combination therapy (anti-PD-1 + E7386). Tumor isografts were harvested 11 days post initiation of treatments and processed for histopathology, immunohistochemistry, and immunofluorescence analyses. Measurements of tumor volumes showed that combination therapy resulted in a statistically significant reduction by ~80% compared to control mice and that it surpassed tumor decrease by either monotherapy. These results were confirmed by histopathology analyses. Evaluation of tumor tissues by immunofluorescence revealed statistically significant decreases in Cbp, H3K4me3, Keratin 14, and Podoplanin expression in response to E7386 alone and in combination with anti PD-1. Collectively our results suggest that a combination therapy comprising anti PD-1 treatment with E7386-targeting β-catenin/CBP epigenetic signaling is likely to impede OSCC evolution to advanced disease. Supported by Eisai, Co Ltd Research Award (MAK), NIH 5R01 DE030350 (MAK, SM, XV) and 5R01 DE031413 (MVB). Citation Format: Emily Fisher, Anthony Spinella, Junji Matsui, Kenichi Nomoto, Xaralabos Varelas, Manish Bais, Maria Kukuruzinska. Targeting epigenetic activity of beta-catenin/CBP impedes oral cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB156.

Abstract LB377: The combination of epigallocatechin gallate (EGCG) and resveratrol prevents 4-NQO-induced oral carcinogenesis in mice

Abstract Head and neck cancer (HNC), commonly known as oral cancer, is a devastating disease and the 6th most common cancer worldwide. Most HNC patients are diagnosed with advanced-stage disease for which the 5-year survival is below 50%, stressing the need for chemoprevention. Recently, we have reported that the combination of resveratrol and EGCG induces synergistic apoptosis and inhibits xenografted HNC growth by inhibiting the AKT-mTOR pathway. This study investigated the chemopreventive efficacy of resveratrol, EGCG, and their combination using the 4NQO-induced oral carcinogenesis model. C57BL/6 mice were exposed to 4-NQO (50 μg/ml) via drinking water for 10 weeks, followed by treatment with vehicle (50% sweetened condensed milk), resveratrol (30 mg/kg), EGCG (30 mg/kg) and their combination for 8 weeks, 5 days/week. The mice were sacrificed on week 24, and the number of visible and microscopic lesions was counted. Resveratrol alone and in combination with EGCG significantly inhibited the number of visible lesions. In contrast, the number of microscopic lesions and lesion area was significantly inhibited only in the combination group. The expression of Ki-67 was also significantly inhibited in resveratrol and combination groups when compared with untreated control. Furthermore, RNASeq and qPCR analysis using an HNC cell line identified GDF15, ATF3, p21, p27, and Bim as significantly upregulated genes, with GDF15 being the most upregulated one. Expression of GDF15 and ATF3 proteins was confirmed by Western blotting. Taken together, our data strongly demonstrate the chemopreventive potential of the combination of EGCG and resveratrol and pave the way for further clinical developments. (Supported by NIH Grants R15DE032063 and P20GM103434) Citation Format: Adeoluwa A. Adeluola, Lukmon Raji, Saroj Sigdel, ASM Anisuzzaman, A.R.M. Ruhul Amin. The combination of epigallocatechin gallate (EGCG) and resveratrol prevents 4-NQO-induced oral carcinogenesis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB377.

Serum Raman spectroscopy: Evaluation of tumour load variations in experimental carcinogenesis.

Several serum Raman spectroscopy (RS) studies have demonstrated its potential as an oral cancer screening tool. This study investigates influence of low tumour load (LTL) and high tumour load (HTL) on serum RS using hamster buccal pouch model of experimental oral carcinogenesis. Sera of untreated control, LTL, and HTL groups at week intervals during malignant transformation were employed. Serum Raman spectra were subjected to multivariate analyses-principal component analysis, principal component-based linear discriminant analysis (for stratification of study groups), and multivariate curve resolution-alternating least squares (MCR-ALS) (to comprehend biomolecular differences). Multivariate analysis revealed misclassifications between LTL and HTL at all week intervals. MCR-ALS components showed statistically significant abundances between control versus LTL and control versus HTL, but could not discern LTL and HTL. MCR-ALS components exhibited spectral mixtures of proteins, lipids, heme and nucleic acids. Thus, these findings support use of serum RS as a screening tool as varying tumour load is not a confounding factor influencing the technique.

Mass cytometry and transcriptomic profiling reveal PD1 blockade induced alterations in oral carcinogenesis.

Oral squamous cell carcinoma is the predominant subtype of head and neck squamous cell carcinoma, characterized by a challenging prognosis. In this study, we established a murine model of oral carcinogenesis using 4-nitroquinoline-1-oxide (4-NQO) induction to investigate the impact of immunotherapy on microenvironmental alterations. Mice in the precancerous condition were randomly divided into two groups:one receiving programmed death-1 (PD1) monoclonal antibody treatment and the other, control immunoglobulin G. Our observations showed that while PD1 blockade effectively delayed the progression of carcinogenesis, it did not completely impede or reverse it. To unravel the underlying reasons for the limited effectiveness of PD1 blockade, we collected tongue lesions and applied mass cytometry (CyTOF) and RNA sequencing (RNA-seq) to characterize the microenvironment. CyTOF analysis revealed an increased macrophage subset (expressing high levels of IFNγ and iNOS) alongside a diminished Th1-like subset (exhibiting low expression of TCF7) and three myeloid-derived suppressor cell subsets (displaying low expression of MHC Class II or IFNγ) following anti-PD1 treatment. Notably, we observed an increased presence of cancer-associated fibroblasts (CAFs) expressing collagen-related genes after PD1 blockade. Furthermore, we found a negative correlation between the infiltration levels of CAFs and CD8+ T cells. These findings were validated in murine tongue tissue slides, and publicly available multi-omics datasets. Our results suggest that CAFs may impair the therapeutic efficacy of PD1 blockade in oral carcinogenesis by the remodeling of the extracellular matrix.

Inhibition of PORCN Blocks Wnt Signaling to Attenuate Progression of Oral Carcinogenesis.

Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/β-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis. We performed a model of tobacco-induced oral cancer in vitro, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an in vivo model of oral carcinogenesis and ex vivo samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59. Both in vitro and ex vivo oral carcinogenesis approaches revealed decreased levels of nuclear β-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 in vitro resulted in diminished cell migration, viability, and invasion. Finally, by using an in vivo model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions. The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.

Abstract PO-095: TGF-β carrying exosomes in plasma of HNSCC as potential biomarkers of disease progression in patients with HNSCC

Abstract Background: The contribution of TGFβ+ exosomes, now referred to as small extracellular vesicles (sEV), in plasma of cancer patients to disease progression has been unclear. Levels of TGFβ in plasma often do not correlate with clinicopathological data, and TGFβ can exert dual (yin-yang) effects as a tumor promoter and a tumor inhibitor. We evaluated the potential of tumor-derived sEV dubbed TEX to serve as liquid tumor biopsy in patients with HNSCC. Methods: Plasma was obtained from 3 different cohorts of patients with HNSCC (n=97) and HDs (n=22). Clinicopathology/demographic data were available for all patients. sEV were isolated by ultrafiltration/size exclusion chromatography (SEC) from plasma or cell supernatants. TGF-β levels in plasma were assessed by ELISA; TGF-β expression in tissues was studied by IHC and immunoblots; immunoblots, flow cytometry and LC-MS/MS were used to measure TGF-β in isolated sEV. TGFB1 gene expression was analyzed using the TCGA Head and Neck Cancer database (n=520 cases and 44 NC). TGF-β signaling and activity of EVs were quantified in the reporter cell (MFB-F11) proliferation assays. Changes of TGF-β expression levels in plasma, tissues and sEV during carcinogenesis were studied in an orthotopic 4NQO mouse model. Results: Using the TCGA we found high expression levels of the vesiculation genes regulating EV production/release and of the TGFB1 gene expression in HNSCC. In the 4NQO orthotopic model of OSCC, cancer progression correlated with TGF-β accumulations in tumor tissues and a significant increase in circulating TGF-β+ sEV. In HNSCC patients, TGF-β levels and activity in plasma and TGF-β expression levels in tumor tissues were significantly elevated relative to controls but did not correlate with clinicopathologic data, tumor burden or disease outcome. TEX produced by HNSCC cell lines carried TGF-β and initiated Smad3 signaling in reporter cells. TGF-β inhibitors blocked this signaling. High TGFβ abundance in sEV was confirmed by proteomics and by RNAseq using the TCGA data base. Levels of TGF-β expression in sEV, sEV numbers and TGF-β activity of sEV were significantly elevated in HNSCC patients vs HDs’ sEV and correlated with tumor stage and LN involvement. Conclusions: TEX and sEV from HNSCC patients’ plasma carry TGF-β which reflects disease progression in HNSCC patients and in a mouse model of oral carcinogenesis. Neither plasma levels of TGF-β (ELISA) nor levels of TGF-β expression in the tumor (IHC) correlated with disease progression. Only sEV-associated TGF-β correlates with tumor burden and shows promise as a non-invasive biomarker of HNSCC progression and response to therapy. Citation Format: Nils Ludwig, Saigopalakrishna S. Yerneni, Theresa L. Whiteside. TGF-β carrying exosomes in plasma of HNSCC as potential biomarkers of disease progression in patients with HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-095.

A scientometric study of chemical carcinogen-induced experimental oral carcinogenesis with emphasis on chemopreventive agents

Background/purpose4-Nitroquinoline 1-oxide (4NQO)-induced tongue carcinoma and 7,12-dimethlybenz(a)anthracene (DMBA)-induced cheek pouch carcinoma are the most common and classical chemical carcinogen-induced animal models of oral carcinogenesis. The purpose of this study was to provide the research trends and characteristics of 4NQO- and DMBA-induced experimental oral carcinogenesis. Materials and methodsThe papers on both 4NQO- and DMBA-induced experimental oral carcinogenesis were published since 1962. All the eligible papers were retrieved on 12 May 2023 from the Scopus database. ResultsThere were 506 and 349 papers on 4NQO- and DMBA-induced experimental oral carcinogenesis with 10,152 and 6306 citations, respectively. The common distinctive keywords such as rat, tongue neoplasms, drinking water, tumor microenvironment, and cyclooxygenase (COX)-2 were identified in the papers on 4NQO; and the common keywords such as hamster, cheek pouch, lipid peroxidation, glutathione, antioxidants, and topical drug administration were identified in the papers on DMBA. Importantly, 105 and 65 potential chemopreventive agents were identified from the papers on 4NQO and DMBA, respectively. Furthermore, 15 promising agents such as COX-2 inhibitor, curcumin, garlic were researched concurrently in both the two animal models. ConclusionThis study for the first time reports the scientometric characteristics of 4NQO- and DMBA-induced experimental oral carcinogenesis. Importantly, we identify a valuable profile for oral cancer chemopreventive agents, which will aid researchers and investigators in studying oral cancer chemoprevention.

Proposal of a secure and efficient protocol for a murine oral carcinogenesis model induced by 4-nitroquinoline-1-oxide (4NQO)

An important rat model using the chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) has been described for the study of the process of oral carcinogenesis. This model replicates the gradual progression seen in oral carcinoma patients. However, due to its high level of toxicity, its use in fundamental research is challenging. Here, we propose a secure and efficient modified protocol based on a lower dose of 4NQO concentration as well as an increased water supply and hypercaloric diet, in order to reduce the damage caused to the animals during the process of oral carcinogenesis. Twenty-two male Wistar rats were exposed to 4NQO, evaluated clinically once a week and euthanized at 12 and 20 weeks for histopathological analysis. The protocol involves a staggered dose of 4NQO up to a concentration of 25 ppm, associated with two days of pure water, a 5% glucose solution once a week and a hypercaloric diet. This modified protocol prevents the immediate consequences of the carcinogen. At week 7, all animals displayed clinically evident tongue lesions. From a histological perspective, after 12 weeks of 4NQO exposure, 72.7% of the animals developed epithelial dysplasia and 27.3% developed in situ carcinoma. In the group exposed for 20 weeks, epithelial dysplasia and in situ carcinoma were diagnosed in one case each, whereas invasive carcinoma was diagnosed in 81.8% of the cases. Nonsignificant modification of animal’s behavior and weight was observed. This new proposed 4NQO protocol was secure and effective for studying oral carcinogenesis and can be used to conduct lengthy investigations.

Abstract 4743: Wnt/beta-catenin mediated epigenetic modifications drive age-dependent oral squamous cell carcinoma evolution

Abstract Oral Squamous Cell Carcinoma (OSCC) is a devastating disease associated with high morbidity, poor survival, and few therapeutic options. This malignancy is driven, in part, by epigenetic reprogramming of the chromatin landscape directed by β-catenin in complex with CREB-binding protein (CBP) and mixed lineage leukemia methyltransferase 1 (MLL1). The β-catenin/CBP/MLL1 complex promotes an open chromatin structure by enabling transcription of genes associated with cell plasticity, including cancer stem cells and cells with partial EMT (p-EMT) phenotypes. Growing evidence indicates that cell plasticity and cellular senescence are integral processes shared by cancer and aging. Given that the median age of OSCC diagnosis is 66 years, it is likely that aging promotes OSCC evolution to advanced disease. To examine the effects of aging on OSCC evolution, we adapted a syngeneic mouse model of tobacco-associated oral carcinogenesis. This model utilizes a mouse cell line, 4MOSC1, derived from 4-nitroquinoline-1 oxide- (4NQO)-induced tongue tumor which, when implanted into mouse tongues, generates tumors that recapitulate human OSCC mutanome. The growth of 4MOSC1 derived orthotopic tumors was studied in 6- and 80-week-old mice. Tumors were allowed to develop for 18 days, after which they were harvested and processed for single cell RNA sequencing, histopathology and immunofluorescence (IF) analyses. Results showed that tumors grew at a faster rate and to a greater overall size in old mice compared to their young cohorts. OSCC harvested from the old mice displayed statistically significant reduction in membranous E-cadherin concomitant with increased Cbp and H3K4me3 abundance, suggesting an age-associated upregulation of β-catenin/Cbp/Mll1 epigenetic activity. Increased expression of Bmi1, keratin 14, and podoplanin supported increased cell plasticity in OSCC from aged mice. Further, these tumors exhibited augmented cellular senescence, as judged by the disruption of lamin B1-associated nuclear membrane integrity, and by an increased cell population with markers of senescence from single cell sequencing analyses. These data suggest that aging is associated with increased β-catenin/Cbp/Mll1 epigenetic signaling, cell plasticity and cellular senescence, which collectively contribute to the evolution of OSCC. Citation Format: Emily R. Fisher, Anthony Spinella, Nina C. Hardy, Xaralabos Varelas, Manish Bais, Maria A. Kukuruzinska. Wnt/beta-catenin mediated epigenetic modifications drive age-dependent oral squamous cell carcinoma evolution. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4743.

The participation of tumor residing pericytes in oral squamous cell carcinoma

Pericytes are perivascular cells related to vessel structure and angiogenesis that can interact with neoplastic cells, interfering with cancer progression and outcomes. This study focused on the characterization of pericytes in oral squamous cell carcinoma (OSCC) using clinical samples and a transgenic mouse model of oral carcinogenesis. Nestin-/NG2+ (type-1) and nestin+/NG2+ (type-2) pericytes were analyzed by direct fluorescence after induction of oral carcinogenesis (4-nitroquinoline-1-oxide). Gene expression of neuron glial antigen-2 (NG2), platelet-derived growth factor receptor beta (PDGFR-β), and cluster of differentiation 31 (CD31) was examined in human OSCC tissues. The protein expression of von Willebrand factor and NG2 was assessed in oral leukoplakia (i.e., oral potentially malignant disorders) and OSCC samples. Additionally, clinicopathological aspects and survival data were correlated and validated by bioinformatics using The Cancer Genome Atlas (TCGA). Induction of carcinogenesis in mice produced an increase in both NG2+ pericyte subsets. In human OSCC, advanced-stage tumors showed a significant reduction in CD31 mRNA and von Willebrand factor-positive vessels. Low PDGFR-β expression was related to a shorter disease-free survival time, while NG2 mRNA overexpression was associated with a reduction in overall survival, consistent with the TCGA data. Herein, oral carcinogenesis resulted in an increase in NG2+ pericytes, which negatively affected survival outcomes.

A Novel Preclinical In Vitro 3D Model of Oral Carcinogenesis for Biomarker Discovery and Drug Testing.

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents.

Abstract A004: Chemoprevention of head and neck cancers by the combination of epigallocatechin gallate (EGCG) and resveratrol

Abstract Head and neck cancer (HNC) is a devastating disease and the 6th most common cancer worldwide. Most HNC patients are diagnosed with advanced stage disease for which the 5-year survival is below 50%, stressing the need for chemoprevention. Recently, we have reported that the combination of resveratrol and EGCG induces synergistic apoptosis and inhibits xenografted HNC growth by inhibiting AKT-mTOR pathway. In this study, we have investigated the chemopreventive efficacy of resveratrol, EGCG and their combination using 4NQO-induced oral carcinogenesis model. C57BL/6 mice were exposed to 4-NQO (50 μg/ml) via drinking water for 10 weeks, followed by treatment with vehicle (50% sweetened condense milk), resveratrol (30 mg/kg), EGCG (30 mg/kg) and their combination for 8 weeks, 5 days/week. The mice were sacrificed on week 24 and the number of visible and microscopic lesions were counted. Resveratrol alone and in combination with EGCG significantly inhibited the number of visible lesions whereas the number of microscopic lesions and lesion area were significantly inhibited only in the combination group. Furthermore, RNASeq and qPCR analysis using a HNC cell line identified GDF15, ATF3, p21, p27 and Bim as significantly upregulated genes with GDF15 being the most upregulated one. Expression of GDF15 and ATF3 proteins were confirmed by western blotting. Taken together, our data strongly demonstrate the chemopreventive potential of the combination of EGCG and resveratrol and paves the way for further clinical developments. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence) Citation Format: Adeoluwa Adeluola, Lukmon Raji, Saroj Sigdel, A.S.M. Anisuzzaman, A. R. M. Ruhul Amin. Chemoprevention of head and neck cancers by the combination of epigallocatechin gallate (EGCG) and resveratrol [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A004.

Prevention of carcinogen-induced oral cancers by polymeric black tea polyphenols via modulation of EGFR-Akt-mTOR pathway

The overexpression of Epidermal Growth Factor Receptor (EGFR) and dysregulation of its downstream effector pathways are important molecular hallmarks of oral cancers. Present study investigates the chemopreventive potential of polymeric black tea polyphenols (PBPs)/thearubigins (TRs) in the hamster model of oral carcinogenesis as well as determine the effect of PBPs on EGFR and the molecular players in the EGFR pathway. In dose-dependent manner, pre and concurrent treatment with PBPs (1.5%, 5%, 10%) decreased the number and volume of macroscopic tumors as well as the number and area of microscopic lesions. Interestingly, at 10% dose of PBPs, no macroscopic or microscopic tumors were observed. We observed PBPs mediated dose-dependent decrease in oxidative DNA damage (8OHdG); inflammation (COX-2); proliferation (PCNA, Cyclin D1); expression of EGFR, and its downstream signaling kinases (pAkt, Akt, and mTOR); hypoxia (HIF1α) and angiogenesis (VEGF). There was also a PBPs mediated dose-dependent increase in apoptosis (Bax). Thus, our data clearly indicate that the observed chemopreventive potential of PBPs was due to modulation in the EGFR pathway associated with cell proliferation, hypoxia, and angiogenesis. Taken together, our results demonstrate preclinical chemopreventive efficacy of PBPs and give an insight into its mechanistic role in the chemoprevention of experimental oral cancer.

Oroxylum indicum Stem Bark Extract Reduces Tumor Progression by Inhibiting the EGFR-PI3K-AKT Pathway in an In Vivo 4NQO-Induced Oral Cancer Model

Objectives Oral squamous cell carcinoma (OSCC) is the predominant type of oral cancer. Its incidence is high in certain geographic regions, and it is correlated with chewing tobacco. Epidermal growth factor receptor (EGFR), induced by tobacco carcinogens, is overexpressed in OSCC, leading to poor prognosis. Thus, EGFR inhibitors are promising agents against OSCC. High cost and toxicity of existing EGFR inhibitors necessitate alternative EGFR-targeted therapy. Here, we tested the antitumor potential of ethyl acetate fraction of an ethnomedicinal tree, Oroxylum indicum stem bark extract (OIEA) in a 4-nitroquinoline-1-oxide (4NQO)-induced oral carcinogenesis model. Methods OIEA was prepared by solvent extraction method, and subsequently its in vitro radical scavenging activities were measured. High-performance liquid chromatography (HPLC) analysis of OIEA was done to identify the constituent active compounds. Hemolytic, trypan blue exclusion, and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assays were performed in normal and cancer cells to select an optimum dose of OIEA for antitumor activity study in 4NQO-induced oral cancer in F344 rats. Measurement of tumor volume, weight, and cell count was followed by tumor cell cycle analysis and comet and annexin V/Propidium Iodide (PI) assay. Pro-apoptotic markers were detected by western blot testing. Molecular docking was done to predict the interaction between OIEA active component and EGFR or phosphatidylinositol-3-kinase (PI3K), which was further validated biologically. Finally, hepatic and renal function testing and histopathology were performed. Results OIEA reduced tumor burden and increased survivability of the tumor-bearing rats significantly as compared to untreated tumor bearers. HPLC revealed oroxylin A as the predominant bioactive component in OIEA. Molecular docking predicted significant binding between oroxylin A and EGFR as well as PI3K, which was confirmed by western blot analysis of in vivo samples. OIEA also ameliorated hepato-, renal- and myelotoxicity induced by 4NQO. Conclusion OIEA reduces 4NQO-induced OSCC by modulating the EGFR/PI3K/AKT signaling cascade and also ameliorated toxicity in tumor bearers.

Three-Dimensional Printing of an Apigenin-Loaded Mucoadhesive Film for Tailored Therapy to Oral Leukoplakia and the Chemopreventive Effect on a Rat Model of Oral Carcinogenesis.

Oral leukoplakia, which presents as white lesions in the oral cavity, including on the tongue, is precancerous in nature. Conservative treatment is preferable, since surgical removal can markedly reduce the patient’s quality of life. In the present study, we focused on the flavonoid apigenin as a potential compound for preventing carcinogenesis, and an apigenin-loaded mucoadhesive oral film was prepared using a three-dimensional (3D) bioprinter (semi-solid extrusion-type 3D printer). Apigenin-loaded printer inks are composed of pharmaceutical excipients (HPMC, CARBOPOL, and Poloxamer), water, and ethanol to dissolve apigenin, and the appropriate viscosity of printer ink after adjusting the ratios allowed for the successful 3D printing of the film. After drying the 3D-printed object, the resulting film was characterized. The chemopreventive effect of the apigenin-loaded film was evaluated using an experimental rat model that had been exposed to 4-nitroquinoline 1-oxide (4NQO) to induce oral carcinogenesis. Treatment with the apigenin-loaded film showed a remarkable chemopreventive effect based on an analysis of the specimen by immunohistostaining. These results suggest that the apigenin-loaded mucoadhesive film may help prevent carcinogenesis. This successful preparation of apigenin-loaded films by a 3D printer provides useful information for automatically fabricating other tailored films (with individual doses and shapes) for patients with oral leukoplakia in a future clinical setting.

Development of a murine model of oral carcinogenesis: an accelerated tool for biomarker and anti-tumour drug discovery.

Oral squamous cell carcinoma (OSCC) is the most common cancer in Pakistani men and the second most common cancer in women. The objective of our study was to devise a novel accelerated murine model of oral carcinogenesis that can be exploited as a tool to investigate the cancer circuitry involved in OSCC and to identify molecules of diagnostic, therapeutic and prognostic significance. A total of 40 healthy male, 6–8 weeks old, 22 ± 2 gram, Naval Medical Research Institute (NMRI) outbred strain mice were recruited in the experiment. NMRI mice are commonly used for animal experiments in various fields of biology and for drug toxicity. Of these, 25 mice underwent the oral carcinogenesis regimen via topical application of 0.5% 9,10-dimethyl-1,2-benzanthracene (DMBA) on the lower left lip for a maximum of 20 weeks and 15 mice were used as controls (without the carcinogenic regimen). Exophytic tissue masses were harvested, fixed in 10% formalin and stained with haematoxylin and eosin (H&E) for microscopic diagnosis. Additionally, the expression levels of CK 5/6, p53 and Ki-67 were investigated using immunohistochemistry. Of the 25 mice which underwent the carcinogenic regimen, 21 developed moderately differentiated squamous cell carcinoma and 1 showed dysplastic features with foci of invasion. Three mice were found dead with lesion(s). CK 5/6 showed strong positivity (100%) and p53 and Ki-67 showed patchy (<30%) strong positivity in OSCC, suggesting the similarity of our model to human OSCC. We present an accelerated, close-to-human carcinogenesis, model of oral carcinogenesis using DMBA in NMRI mice that can be exploited to study the pathogenesis of oral squamous cell carcinoma and subsequently devise immunotherapy or targeted therapy.