Oral Cancer Model Research Articles

Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately mimic the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report a mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we find that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform to accelerate the development of immunotherapeutic options for HNSCC.

Cancer stem cells enrichment with surface markers CD271 and CD44 in human head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) has a poor 5-year survival rate of 50%. One potential reason for treatment failure is the presence of cancer stem cells (CSCs). Several cell markers, particularly CD44, have been used to isolate CSCs. However, isolating a pure population of CSC in HNSCC still remains a challenging task. Recent findings show that normal oral stem cells were isolated using CD271 as a marker. Thus, we investigated the combined use of CD271 and CD44 to isolate an enriched subpopulation of CSCs, followed by their characterization in vitro, in vivo, and in patients' tissue samples. Fluorescent-activated cell sorting was used to isolate CD44+/CD271+ and CD44+/CD271- from two human HNSCC cell lines. Cell growth and self-renewal were measured with MTT and sphere/colony formation assays. Treatment-resistance was tested against chemotherapy (cisplatin and 5-fluorouracil) and ionizing radiation. Self-renewal, resistance, and stemness-related genes expression were measured with qRT-PCR. In vivo tumorigenicity was tested with an orthotopic immunodeficient mouse model of oral cancer. Finally, we examined the co-localization of CD44+/CD271+ in patients' tissue samples. We found that CD271+ cells were a subpopulation of CD44+ cells in human HNSCC cell lines and tissues. CD44+/CD271+ cells exhibited higher cell proliferation, sphere/colony formation, chemo- and radio-resistance, upregulation of CSCs-related genes, and in vivo tumorigenicity when compared to CD44+/CD271- or the parental cell line. These cell markers showed increased expression in patients with the increase of the tumor stage. In conclusion, using both CD44 and CD271 allowed the isolation of CSCs from HNSCC. These enriched CSCs will be more relevant in future treatment and HNSCC progression studies.

Abrus agglutinin inhibits oral carcinogenesis through inactivation of NRF2 signaling pathway

Abrus agglutinin (AGG), a heterotetrameric type II ribosome inactivating protein isolated from the seeds of Abrus precatorius shows potent antitumor activity in different cancer models. We examined the role of antioxidant system in modulation of the anticancer activity of AGG in in vitro and in hamster model of oral cancer. AGG promotes apoptosis through accumulation of ROS in CAL33 cells. Interestingly, our data showed that AGG decreases the activity of antioxidant enzymes including superoxide dismutase, catalase, glutathione peroxidase in CAL33 cells indicating antioxidant enzyme inhibition leads to AGG-induced ROS accumulation. Moreover, AGG inhibits expression of NRF2, transcription factor which regulates the expression of antioxidant enzymes in CAL33 cells. We found that AGG induces autophagy stimulation and loss of p62 expression in CAL33 cells. Furthermore, it showed that NRF2 expression is restored in the presence of 3-methyladenine and Baficomycin-A1 establishing role of autophagy in modulation of NRF2 through p62. Our study showed that AGG significantly inhibited tumor growth in DMBA-induced carcinogenesis. In immunohistochemical analysis, AGG-treated tumor displays higher caspase 3 expression and less p62 and NRF2 expression in comparison to the control. In conclusion, AGG-induced degradation of NRF2 through autophagy leads to ROS accumulation dependent apoptosis which might be used for treatment of oral cancer.

COTI-2, A Novel Thiosemicarbazone Derivative, Exhibits Antitumor Activity in HNSCC through p53-dependent and -independent Mechanisms.

TP53 mutations are highly prevalent in head and neck squamous cell carcinoma (HNSCC) and associated with increased resistance to conventional treatment primarily consisting of chemotherapy and radiation. Restoration of wild-type p53 function in TP53-mutant cancer cells represents an attractive therapeutic approach and has been explored in recent years. In this study, the efficacy of a putative p53 reactivator called COTI-2 was evaluated in HNSCC cell lines with different TP53 status.Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine in vitro and in vivo sensitivity of HNSCC cell lines with either wild-type, null, or mutant TP53 to COTI-2 alone, and in combination with cisplatin and/or radiation. Western blotting, cell cycle, live-cell imaging, RNA sequencing, reverse-phase protein array, chromatin immunoprecipitation, and apoptosis analyses were performed to dissect molecular mechanisms. COTI-2 decreased clonogenic survival of HNSCC cells and potentiated response to cisplatin and/or radiation in vitro and in vivo irrespective of TP53 status. Mechanistically, COTI-2 normalized wild-type p53 target gene expression and restored DNA-binding properties to the p53-mutant protein in HNSCC. In addition, COTI-2 induced DNA damage and replication stress responses leading to apoptosis and/or senescence. Furthermore, COTI-2 lead to activation of AMPK and inhibition of the mTOR pathways in vitro in HNSCC cells. COTI-2 inhibits tumor growth in vitro and in vivo in HNSCC likely through p53-dependent and p53-independent mechanisms. Combination of COTI-2 with cisplatin or radiation may be highly relevant in treating patients with HNSCC harboring TP53 mutations.

Metabolic Regulation of Glycolysis and AMP Activated Protein Kinase Pathways during Black Raspberry-Mediated Oral Cancer Chemoprevention.

Oral cancer is a public health problem with an incidence of almost 50,000 and a mortality of 10,000 each year in the USA alone. Black raspberries (BRBs) have been shown to inhibit oral carcinogenesis in several preclinical models, but our understanding of how BRB phytochemicals affect the metabolic pathways during oral carcinogenesis remains incomplete. We used a well-established rat oral cancer model to determine potential metabolic pathways impacted by BRBs during oral carcinogenesis. F344 rats were exposed to the oral carcinogen 4-nitroquinoline-1-oxide in drinking water for 14 weeks, then regular drinking water for six weeks. Carcinogen exposed rats were fed a 5% or 10% BRB supplemented diet or control diet for six weeks after carcinogen exposure. RNA-Seq transcriptome analysis on rat tongue, and mass spectrometry and NMR metabolomics analysis on rat urine were performed. We tentatively identified 57 differentially or uniquely expressed metabolites and over 662 modulated genes in rats being fed with BRB. Glycolysis and AMPK pathways were modulated during BRB-mediated oral cancer chemoprevention. Glycolytic enzymes Aldoa, Hk2, Tpi1, Pgam2, Pfkl, and Pkm2 as well as the PKA-AMPK pathway genes Prkaa2, Pde4a, Pde10a, Ywhag, and Crebbp were downregulated by BRBs during oral cancer chemoprevention. Furthermore, the glycolysis metabolite glucose-6-phosphate decreased in BRB-administered rats. Our data reveal the novel metabolic pathways modulated by BRB phytochemicals that can be targeted during the chemoprevention of oral cancer.

Cisplatin and oxaliplatin induce similar immunogenic changes in preclinical models of head and neck cancer

ObjectivesPrior studies suggest that oxaliplatin is unique among platinum chemotherapy drugs in its ability to enhance anti-tumor immunity, but the immune mechanisms of different platinum chemotherapy drugs have not been previously compared in preclinical models of head and neck squamous cell carcinoma (HNSCC). Materials and methodsHuman HNSCC cell lines were treated with cisplatin or oxaliplatin, then assessed for markers associated with immunogenic cell death (ICD) and antigen processing. A syngeneic mouse model of oral cancer was then used to compare the effects of cisplatin vs. oxaliplatin, alone or in combination with anti-PD-1 immunotherapy, on tumor growth and survival. A subset of spleens and tumors were analyzed for ICD markers and immune cell infiltrates by flow cytometry. ResultsCisplatin and oxaliplatin both increased cell surface levels of calreticulin, HSP70, MHC class I and PD-L1 in multiple cell lines. Inoculation of immunocompetent mice with cells killed in vitro by either drug resulted in failure of subsequently-injected live tumor cells to establish and grow in a small proportion of animals. Systemic cisplatin and oxaliplatin induced similar tumor growth delay when combined with anti-PD-1 therapy. ConclusionsTreatment of HNSCC cells with platinum chemotherapy appears to induce some features of anti-tumor immunity, which may be enhanced by anti-PD-1 therapy. Cisplatin, the standard drug for HNSCC, appears to affect anti-tumor immunity in a similar fashion to oxaliplatin in these preclinical models.

Different oral cancer scenarios to personalize targeted therapy: Boron Neutron Capture Therapy translational studies.

Boron neutron capture therapy (BNCT) is a targeted therapy, which consists of preferential accumulation of boron carriers in tumor followed by neutron irradiation. Each oral cancer patient has different risks of developing one or more carcinomas and/or oral mucositis induced after treatment. Our group proposed the hamster oral cancer model to study the efficacy of BNCT and associated mucositis. Translational studies are essential to the advancement of novel boron delivery agents and targeted strategies. Herein, we review our work in the hamster model in which we studied BNCT induced mucositis using three different cancerization protocols, mimicking three different clinical scenarios. The BNCT-induced mucositis increases with the aggressiveness of the carcinogenesis protocol employed, suggesting that the study of different oral cancer patient scenarios would help to develop personalized therapies.

Electroporation optimizes the uptake of boron-10 by tumor for boron neutron capture therapy (BNCT) mediated by GB-10: a boron biodistribution study in the hamster cheek pouch oral cancer model.

Boron neutron capture therapy (BNCT) is a promising cancer binary therapy modality that utilizes the nuclear capture reaction of thermal neutrons by boron-10 resulting in a localized release of high- and low-linear energy transfer (LET) radiation. Electrochemotherapy (ECT) is based on electroporation (EP) that induces opening of pores in cell membranes, allowing the entry of compounds. Because EP is applied locally to a tumor, the compound is incorporated preferentially by tumor cells. Based on the knowledge that the therapeutic success of BNCT depends centrally on the boron content in tumor and normal tissues and that EP has proven to be an excellent facilitator of tumor biodistribution of an anti-tumor agent, the aim of this study was to evaluate if EP can optimize the delivery of boronated compounds. We performed biodistribution studies and qualitative microdistribution analyses of boron employing the boron compound sodium decahydrodecaborate (GB-10) + EP in the hamster cheek pouch oral cancer model. Syrian hamsters with chemically induced exophytic squamous cell carcinomas were used. A typical EP treatment was applied to each tumor, varying the moment of application with respect to the administration of GB-10 (early or late). The results of this study showed a significant increase in the absolute and relative tumor boron concentration and optimization of the qualitative microdistribution of boron by the use of early EP + GB-10 versus GB-10 without EP. This strategy could be a tool to improve the therapeutic efficacy of BNCT/GB-10 in vivo.

MiR-31-5p Is a Potential Circulating Biomarker and Therapeutic Target for Oral Cancer

MicroRNAs have been proposed as novel biomarkers for the diagnosis and treatment of many types of cancer. The levels of five candidate microRNAs (miRNAs) (miR-99a-5p, miR-31-5p, miR-138-5p, miR-21-5p, and miR-375-3p) in sera from oral cancer patients and paired tumor and normal tissues were detected by real-time qPCR. The diagnostic power of these miRNAs was analyzed by receiver operating characteristic (ROC) curves. Patient-derived xenograft (PDX) models of oral cancer were established and utilized to verify the potential therapeutic effect of miR-31-5p. Candidate miRNAs were screened from our previous studies and verified in 11 paired oral cancer and adjacent normal tissues. Only serum miR-31-5p levels were significantly different between oral cancer patients and healthy controls and between pre- and postoperative patients. Based on the logistic regression model, this panel of five miRNAs distinguished oral cancer patients from healthy control, with an area under the ROC curve (AUC) of 0.776 (sensitivity = 76.8% and specificity = 73.6%). Furthermore, a miR-31-5p mimic enhanced the proliferation of normal epithelial cells, and antagomiR-31-5p inhibited the proliferation of oral cancer cells in vitro. In vivo, antagomiR-31-5p significantly inhibited tumor growth in oral cancer PDX models. Our findings suggest that circulating miR-31-5p might act as an independent biomarker for oral cancer diagnosis and could serve as a therapeutic target for oral cancer.

Characterization of Lymphoid Cells in the Lungs of Mice Following Oral Administration of 4NQO

IntroductionChronic inflammation is a risk factor in development and progression of lung cancer. The carcinogen 4‐nitroquinoline‐1‐oxide (4NQO) induces oral cancer in mice and mimics the effects of tobacco use. Here we investigated whether oral administration of 4NQO could cause injury and inflammatory changes in the lung. We also examined the possible effect of sex and a high or low in saturated fat diet on lung inflammation as manifested by the presence of lymphoid cells. The number and pattern of distribution of T lymphocytes, macrophages, and neutrophils were evaluated in lung tissues. We hypothesized that treatment with 4NQO would induce inflammation and cancer in lungs, and that sex and dietary fat content would affect 4NQO‐induced lung pathology.Materials and methodsMale and female C57Bl/6 mice (n=36 each gender, 5 weeks old), were divided into low fat (10kcal% fat; LF) and high fat (60 kcal% fat; HF) diet groups. After one week mice in the experimental groups began a treatment with 50μg/mL of 4NQO in 1.25% propylene glycol (PG) in water (4NQO group). Mice in control groups were provided with water or PG in water. After 17 weeks of treatment all mice were given water alone for 6 weeks prior to euthanasia. Lung tissues were harvested, fixed in formalin, and processed for histological evaluation. Lung tissue sections were stained with antibodies against CD3 to detect T lymphocytes, CD68 to detect macrophages, and Ly6‐g to detect neutrophils. Positively‐stained cells were counted in 4 randomly chosen areas at 10× magnification using ImageJ software. Analysis was done using GraphPad Prism 7.0. All animal work was in compliance with the regulations of IACUC at Midwestern University, Downers Grove, IL.ResultsLymphoid cells were present in the control lung tissue, however, 4NQO treatment resulted in a significant increase in all three cell populations (T lymphocytes 194 vs. 283, macrophages 136 vs. 195, and neutrophils 102 vs. 266 cells/per mm2, p<0.002). Neutrophils and macrophages were primarily located in lung parenchyma, while T cells were located both in parenchyma and lymphoid nodules. In 4NQO mice the number of neutrophils and macrophages, but not T cells, was significantly higher (p<0.001) in males than in females (354 vs. 179, 250 vs. 138, and 177 vs. 288 cells/per mm2, respectively). HF diet resulted in significantly more neutrophils in the 4NQO male lungs (324 vs. 100 cells/mm2, p<0.001), but had no effect on the number of macrophages and T cells.ConclusionOral administration of 4NQO induced lung inflammation as manifested by a significant increase in the presence of lymphoid cells. The inflammatory responses in the lungs may be affected by dietary fat and sex. This mouse model of oral cancer that was developed to mimic the effects of smoking may be extended to serve as a model to study the effects of tobacco and diet on the lungs.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of a Low Fat Diet versus a High Fat Diet on Lymphoid Cell Infiltration in a Mouse Model of Oral Cancer

IntroductionRisk factors for oral cancer include tobacco use, male sex, and possibly dietary fat. Exposure to 4‐nitroquinoline 1‐oxide (4NQO) in the drinking water causes oral cancer in mice and models the effects of tobacco use. This study was undertaken to investigate the effects of a high saturated fat (HF) or low fat (LF) diet and sex on presence of lymphoid cells in cancerous tongues. We hypothesized that inflammation, characterized by the increased presence of lymphoid cells, would accompany development of tumor lesions and that sex and diet would affect the levels of inflammation.MethodsMale and female C57Bl/6 mice (36 each gender), 5 weeks old, were divided into a low fat (10kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H2O); or 4NQO in PG‐H2O (50 mg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 more weeks. Tongues harvested from the euthanized animals were fixed in formalin and processed for histological examination. T cells (CD3+ cells), macrophages (CD68+ cells), and neutrophils (Ly6+ cells) were detected by immunohistochemistry, counted, and normalized to tissue area. Analysis was done using GraphPad Prism 7.0. All animal work was in compliance with the regulations of IACUC at Midwestern University, Downers Grove, IL.ResultsOral cancer developed in all mice exposed to 4NQO and was absent in all controls. All three lymphoid cell types were significantly higher (p<0.05) in the 4NQO mice versus the control mice: CD3+ (11.2 vs 0.9 cells/mm2), CD68+ (3.6 vs 1.5 cells/mm2), and Ly6+ (9.4 vs 0.38 cells/mm2). Among the 4NQO treated mice, there were no significant differences between males and females, or between HF and LF mice for T cells or macrophages. 4NQO treated males had a significantly higher numbers of neutrophils than females (12 vs 6.6 cells/mm2). LF diet increased the levels of neutrophils in both males and females (15 and 7.7 cells/mm2 respectively) when compared to the HF diet in both males and females (9.3 and 5.3 cells/mm2 respectively); however, this trend was not statistically significant. We speculate that the higher carbohydrate content of the LF diet may support increased anaerobic glycolytic metabolism via the Warburg effect.ConclusionDevelopment of 4NQO‐induced oral cancer was accompanied by inflammatory responses as manifested by the presence of T cells, macrophages, and neutrophils. Neutrophils were the only cell type affected by sex and possibly by the content of dietary fat.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

No difference in 4-nitroquinoline induced tumorigenesis between germ-free and colonized mice.

Variations in oral bacterial communities have been linked to oral cancer suggesting that the oral microbiome is an etiological factor that can influence oral cancer development. The 4-nitroquinoline 1-oxide (4-NQO)-induced murine oral and esophageal cancer model is frequently used to assess the effects of preventive and/or therapeutic agents. We used this model to assess the impact of the microbiome on tumorigenesis using axenic (germ-free) and conventionally housed mice. Increased toxicity was observed in germ-free mice, however, no difference in tumor incidence, multiplicity, and size was observed. Transcriptional profiling of liver tissue from germ-free and conventionally housed mice identified 254 differentially expressed genes including ten cytochrome p450 enzymes, the largest family of phase-1 drug metabolizing enzymes in the liver. Gene ontology revealed that differentially expressed genes were enriched for liver steatosis, inflammation, and oxidative stress in livers of germ-free mice. Our observations emphasize the importance of the microbiome in mediating chemical toxicity at least in part by altering host gene expression. Studies on the role of the microbiome in chemical-induced cancer using germ-free animal models should consider the potential difference in dose due to the microbiome-mediated changes in host metabolizing capacity, which might influence the ability to draw conclusions especially for tumor induction models that are dose dependent.

Immunotherapeutic Potential of Mollusk Hemocyanins in Combination with Human Vaccine Adjuvants in Murine Models of Oral Cancer.

Mollusk hemocyanins have been used for decades in immunological and clinical applications as natural, nontoxic, nonpathogenic, and nonspecific immunostimulants for the treatment of superficial bladder cancer, as carriers/adjuvants of tumor-associated antigens in cancer vaccine development and as adjuvants to dendritic cell-based immunotherapy, because these glycoproteins induce a bias towards Th1 immunity. Here, we analyzed the preclinical therapeutic potential of the traditional keyhole limpet hemocyanin (KLH) and two new hemocyanins from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH) in mouse models of oral squamous cell carcinoma. Due to the aggressiveness and deadly malignant potential of this cancer, the hemocyanins were applied in combination with adjuvants, such as alum, AddaVax, and QS-21, which have been shown to be safe and effective in human vaccines, to potentiate their antitumor activity. The immunogenic performance of the hemocyanins in combination with the adjuvants was compared, and the best formulation was evaluated for its antitumor effects in two murine models of oral cancer: MOC7 cells implanted in the flank (heterotopic) and bioluminescent AT-84 E7 Luc cells implanted in the floor of the mouth (orthotopic). The results demonstrated that the hemocyanins in combination with QS-21 showed the greatest immunogenicity, as reflected by a robust, specific humoral response predominantly characterized by IgG2a antibodies and a sustained cellular response manifesting as a delayed hypersensitivity reaction. The KLH- and FLH-QS-21 formulations showed reduced tumor development and greater overall survival. Hemocyanins, as opposed to QS-21, had no cytotoxic effect on either oral cancer cell line cultured in vitro, supporting the idea that the antitumor effects of hemocyanins are associated with their modulation of the immune response. Therefore, hemocyanin utilization would allow a lower QS-21 dosage to achieve therapeutic results. Overall, our study opens a new door to further investigation of the use of hemocyanins plus adjuvants for the development of immunotherapies against oral carcinoma.

Mucosal HPV E6/E7 Peptide Vaccination in Combination with Immune Checkpoint Modulation Induces Regression of HPV+ Oral Cancers.

High-risk human papillomavirus (HPV)-associated squamous cell carcinomas of the oropharynx (SCCOP) are among the fastest growing cancers. After standard-of-care treatment, however, patients with HPV+ SCCOP have better overall and disease-specific survival than patients with HPV- SCCOP, suggesting the importance of HPV-specific immunity. We reasoned that therapeutic vaccination targeting the HPV-16 E6 and E7 oncogenes could elicit high-affinity, high-frequency tumor antigen-specific T-cell responses, which could then be augmented and shielded from suppression in the tumor microenvironment by immune checkpoint modulation. In this study, we used a preclinical syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER) to identify combinations of vaccination and checkpoint antibodies capable of promoting tumor regression. Intranasal HPV E6/E7 peptide vaccination and single checkpoint antibodies failed to elicit responses in more than half of animals; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors. The combination of intranasal HPV peptide vaccine and α4-1BB and αCTLA-4 antibodies produced curative efficacy and a better safety profile against orally implanted mEER tumors. Correlates of protective immunity included enhanced intratumoral levels of CD8 T cells relative to immunosuppressive regulatory T cells and myeloid-derived suppressor cells. Overall, our results demonstrate combination vaccine-immunotherapy modalities as novel treatment options for HPV+ SCCOP.Significance: Combinations of vaccine and checkpoint modulation are effective and safe treatment options for HPV+ oral cancers. Cancer Res; 78(18); 5327-39. ©2018 AACR.

Trojan Horse nanotheranostics with dual transformability and multifunctionality for highly effective cancer treatment

Nanotheranostics with integrated diagnostic and therapeutic functions show exciting potentials towards precision nanomedicine. However, targeted delivery of nanotheranostics is hindered by several biological barriers. Here, we report the development of a dual size/charge- transformable, Trojan-Horse nanoparticle (pPhD NP) for delivery of ultra-small, full active pharmaceutical ingredients (API) nanotheranostics with integrated dual-modal imaging and trimodal therapeutic functions. pPhD NPs exhibit ideal size and charge for drug transportation. In tumour microenvironment, pPhD NPs responsively transform to full API nanotheranostics with ultra-small size and higher surface charge, which dramatically facilitate the tumour penetration and cell internalisation. pPhD NPs enable visualisation of biodistribution by near-infrared fluorescence imaging, tumour accumulation and therapeutic effect by magnetic resonance imaging. Moreover, the synergistic photothermal-, photodynamic- and chemo-therapies achieve a 100% complete cure rate on both subcutaneous and orthotopic oral cancer models. This nanoplatform with powerful delivery efficiency and versatile theranostic functions shows enormous potentials to improve cancer treatment.

Abstract 1032: Novel mouse models of high-risk HPV-related oral cancers

Abstract The rate of HPV-induced head and neck squamous cell carcinoma (HNSCC) is steadily increasing and implicated in approximately 60% of all oropharyngeal carcinomas. The advent of whole genome, transcriptome, and proteome analyses have aided in identifying altered signaling pathways in HPV-induced HNSCCs, however, additional tools such as mouse models are needed to study the role of HPV oncogenes in oral tumor initiation and progression. Current inducible models of HPV-driven oral cancer do not accurately recapitulate the levels, stoichiometric ratios, or anatomic location of oncoprotein expression. To address these limitations, we developed a tractable genetically engineered mouse model (GEMM) that enables directed expression of high-risk HPV16 E6 and E7 oncogenes (Rosa26-loxP-STOP-loxP-E7iresE6; “H”) in the oral epithelium. Analysis of mouse embryonic fibroblasts treated with Cre recombinase confirmed E6 and E7 expression and changes in mRNA expression of known E6 and E7 targets. We crossed our H mice to several tissue-specific transgenic Cre-driver mouse strains including the EBV lytic promoter (ED-L2-Cre recombinase; “L”) to drive robust expression in the oropharyngeal squamous epithelia. Analysis of epithelial tissues isolated from LH mice displayed increased oral volumes and cutaneous epithelial thickening associated with hyperplasia, dysplasia, accumulation of neutrophils and recruitment of cytotoxic and regulatory T cells. A second cross to the keratin 14 promoter (KRT14-Cre; “K”) line enabled targeted expression to the basal epithelial layer (KH) and confirmed increased oral volumes and cutaneous epithelial thickening associated with increased suprabasal proliferation and expression of canonical E7 targets. Lastly, a third cross to a tamoxifen-inducible Cre (CreERT2) line driven by the keratin 14 promoter (KRT14-CreERT2; “iK”) enabled conditional and inducible post-natal E6 and E7 expression in basal epithelia (iKH). To prevent systemic oncogene expression in K14+ cells, we validated a method for submucosal delivery of tamoxifen to the tongue. We confirmed that this approach prevents tamoxifen spread and anatomically restricts activation oncogene expression to intra-lingual regions using an optical reporter mouse recently generated by the Amelio lab (Rosa26-loxP-STOP-loxP-LumiFluor; “F”) crossed to the iK line (“iKF”). Notably, direct intra-lingual injection yields higher recombination and reporter activation using 40% less overall tamoxifen than traditional i.p. administration routes. Moreover, immunofluorescent staining revealed mosaic transgene activation in the oropharynx, an important feature for modeling HPV-induced OSCC. In fact, intra-lingual injection of tamoxifen in iKH mice coupled with 4-nitroquniloline 1-oxide (4NQO) administration led to E6 and E7 expression and dysplasia in the tongue epithelia. Ongoing studies are evaluating cooperating mutations in driving HNSCC development. Citation Format: Miranda B. Carper, Scott Troutman, Kevin M. Byrd, Bethany Wagner, Erin C. Henry, Stephanie A. Montgomery, Scott E. Williams, Joseph L. Kissil, Antonio L. Amelio. Novel mouse models of high-risk HPV-related oral cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1032.

Abstract 1057: Characterization and functional consequences of neuronal invasion in oral cancer

Abstract Most oral cancer patients suffer severe, chronic pain promoted by oral cancer-induced changes in sensory nerves innervating the oral cavity. However, the impact of sensory neurons on carcinogenesis beyond nociceptive signaling is an understudied problem in oral cancer. We hypothesize that oral cancer and sensory neurons interact, such that, oral cancer induces sensitization, sprouting and plasticity in neurons, and efferent neuronal activity promotes oral carcinogenesis. Using an oral cancer subcutaneous xenograft model, we tested the impact of peripheral nerve presence on squamous cell carcinoma tumor growth in the mouse hindpaw. Mice that received a sciatic nerve axotomy one week prior to HSC-3 oral cancer cell inoculation developed significantly smaller tumors (28.23 ± 6.6 % of total paw area) compared to mice that received sham surgery (50.71 ± 2.48 %). These data suggest that peripheral neuron innervation supports oral cancer growth. Additionally, neuropeptides are released from primary afferent neurons and suppression of the immune response has been linked to peripheral neurotransmission. Using flow cytometry, we tested whether inhibition of peripheral sensory neurons impacted inflammation in response to oral cancer. Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to injection of supernatant from HSC-3 oral cancer cells resulted in significantly more CD45+ immune cell infiltration (21.66 ± 3.4 %) into the tongue compared to HSC-3 supernatant with intact nerve function (9.10 ± 1.2 %). These results suggest neuron-mediated suppression of cancer-evoked inflammation and support the premise that neurons and neuronal activity modulates the oral cancer microenvironment. High-resolution imaging of neuronal innervation into tongue cancer and its remodeling in response to pathological changes is challenging due to dense neuronal innervation in the tongue. We utilized a recently developed optical clearing protocol (CLARITY) that preserves protein integrity and three-dimensional structure of tissue and high-resolution microscopy to visualize and characterize sensory neuron innervation in thick 1 mm tongue sections from an orthotopic xenograft mouse model of oral cancer. In a preliminary study of neuronal innervation in a tongue HSC-3 xenograft model, we observed that PGP9.5+ neuronal fiber density was greater in the cancer environment and diminished with distance from the cancer. These data suggest that oral cancer induces plasticity and sprouting of innervating peripheral neurons. A better understanding of the nerve-cancer interaction has potential for development of new therapeutic strategies to treat oral cancer and oral cancer pain by targeting sensory neurons. Citation Format: Nicole N. Scheff, Richard Klares, Brian L. Schmidt. Characterization and functional consequences of neuronal invasion in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1057.

Abstract 3064: Oral cancer stem cells modulate Fusobacterium nucleatum to acquire the capability to induce tumor stemness switch

Abstract Background: Fusobacterium nucleatum is an oral bacteria that contributes to oral carcinogenesis. Its potential role in oral cancer is not known. Fusobacterium nucleatum (henceforth FN) is a gram-negative anaerobe associated with periodontal diseases and also colon cancer. The bacteria modulates the host cell signaling mechanism including the activation of beta-catenin signaling and p38 pathway. The Fap2 is the outer membrane proteins of FN that inhibit lymphocytes and NK cells. The bacteria also directly binds to NKp46 receptor of NK cells. These properties of FN may be exploited by oral CSCs to defend their own niche as a part of niche-defense mechanism against immune system. Hence, it is not surprising that oral CSCs may hijack this specific oral bacterium to enhance tumor stemness. We speculate that oral CSCs may hijack specific bacteria type present in oral mucosa, and enhance their stemness-inducing mechanisms to switch non-CSCs to CSCs. Methods: We have established an in vitro model of oral cancer cells/FN host/pathogen interaction model. Using this model, we wanted to study potential role of the bacteria in cancer stemness. We also used the saliva of subjects with oral cancer to infect oral cancer stem cells and non-stem cancer cells. Results: When the SCC-25 oral cancer cell line was treated with the saliva of subjects with oral cancer, the EpCAM+/ABCG2+ oral CSCs population was increased and showed the presence of FN. The FN recovered from CSCs exhibited high expression of FadA and Fap2, two surface proteins present in FN that can modulate signaling pathways of host cells. Importantly, FN recovered from the infected CSCs induced stemness in the non-stem cancer cell population of SCC-25, suggesting that CSCs may modulate the pathogen. Indeed, our preliminary observation indicates that FN recovered from the FN strain ATCC 25586 infected SCC-25 ABCG2+ cells showed increased expression of FADA and Fap2 as compared to the parental strain ATCC 25586. Conclusion: Our data suggests that CSCs have the capability to reprogram F. nucleatum genome in order to make the bacteria a potent inducer of tumor stemness in non-stem cancer cells. Citation Format: Bidisha Pal, Seema Bhuyan, Debabrat Baishya, Bikul Das. Oral cancer stem cells modulate Fusobacterium nucleatum to acquire the capability to induce tumor stemness switch [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3064.

Abstract 1268: Anti-PD-1 monotherapy outperforms multiple immunotherapy combination strategies in an oral cancer prevention mouse model

Abstract Oral premalignant lesion (OPL) expression of PD-L1 is associated with increased cancer risk. These data suggest that immune evasion is already present at the OPL stage and warrant an evaluation of immune modulatory therapies for cancer prevention. We conducted this preclinical study in a carcinogen-induced mouse model of oral cancer to evaluate the efficacy of multiple immunomodulatory strategies, given at a time point when only OPLs are present, to reduce the incidence of oral squamous cell carcinoma (OSCC). We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide (4-NQO) for 8 weeks in drinking water. Eight weeks after discontinuing 4-NQO treatment, a group of mice (N=6) was sacrificed for assessment of invasive and non-invasive tongue lesions. A second group of mice was treated with either anti-PD-1 (N=40), anti-CTLA-4 (N=20), anti-OX40 (N=20), anti-PD-1 + anti-CTLA-4 (N=20), anti-PD-1 + anti-OX40 (N=20) antibodies, or IgG (N=40) for a total of 3 doses every 3 days, initiating 8 weeks after the cessation of 4-NQO. Antibodies used in combination were given on the same day . Mice were sacrificed 56 days after the last dose of treatment. We assessed serial H&E-stained cross sections of the tongues harvested at that same time point for development of OPLs and cancer and measured the OSCC area of each tongue using aperio imagescope software. Mann-Whitney and Fisher's exact tests were used for statistical comparisons between groups. Eight weeks after 4NQO cessation, 100% of mice developed tongue hyperplasia or dysplasia and invasive lesions were not identified, indicating this to be an ideal time point to initiate treatment strategies addressing OPLs. Tongues from mice treated with anti-PD-1 antibody displayed a decrease in the mean OSCC area when compared with mice treated with IgG (mean 3.53 mm2 versus 6.62 mm2, respectively; p= 0.018). At this time point, invasive oral cancers had developed in 75% versus 50% of IgG and anti-PD-1 treated mice, respectively (p=0.03). There were also non-significant decreases in the mean OSCC area in tongues from mice treated with anti-CTLA-4 (mean = 5.07 mm2), anti-OX40 (mean = 3.79 mm2) and anti-PD-1 + anti-CTLA-4 (mean = 3.86 mm2) antibodies when compared to mice treated with IgG (mean = 6.62 mm2) control. In the group treated with anti-PD-1 + anti-OX40 combination therapy, there was an increase in the mean OSCC area when compared to anti-PD-1 monotherapy (mean 3.53 mm2 versus 9.03 mm2), respectively (p= 0.01). Short-term anti-PD-1 immune checkpoint inhibitor therapy in the context of OPLs led to a reduction in the incidence of oral cancer. When comparing the mean area of OSCC in each group, anti-PD-1 monotherapy was superior to IgG and all other treatment strategies. Paradoxically, combining anti-OX40 and anti-PD-1 antibodies created an antagonizing effect on the therapy and increased total tumor burden when compared with anti-PD-1 monotherapy. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Marlese A. Pisegna, Alissa Poteete, Fahao Zhang, John V. Heymach, William N. William. Anti-PD-1 monotherapy outperforms multiple immunotherapy combination strategies in an oral cancer prevention mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1268.

Abstract 1270: Potential metabolic and molecular mechanisms of black raspberry-mediated oral cancer chemoprevention

Abstract Oral cancer accounts for about 50,000 new cases and 10,000 new deaths in the U.S. every year. This amounts to about one person every hour every day. To combat this public health challenge, there is a need to identify agents that prevent oral cancer development and fully characterize their mechanisms of action. Preclinical and clinical studies demonstrate the ability of black raspberries (BRBs) to inhibit oral carcinogenesis. We recently showed that in an experimental model of rat oral carcinogenesis using the carcinogen 4 nitroquinoline-1-oxide (4NQO), BRBs reduce oral lesion incidence and multiplicity. However, understanding how the bioactive compounds in BRBs drive the metabolic and molecular pathways that lead to oral cancer chemoprevention remains unclear. In this study, we determined the potential metabolic and molecular mechanisms associated with BRB-mediated chemoprevention of oral carcinogenesis using the well-established carcinogen-induced rat oral cancer model. Male F344 rats were divided into 4 groups: sentinel group, 4NQO only group, 4NQO + 5% BRB group, and 4NQO + 10% BRB group. 4NQO was administered in drinking water (20ug/mL) for 10 weeks after which regular drinking water was provided for 6 weeks. BRBs were incorporated into rat diets at 5% or 10% concentrations and fed to the rat treatment groups for the last 6 weeks after 4NQO administration. Sentinel animals did not receive 4NQO or BRB. At terminal sacrifice, urine samples from all groups were harvested and analyzed by NMR-based metabolomics. We identified metabolites in rat urine samples by "Complex Mixture Analysis by NMR" (COLMAR) followed by 2D 13C-1H HSQC NMR quantitative analysis. RNA was extracted from rat tongue samples and used for RNA sequencing analysis. 4NQO only administered rats showed highest overall intensity, which was about 2.5 times higher than 4NQO + BRB treated rats. 171 metabolites were identified in urine samples of 4NQO only administered rats while 101 and 90 metabolites were identified in 4NQO administered rats that were treated with 10% and 5% BRB, respectively. About 31 "unique" metabolites were identified in 4NQO only administered rat urine samples but were absent in BRB treated groups. Fourteen additional metabolites were increased and 11 metabolites were decreased following BRB treatment of 4NQO administered rats compared to rats administered with 4NQO only. Our metabolomic and transcriptomic analyses reveal distinct molecular and metabolic markers and pathways that drive oral carcinogenesis and BRB-mediated oral chemoprevention, which can potentially be exploited in oral cancer treatment strategies. Citation Format: Steve Oghumu, Thomas J. Knobloch, Logan C. Weghorst, Lei Bruschweiler-Li, Cheng Wang, Rafael Bruschweiler, Christopher M. Weghorst. Potential metabolic and molecular mechanisms of black raspberry-mediated oral cancer chemoprevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1270.