Oral Cancer Mouse Research Articles

Periodontitis promotes tumor growth and immune evasion via PD-1/PD-L1.

Our study investigated the role of experimental periodontitis on tumor growth, local and systemic immunosuppressive status, and programmed death receptor 1 (PD-1) / programmed death ligand 1 (PD-L1) expression in oral squamous cell carcinoma (OSCC) and prostate cancer. Mouse oral or prostate cancer xenograft models were divided into control, periodontitis and periodontitis + anti-PD-1 groups. Tumor volume and weight were recorded and the levels of relevant immune-suppressive cells and T cells were detected by flow cytometry or immunofluorescence. THP-1 cells were stimulated using conditioned media of LPS-stimulated Cal-27 cells and PD-L1 expression was measured by quantitative real-time PCR, western blotting and immunofluorescence. Tumor specimens from OSCC patients with or without periodontitis were also collected for immunofluorescence. Periodontitis significantly promoted tumor volume and weight. Compared to the control, the proportions of tumor-associated macrophages (TAMs), regulatory T cells (Tregs), PD-L1+TAMs and PD-1+CD8+T cells increased, while CD8+T cells decreased in the periodontitis group. Immunofluorescence demonstrated that there was an increase in PD-L1+TAMs and PD-1+CD8+T cells, but a decrease in IFN-γ+CD8+T cells in both xenografts and clinical OSCC samples with periodontitis. In vitro, LPS-stimulated Cal-27 cells had a stronger potential to induce PD-L1 expression in macrophages compared with unstimulated Cal-27 cells. And the promoting effect of periodontitis on tumor growth and immune evasion was significantly attenuated after anti-PD-1 therapy. Periodontitis may facilitate tumor growth and immune escape evidenced by the increased immune-suppressive cells and the decreased functional T cells, via enhancing PD-1/PD-L1 expression in the tumor microenvironment.

Roles of ZEB1 and ZEB2 in E-cadherin expression and cell aggressiveness in head and neck cancer.

Zinc finger E-box binding homeobox 1 (ZEB1) has been identified as a key factor in cancer cell differentiation and metastasis, and has been well studied in the field of cancer cell biology. ZEB2 has a highly similar conformation to ZEB1, but its role in head and neck squamous cell carcinoma (HNSCC) cells is not fully understood. Here, we separately overexpressed ZEB1 and ZEB2 in C57BL/6 mouse oral cancer (MOC) cells and investigated their cellular characteristics, including E-cadherin levels, motile properties, chemoresistance, and metastatic ability in immunocompetent mice. Both ZEB1 and ZEB2 overexpression reduced epithelial traits and converted cells to an aggressive phenotype. Surprisingly, ZEB1 overexpression increased the endogenous level of ZEB2 in MOC cells, and vice versa. The molecular mechanisms underlying these findings remain unclear. However, the in vitro anchorage-independent growth of MOC cells overexpressing ZEB2 was considerably greater than that of MOC cells overexpressing ZEB1. These findings suggest that ZEB2, like ZEB1, has the ability to induce the differentiation of cancer cells into those with highly aggressive traits.

Targeting CD36-Mediated Lipid Metabolism by Selective Inhibitor-Augmented Antitumor Immune Responses in Oral Cancer.

The fatty acid receptor CD36 is expressed on various malignant cells and is suggested to contribute to tumor progression. CD36 is also expressed by several immune cells and involved in immune responses and may be a potential target in cancer immunotherapy. In this study, we investigated whether the selective inhibition of CD36 can inhibit tumor progression and facilitate an antitumor immune response in oral squamous carcinoma cells (OSCCs). We assessed the effects of sulfosuccinimidyl oleate sodium (SSO), a CD36 inhibitor, on the proliferation apoptosis and alteration in tumor cell surface expression levels of immune accessory molecules in vitro. We also assessed whether SSO-treated OSCCs could promote a T cell response via a Mixed Lymphocyte Reaction (MLR) assay. We also investigated the direct antitumor effects and immunomodulatory effects of SSO using a mouse oral cancer OSCC model. SSO treatment significantly inhibited OSCC proliferation, increased apoptotic cell death, and upregulated the cell surface expression of several immune accessory molecules, including CD83, MHC-Class II, and PD-L1. SSO-treated OSCCs augmented T cell proliferation following MLR. In vivo SSO administration significantly attenuated mouse tumor growth with an increased proportion of immune cells, including CD4+ T, CD8+ T, and dendritic cells; it also decreased the proportion of immune suppressive cells, such as myeloid-derived suppressor and regulatory T cells. These results suggest that the selective inhibition of CD36 can induce direct and indirect antitumor effects by facilitating host antitumor immune responses in OSCCs.

MHC-I and PD-L1 Expression is Associated with Decreased Tumor Outgrowth and is Radiotherapy-inducible in the Murine Head and Neck Squamous Cell Carcinoma Model MOC1

IntroductionCombined radiotherapy and immune checkpoint inhibition is a potential treatment option for head and neck squamous cell carcinoma (HNSCC). Immunocompetent mouse models can help to successfully develop radio- immunotherapy combinations and to increase our understanding of the effects of radiotherapy on the tumor microenvironment for future clinical translation. Therefore, the aim of this study was to develop a homogeneous, reproducible HNSCC model originating from the Mouse Oral Cancer 1 (MOC1) HNSCC cell line, and to explore the radiotherapy-induced changes in its tumor microenvironment, using flow cytometry and PD-L1 microSPECT/CT imaging.Materials and MethodsIn vivo growing tumors originating from the parental MOC1 line were used to generate single cell derived clones. These clones were screened in vitro for their ability to induce programmed cell death ligand 1 (PD-L1) and major histocompatibility complex class I (MHC-I) following IFNγ exposure. Clones with different IFNγ sensitivity were inoculated in C57BL/6 mice and assessed for tumor outgrowth. The composition of the tumor microenvironment of a stably growing (non)irradiated MOC1-derived clone was assessed by immunohistochemistry, flow cytometry and PD-L1 microSPECT/CT.ResultsLow in vitro inducibility of MHC-I and PD-L1 by IFNγ was associated with increased tumor outgrowth of MOC1 clones in vivo. Flow cytometry analysis of cells derived from a stable in vivo growing MOC1 clone MOC1.3D5low showed expression of MHC-I and PD-L1 on several cell populations within the tumor. Upon irradiation, MHC-I and PD-L1 increased on leukocytes (CD45.2+) and cancer associated fibroblasts (CD45.2−/EpCAM−/CD90.1+). Furthermore, PD-L1 microSPECT/CT showed increased tumor uptake of radiolabeled PD-L1 antibodies with a heterogeneous spatial distribution of the radio signal, which co-localized with PD-L1+ and CD45.2+ areas.DiscussionPD-L1 and MHC-I inducibility by IFNγ in vitro is associated with tumor outgrowth of MOC1 clones in vivo. In tumors originating from a stably growing MOC1-derived clone, expression of these immune-related markers was induced by irradiation shown by flow cytometry on several cell populations within the tumor microenvironment such as immune cells and cancer associated fibroblasts. PD-L1 microSPECT/CT showed increased tumor uptake following radiotherapy, and autoradiography showed correlation of uptake with areas that are heavily infiltrated by immune cells. Knowledge of radiotherapy-induced effects on the tumor microenvironment in this model can help optimize timing and dosage for radio- immunotherapy combination strategies in future research.

Characterization of the tumor microenvironment in the mouse oral cancer (MOC1) model after orthotopic implantation in the buccal mucosa.

Preclinical models are invaluable for studies of head and neck cancer. There is growing interest in the use of orthotopic syngeneic models, wherein cell lines are injected into the oral cavity of immunocompetent mice. In this brief report, we describe injection of mouse oral cancer 1 (MOC1) cells into the buccal mucosa and illustrate the tumor growth pattern, lymph node response, and changes in the tumor immune microenvironment over time. MOC1 cells were injected into the buccal mucosa of C57BL6 mice. Animals were sacrificed at 7, 14, 21, or 27 days. Tumors and lymph nodes were analyzed by flow cytometry. All mice developed tumors by day 7 and required euthanasia for tumor burden and/or weight loss by day 27. Lymph node mapping showed that these tumors reliably drain to a submandibular lymph node. The proportion of intratumoral CD8+ T cells decreased over time, while neutrophilic myeloid cells increased dramatically. Growth of orthotopic MOC2 and MOC22 also showed similar growth patterns versus published data in flank tumors. When used orthotopically in the buccal mucosa, the MOC1 model induces a robust lymph node response and distinct pattern of immune cell infiltration, with peak immune infiltration by day 14.

Inhibition of transforming growth factor-β signals suppresses tumor formation by regulation of tumor microenvironment networks.

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-β (TGF-β) promotes tumor progression by inducing epithelial-mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-β receptor containing both TGF-β type I (TβRI) and type II (TβRII) receptors (TβRI-TβRII-Fc), which trapped all TGF-β isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TβRI-TβRII-Fc protein effectively suppressed invitro EMT of oral cancer cells and invivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TβRI-TβRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TβRI-TβRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1β (IL-1β) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1β and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-β signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1β/EREG pathways and that TβRI-TβRII-Fc protein is a promising tool for targeting the TME networks.

A new in vitro study demonstrates that electronic cigarettes promote cellular carcinogenic characteristics.

Electronic cigarettes have been used as a new form of cigarettes, especially among young people, but the impact of these effects on mouth cancer is unknown. Therefore, there is a need for studies to evaluate their impact on health and oral mucosa. In this way, this study evaluates the risk of electronic cigarette liquid on in vitro cells of a panel: normal oral epithelium cell lines (NOE and HMK), oral squamous cell carcinoma human cell lines (CAL27 and HSC3), and a mouse oral cancer cell line (AT84). It was demonstrated that electronic cigarettes promote proliferation and anchorage-independent growth and induces morphological changes associated with enhanced motility and invasive phenotypes. Also, it was observed that the enhanced invasive migratory activity associated with the loss of epithelial markers, such as E-cadherin, and the acquisition of mesenchymal markers, strongly suggest that epithelial cells are undergoing to an aggressive phenotype within the framework of epithelial-mesenchymal transition. In conclusion, the liquid can promote carcinogenesis, in addition to promoting an aggressive phenotype in pre-existing lesions.

Abstract PO-089: Opioid-mediated suppression of anti-tumor immunity via peripheral OPRM1 signaling in head and neck squamous cell carcinoma

Abstract Head and Neck squamous cell carcinoma (HNSCC) causes severe pain, beyond what is reported in other cancer types. Opioids are the current backbone of HNSCC pain treatment but may have an immunomodulatory effect in the presence of cancer; T cells express opioid receptors. We recently demonstrated that opioids may affect the efficacy of anti-PD1 monoclonal antibody (mAb) treatment in recurrent/metastastic HNSCC patients; opioid usage was associated with significantly lower progression free survival and overall survival as well as decreased CD8 T cells in the tumor microenvironment. We hypothesized that cancer immunosurveillance is impaired by the immunosuppressive actions of exogenous opioids via peripheral mu-opioid receptor (OPRM1) signaling. A syngeneic mouse oral cancer model was used to determine if analgesic morphine impacted the tumor-associated immune response during tumor progression and in response to anti-PD1 mAb; mouse oral cancer cell line 1 (MOC1) tumor-bearing mice were treated with vehicle or morphine (10mg/kg i.p., 2x daily for 4.5 days) and the tumor-associated immune infiltrate was assessed between groups using flow cytometry. Anti-PD1 mAb (250µg/mouse, 3 × 2 day interval) was initiated directly following morphine treatment. Co-administration of methylnaltrexone (MNTX), a peripheral OPRM1 antagonist used to confirm specificity and site of action of the morphine-induced effects. Finally, any direct effects of morphine on tumor cells were investigated in oral cancer cell lines using quantitative PCR and colorimetric proliferation assays. Morphine induced a 75±5% reduction in CD8+ T cells and a 90±3% reduction in CD19+ B cells in MOC1 compared to sham mice (n=6/group); morphine-induced immunosuppression was completely blocked with co-treatment of MNTX. Studies involving morphine administration prior to anti-PD1 treatment are currently underway but given the substantial suppression in infiltrating lymphocyte populations, efficacy is expected to be lessened. Lastly, Oprm1 expression was not detected at the mRNA level in mouse oral cancer cell lines. In vitro, 10µM morphine did not induce cell proliferation (proliferation relative to vehicle after 48hr; vehicle=100±8.3%, morphine=115.7±9.6%) or improve wound healing (wound closure after 24hr; vehicle=76.2±11.6%, morphine=78.5±11.4%). Taken together, our data suggest that morphine suppresses anti-tumor immunity via peripheral OPRM1 signaling in immune cells but not tumor cells. Further investigation is warranted to determine whether peripheral opioid blockade (i.e. MNTX) can slow tumor progression and improve response to cancer treatments while preserving centrally-mediated analgesia. Citation Format: Lisa A. McIlvried, Mona Yuan, Nicole L. Horan, Megan A. Atherton, Marci L. Nilsen, Dan P. Zandberg, Nicole N. Scheff. Opioid-mediated suppression of anti-tumor immunity via peripheral OPRM1 signaling in head and neck squamous cell carcinoma [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-089.

Abstract 6422: Combined TGFβ/PD-L1 blockade enhances systemic antitumor immunity in head and neck cancer

Abstract Head and neck squamous cell carcinoma (HNSCC) unrelated to human papillomavirus (HPV) confers poor prognosis, with half of patients experiencing disease relapse in advanced stages. Response rates to traditional immunotherapies are low and novel approaches such as the bifunctional fusion protein Bintrafusp alfa (BA) that concurrently blocks PD-L1 signaling and neutralizes TGFβ are actively under investigation. Our group showed that when HNSCC patients received neoadjuvant BA therapy, it reversed exhaustion of tumor-infiltrating lymphocytes (TILs) by promoting antigen-mediated signaling. Additionally, we observed that BA promoted recirculation of TILs into peripheral blood possibly through decreased expression of the TGF-β-driven tissue-retention marker CD103. We hypothesize that addition of TGFβ neutralization to PD-L1 blockade can enhance systemic anti-tumor immunity compared to PD-L1 blockade alone. We assessed immune responses to BA in wild-type mice bearing established mouse oral cancer (MOC)-1 tumors that were randomized into the following groups: 1) vehicle; 2) anti-PD-L1 antibody (Avelumab); 3) TGFβ neutralizing construct with a mutated PD-L1 antibody (BA-M); 4) TGFβ neutralizing construct with a functional PD-L1 antibody (BA). Additionally, since our previous data suggests that BA responsiveness depends on CD8 T cell activity, we included two extra groups 5) CD8 depleting antibody alone or 6) in combination with BA. Three days after completing treatment, MOC-1 tumor cells were injected into the contralateral flank and engraftment and progression of secondary challenge tumors was analyzed. Primary tumor progression was significantly inhibited by BA and to a lesser extent avelumab compared to control or BA-M. Engraftment or progression of secondary challenge tumors was significantly inhibited by BA and to a lesser extent avelumab compared to control or BA-M. The growth inhibitory effects of BA were abrogated by CD8 depletion in both the primary and challenge tumors. Flow cytometry analysis of tissue collected on day 7 post-tumor challenge showed that BA-mediated clearance of the tumors was associated with a proportional increase of CD4+ and CD8+ TILs. We also observed that the frequency of CD103 expression among CD4+ TILs was reduced both in the primary and challenge tumors. Although TGFβ neutralization alone fails to induce anti-tumor immunity, the addition of TGFβ neutralization to PD-L1 blockade provides greater control of primary tumor growth and protection from engraftment of a challenge tumor compared to PD-L1 blockade alone. Through adoptive transfer of tumor-specific T cell receptor-engineered T cells, ongoing experiments aim to determine if TGFβ-driven CD103 expression is required for T cell tumor residency and if neutralization of TGFβ in the setting of PD-L1 immune checkpoint blockade allows for recirculation of tumor-specific T cells into the periphery. Citation Format: Magdalena Rainey, Marco Craveiro, Yvette Robbins, Cem Sievers, Clint T. Allen. Combined TGFβ/PD-L1 blockade enhances systemic antitumor immunity in head and neck cancer [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 6422.

Dual Axl/MerTK inhibitor INCB081776 creates a proinflammatory tumor immune microenvironment and enhances anti-PDL1 efficacy in head and neck cancer.

The tyrosine kinase receptors Axl and MerTK are highly overexpressed in head and neck cancer (HNC) cells, where they are critical drivers of survival, proliferation, metastasis, and therapeutic resistance. We investigated the role of Axl and MerTK in creating an immunologically "cold" tumor immune microenvironment (TIME) by targeting both receptors simultaneously with a small molecule inhibitor of Axl and MerTK (INCB081776). Effects of INCB081776 and/or anti-PDL1 on mouse oral cancer (MOC) cell growth and on the TIME were evaluated. Targeting Axl and MerTK can reduce M2 and induce M1 macrophage polarization. In vivo, INCB081776 treatment alone or with anti-PDL1 appears to slow MOC tumor growth, increase proinflammatory immune infiltration, and decrease anti-inflammatory immune infiltration. This data indicates that simultaneous targeting of Axl and MerTK with INCB081776, either alone or in combination with anti-PDL1, slows tumor growth and creates a proinflammatory TIME in mouse models of HNC.

HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer.

The membrane-associated RING-CH-type finger ubiquitin ligase MARCHF8 is a human homolog of the viral ubiquitin ligases Kaposi's sarcoma herpesvirus K3 and K5 that promote host immune evasion. Previous studies have shown that MARCHF8 ubiquitinates several immune receptors, such as the major histocompatibility complex II and CD86. While human papillomavirus (HPV) does not encode any ubiquitin ligase, the viral oncoproteins E6 and E7 are known to regulate host ubiquitin ligases. Here, we report that MARCHF8 expression is upregulated in HPV-positive head and neck cancer (HNC) patients but not in HPV-negative HNC patients compared to normal individuals. The MARCHF8 promoter is highly activated by HPV oncoprotein E6-induced MYC/MAX transcriptional activation. The knockdown of MARCHF8 expression in human HPV-positive HNC cells restores cell surface expression of the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2, and enhances apoptosis. MARCHF8 protein directly interacts with and ubiquitinates the TNFRSF death receptors. Further, MARCHF8 knockout in mouse oral cancer cells expressing HPV16 E6 and E7 augments cancer cell apoptosis and suppresses tumor growth in vivo. Our findings suggest that HPV inhibits host cell apoptosis by upregulating MARCHF8 and degrading TNFRSF death receptors in HPV-positive HNC cells.

E-liquid alters oral epithelial cell function to promote epithelial to mesenchymal transition and invasiveness in preclinical oral squamous cell carcinoma

The gaining popularity of tobacco and nicotine delivery products, such as electronic cigarettes (e-cigarettes) being perceived as relatively safe is of a medical concern. The long-term safety of these new products remains uncertain for oral health. In this study, in vitro effects of e-liquid were assessed in a panel of normal oral epithelium cell lines (NOE and HMK), oral squamous cell carcinoma (OSCC) human cell lines (CAL27 and HSC3), and a mouse oral cancer cell line (AT84) using cell proliferation, survival/cell death, and cell invasion assays. In addition, signaling pathways underlying the pro-invasive activity of e-cigarettes were evaluated by gene and protein expression analysis. We demonstrated that e-liquid promotes proliferation and anchorage-independent growth of OSCC and induces morphological changes associated with enhanced motility and invasive phenotypes. Furthermore, e-liquid-exposed cells express significantly reduced cell viability, regardless of e-cigarette flavour content. At the gene expression level, e-liquid induces changes in gene expression consistent with epithelial to mesenchymal transition (EMT) revealed by reduced expression of cell epithelial markers such as E-cadherin and enhanced expression of mesenchymal proteins like vimentin and B-catenin seen both in OSCC cell lines and normal oral epithelium cells. In summary, the ability of e-liquid to induce proliferative and invasive properties along the activation of the EMT process can contribute to the development of tumorigenesis in normal epithelial cells and promote aggressive phenotype in pre-existing oral malignant cells.

P. gingivalis Infection Upregulates PD-L1 Expression on Dendritic Cells, Suppresses CD8+ T-cell Responses, and Aggravates Oral Cancer.

Accumulating evidence shows that PD-L1 expression on dendritic cells (DC) is critical for cancer immunotherapy and that Porphyromonas gingivalis (Pg) colonization aggravates the progression of upper gastrointestinal cancers. However, the effects of Pg infection on PD-L1 expression on DCs and related immune consequences in the infection milieu of oral cancer remain unexplored. Here, we found that Pg infection robustly enhanced PD-L1 expression on DCs in a gingipain-dependent manner in cultured cell and systemic infection assays. Pg infection suppressed antigen-specific CD8+ T cells through upregulation of PD-L1 expression on ovalbumin (OVA)-pulsed DCs. This suppression was manifested by decreased IFNγ, perforin, granzyme B, and CD107a. Further analysis showed that Pg drastically reduced CD8+ T cells' ability to lyse OVA-pulsed target cells. Additionally, Pg infection increased the phosphorylation of Akt and STAT3, leading to a significant increase in PD-L1 expression. This was substantiated by using siRNA, overexpression plasmids, and pharmacologic inhibitors. Consistent with the in vitro observations, in a syngeneic mouse oral cancer model, Pg infection significantly enhanced PD-L1 expression on DCs from intratumoral tissues and cervical lymph nodes and exacerbated oral cancer progression, whereas a Pg lysine-specific, gingipain-defective mutant failed to do so. These influences of Pg were largely diminished when tumor cells were pretreated with antibiotics or a STAT3 inhibitor. Therefore, we demonstrated that Pg infection upregulates PD-L1 expression on DCs through Akt-STAT3 signaling, suppresses CD8+ T-cell cytotoxicity, and aggravates oral cancer growth, suggesting targeting Pg, and/or its mediated signaling, could be a therapeutic strategy to improve the efficacy of checkpoint blockade immunotherapy.

Statin drugs enhance responses to immune checkpoint blockade in head and neck cancer models

BackgroundAnti-PD-1 immune checkpoint blockade is approved for first-line treatment of recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), but few patients respond. Statin drugs (HMG-CoA reductase inhibitors) are associated with...

Chemoprevention of 4NQO-Induced Mouse Tongue Carcinogenesis by AKT Inhibitor through the MMP-9/RhoC Signaling Pathway and Autophagy.

Oral cancer (OC), the most common cancer in the head and neck, which has a poor prognosis, histopathologically follows a stepwise pattern of hyperplasia, dysplasia, and cancer. Blocking the progression of OC in the precancer stage could greatly improve the survival and cure rates. AKT protein plays a critical role in the signal transduction of cancer cells, and we found that AKT was overexpressed in human OC samples through analysis of TCGA database. Therefore, this study is aimed at investigating the chemopreventive effect of an AKT inhibitor (MK2206 2HCl) on OC. In vivo, we established a 4-nitroquinoline-1-oxide- (4NQO-) induced mouse tongue carcinogenesis model to investigate the potential chemopreventive effect of MK2206 2HCl on mouse OC resulting from 4NQO. The results showed that MK2206 2HCl could significantly reduce the incidence rate and growth of OC, inhibit the transformation of dysplasia to cancer in the 4NQO-induced mouse tongue carcinogenesis model, and simultaneously markedly suppress cell proliferation, angiogenesis, and mast cell (MC) infiltration in 4NQO-induced mouse tongue cancers. In vitro, our results revealed that MK2206 2HCl could also inhibit oral squamous cell carcinoma (OSCC) cell malignant biological behaviors, including cell proliferation, colony formation, cell invasion, and migration, while promoting apoptosis. Mechanistic studies revealed that MK2206 2HCl suppressed matrix metalloproteinase 9 (MMP-9) and RhoC expression and promoted autophagy gene LC3 II expression. In summary, our findings demonstrated the chemopreventive effect of MK2206 2HCl on the 4NQO-induced mouse tongue carcinogenesis model, which likely has an underlying mechanism mediated by the MMP-9/RhoC signaling pathway and autophagy.

The EXTREME Regimen Associating Cetuximab and Cisplatin Favors Head and Neck Cancer Cell Death and Immunogenicity with the Induction of an Anti-Cancer Immune Response.

(1) Background: The first line of treatment for recurrent/metastatic Head and Neck Squamous Cell Carcinoma (HNSCC) has recently evolved with the approval of immunotherapies that target the anti-PD-1 immune checkpoint. However, only about 20% of the patients display a long-lasting objective tumor response. The modulation of cancer cell immunogenicity via a treatment-induced immunogenic cell death is proposed to potentially be able to improve the rate of patients who respond to immune checkpoint blocking immunotherapies. (2) Methods: Using human HNSCC cell line models and a mouse oral cancer syngeneic model, we have analyzed the ability of the EXTREME regimen (combination therapy using the anti-EGFR cetuximab antibody and platinum-based chemotherapy) to modify the immunogenicity of HNSCC cells. (3) Results: We showed that the combination of cetuximab and cisplatin reduces cell growth through both cell cycle inhibition and the induction of apoptotic cell death independently of p53. In addition, different components of the EXTREME regimen were found to induce, to a variable extent, and in a cell-dependent manner, the emission of mediators of immunogenic cell death, including calreticulin, HMGB1, and type I Interferon-responsive chemokines. Interestingly, cetuximab alone or combined with the IC50 dose of cisplatin can induce an antitumor immune response in vivo, but not when combined with a high dose of cisplatin. (4) Conclusions: Our observations suggest that the EXTREME protocol or cetuximab alone are capable, under conditions of moderate apoptosis induction, of eliciting the mobilization of the immune system and an anti-tumor immune response in HNSCC.

The impact of tumor immunogenicity on cancer pain phenotype using syngeneic oral cancer mouse models.

Head and neck squamous cell carcinoma (HNSCC) patients report severe function-induced pain at the site of the primary tumor. The current hypothesis is that oral cancer pain is initiated and maintained in the cancer microenvironment due to secretion of algogenic mediators from tumor cells and surrounding immune cells that sensitize the primary sensory neurons innervating the tumor. Immunogenicity, which is the ability to induce an adaptive immune response, has been widely studied using cancer cell transplantation experiments. However, oral cancer pain studies have primarily used xenograft transplant models in which human-derived tumor cells are inoculated in an athymic mouse lacking an adaptive immune response; the role of inflammation in oral cancer-induced nociception is still unknown. Using syngeneic oral cancer mouse models, we investigated the impact of tumor cell immunogenicity and growth on orofacial nociceptive behavior and oral cancer-induced sensory neuron plasticity. We found that an aggressive, weakly immunogenic mouse oral cancer cell line, MOC2, induced rapid orofacial nociceptive behavior in both male and female C57Bl/6 mice. Additionally, MOC2 tumor growth invoked a substantial injury response in the trigeminal ganglia as defined by a significant upregulation of injury response marker ATF3 in tongue-innervating trigeminal neurons. In contrast, using a highly immunogenic mouse oral cancer cell line, MOC1, we found a much slower onset of orofacial nociceptive behavior in female C57Bl/6 mice only as well as sex-specific differences in the tumor-associated immune landscape and gene regulation in tongue innervating sensory neurons. Together, these data suggest that cancer-induced nociceptive behavior and sensory neuron plasticity can greatly depend on the immunogenic phenotype of the cancer cell line and the associated immune response.

5-Methoxytryptophan Sensitizing Head and Neck Squamous Carcinoma Cell to Cisplatitn Through Inhibiting Signal Transducer and Activator of Transcription 3 (STAT3).

Head and neck squamous cell carcinoma (HNSCC) is a common cancer of the oral cavity. Cisplatin (CDDP) is the ideal chemo-radiotherapy used for several tumor types, but resistance to the drug has become a major obstacle in treating patients with HNSCC. 5-methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of tryptophan metabolism, reduces inflammation-mediated proliferation and metastasis. This study aimed to assess the anti-oral cancer activity of 5-MTP when used alone or in combination with CDDP. Results showed that CDDP dose dependently reduced the growth of SSC25 cells but not 5-MTP. The combination of CDDP and 5-MTP exerted additional inhibitory effect on the growth of SSC25 cells by attenuating the phosphorylation of STAT3. In the 4-nitroquinoline-1-oxide-induced oral cancer mouse model, 5-MTP sensitized the reduction effect of CDDP on tumorigenesis, which restricted the tongue tissue in hyperkeratotic lesion rather than squamous cell carcinoma. The combination of CDDP and 5-MTP may be a potent therapeutic strategy for HNSCC patients with radiotherapy.

Inhibition of angiogenesis and tumor progression of MK-0429, an integrin αvβ3 antagonist, on oral squamous cell carcinoma

PurposeIntegrin αvβ3 is an essential molecule for tumor angiogenesis. This study aimed to investigate the anti-tumor effect of MK-0429, an integrin αvβ3 antagonist, on oral squamous cell carcinoma (OSCC) through its inhibitory effect on angiogenesis.MethodsIn this study, we investigated the effect of MK-0429 on cellular function and angiogenesis in vitro with the use of an immortalized human umbilical vein endothelial cell, HUEhT-1, which is immortalized by the electroporatic transfection of hTERT. The effect of MK-0429 on the integrin αvβ3 signaling pathway was examined by FAK, MEK1/2 and ERK 1/2 phosphorylation. The anti-angiogenic effect of MK-0429 was evaluated by in vitro tube formation assay. The anti-tumor effect on OSCC was assessed by administrating MK-0429 to mouse oral cancer xenografts.ResultsMK-0429 inhibited cell proliferation, migration, and adhesion of HUEhT-1 in a dose-dependent manner. FAK, MEK and ERK phosphorylation were significantly blocked by MK-0429 treatment. Tube formation was suppressed by MK-0429 in dose-dependent manner. Tumor progression was significantly suppressed by MK-0429 administration in mouse oral cancer xenografts. Histological study revealed that MK-0429 decreased tumor vascularization.ConclusionThese results indicated integrin αvβ3 as a therapeutic target for OSCC and suggested that MK-0429 might be clinically applicable as an anti-tumor agent with potent anti-angiogenic activity.

Abstract 3535: Simultaneous inhibition of Axl and MerTK enhances anti-PDL1 efficacy and creates a pro-inflammatory tumor immune microenvironment in head and neck cancer

Abstract The tyrosine kinase receptors Axl and MerTK, known for their role on macrophages in regulating clearance of apoptotic cells, are highly overexpressed in head and neck cancer (HNC). Previous studies in our laboratory have shown that Axl is a critical driver of survival, proliferation, metastasis, and therapeutic resistance in HNC, and that MerTK is functionally redundant to Axl. In this study, we investigated the cooperative role of Axl and MerTK in creating an immunologically cold tumor immune microenvironment (TIME) by targeting both receptors simultaneously with a small molecule inhibitor of Axl and MerTK (INCB081776). Because Axl and MerTK are expressed on both macrophages and HNC cancer cells, we examined the effect of INCB081776 treatment on each cell type. In macrophages, Axl and MerTK signaling leads to M2-type polarization, an anti-inflammatory state that leads to the resolution of inflammation and, in cancer settings, promotes tumor growth. Our experiments suggest that treatment with INCB081776 can reduce M2 polarization and increase M1-type polarization, a pro-inflammatory state that promotes inflammation and tumor cell killing. Next, to determine the efficacy of INCB081776 on HNC cancer cells, mouse oral cancer (MOC) tumors were implanted in syngeneic mice and treated with INCB081776 alone or in combination with a monoclonal antibody against PDL1 (anti-PDL1), thereby mimicking current standard-of-care immune checkpoint inhibitor treatment. The results showed a marked effect of INCB081776 single-agent treatment on MOC tumor growth and an increase in several pro-inflammatory cell types (M1 macrophages, CD8+ T cells, total infiltrating leukocytes), suggesting that INCB081776 treatment can create an immunologically hot TIME. Further, in-depth analysis of tumor infiltrating leukocytes following INCB081776 treatment in both immunologically hot (MOC1) and cold (MOC2) HNC tumors suggested that INCB081776 has a greater effect in cold tumors. In cold tumors, levels of pro-inflammatory cells (CD8+ T cells, M1 macrophages, etc.) increased, and levels of anti-inflammatory cells (M2 macrophages, regulatory T cells, etc.) decreased, both to a greater extent than in hot tumors. Finally, the combination of INCB081776 and anti-PDL1 was superior to either treatment alone in slowing tumor growth. Together, these studies indicate that INCB081776 cooperates with anti-PDL1 in a syngeneic mouse model of HNC to slow tumor growth and create a pro-inflammatory environment, especially in immunologically cold tumors, thereby highlighting the potential clinical benefit of this therapeutic combination. Citation Format: Kourtney L. Kostecki, Mari Iida, Anne L. Wiley, Seungpyo Hong, Ravi Salgia, Paul M. Harari, Deric L. Wheeler. Simultaneous inhibition of Axl and MerTK enhances anti-PDL1 efficacy and creates a pro-inflammatory tumor immune microenvironment in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3535.