Oral Cancer Model Research Articles

Abstract 2016: Novel dual cIAP1/XIAP antagonist ASTX660 activity in preclinical models of human papillomavirus(+) and (-) head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer with a five-year survival of ~50%. HNSCC are linked to human papillomavirus (HPV+) or tobacco carcinogenesis (HPV-). The Cancer Genome Atlas recently analyzed 279 HNSCC, revealing that 30% overexpress FADD (Fas-Associated Death Domain), with or without BIRC2/3 genes encoding cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2), which are critical components of the Tumor Necrosis Factor (TNF) Receptor signaling pathways that determine cell death or survival. The frequency of such mutations provides a window for potential therapeutics, as IAP antagonists have been shown to switch cancer cell TNFα signaling from being pro-survival to pro-apoptotic. We recently showed that birinapant, a second mitochondrial activator of caspases (SMAC) mimetic that promotes IAP degradation, sensitizes HNSCC to cell death by TNFα and eradicates tumors in combination with radiation in HPV- HNSCC models overexpressing FADD+/-BIRC2. ASTX660 (developed by Astex Pharmaceuticals) is a novel dual cIAP1/XIAP antagonist currently in clinical trials for treating advanced solid tumors and lymphomas. The objective of the present study is to determine the therapeutic effects of ASTX660 in HPV+/- HNSCC preclinical models. ASTX660 at nanomolar concentrations was found to potently inhibit cell proliferation (measured using XTT assays) and induce apoptosis (Annexin/7-AAD flow cytometry), and displayed combinatorial activity with TNFα, TRAIL, or cisplatin in multiple human HPV+/- HNSCC cell lines. Western blotting showed near complete degradation of cIAP1 expression at nanomolar concentrations in human HNSCC cell lines. Flow cytometry revealed that ASTX660 in combination with either TNFα or cisplatin reduced MHC-I expression and increased PD-L1 expression, suggesting that potential synergistic efficacy could be observed in combination with immune checkpoint inhibitors. Our results demonstrate that ASTX660 is a potential therapeutic agent for both HPV+/- HNSCC. Experiments evaluating the anti-tumor effects of ASTX660 as a monotherapy and in combination with radiation, cisplatin, and anti-PD-1 therapies are ongoing, using both human HNSCC xenograft and syngeneic mouse oral cancer (MOC) models. Supported by NIDCD intramural projects (ZIA-DC-000016, 73 and 74). Citation Format: Roy Xiao, Yi An, Adeeb Derakhshan, Zhong Chen, Nicole C. Schmitt, Carter Van Waes. Novel dual cIAP1/XIAP antagonist ASTX660 activity in preclinical models of human papillomavirus(+) and (-) head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2016. doi:10.1158/1538-7445.AM2017-2016

Abstract 5648: Combined immune checkpoint targeting with anti-PD-1 plus anti-CD40 antibodies as the most effective approach to eradicate head and neck squamous cell carcinomas (HNSCCs) in mouse models

Abstract Introduction: Immunotherapy with single agent anti-PD-1 antibody confers modest overall survival benefits in patients with recurrent/metastatic HNSCCs. Combined immune checkpoint targeting has shown promising results for melanomas and other solid tumors. Thus, evaluation of combination immunotherapy in animal models specific to HNSCCs could inform prioritization and design of human clinical trials for this disease. We conducted this pre-clinical study to test the hypothesis that combining anti-PD-1 with either anti-CTLA-4 or co-stimulatory immune checkpoint agonist antibodies would be more effective than anti-PD-1 monotherapy in a mouse model of oral carcinoma. We also aimed at identifying the combination regimen with highest anti-tumor activity. Methods: C57BL/6 mice were subcutaneously grafted with 2x106 mouse oral cancer 1 (MOC1) cells derived from a carcinogen-induced tumor molecularly similar to human HNSCC. After 25 days, mice were randomized into one of 12 treatment groups (IgG, or antibodies targeting PD-1, CTLA-4, CD40, OX-40, GITR, 4-1BB, PD-1+CTLA-4, PD1+CD40, PD-1+OX-40, PD-1+GITR, or PD-1+4-1BB), N=8-9 mice/group. Antibodies were administered every 4 days for 3 doses. Tumors were measured twice per week. The primary endpoint was overall survival. Mice were sacrificed when tumor diameter was >2 cm or tumor ulceration was >5 mm. Survival curves were computed using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test. Results: In analogy to human clinical trials, anti-PD-1 therapy led to a modest improvement in survival compared to IgG [HR=0.41; 95%CI 0.06-0.53; P=0.015], indicating that this model recapitulates, in part, the effects of immunotherapy in HNSCC patients; none of the animals survived long term. Monotherapy with anti-CTLA-4, anti-CD40, anti-OX-40, or anti-4-1BB also improved survival compared to IgG (P=0.0001, 0.003, 0.02, and 0.003, respectively), but were not more effective than anti-PD-1 alone (P=0.09, 0.12, 0.72, and 0.77, respectively). Combination therapy did not prolong survival compared to anti-PD-1 alone, with the exception of anti-PD-1+ anti-CTLA4 [HR=0.36; 95% CI 0.07-0.6; P=0.016], and anti-PD-1+ anti-CD40 [HR=0.125, 95% CI 0.02-0.24; P=0.0003]. Notably, 50% of mice receiving anti-PD-1+ anti-CD40 antibodies had complete tumor eradication and are alive and disease-free 120 days after tumor grafting. Conclusions: Single agent immunotherapy targeting PD-1, CTLA-4, CD40, OX-40 and 4-1BB modestly improved survival in a mouse model of oral cancer. Combination with anti-PD-1+ anti-CTLA4 and anti-PD-1+ anti-CD40 antibodies outperformed anti-PD-1 monotherapy. Complete responses were exclusively seen with PD-1+CD40 dual targeting. This regimen should be prioritized for evaluation in HNSCC clinical trials. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Alissa R. Poteete, Marlese A. Pisegna, Uma Giri, Fahao Zhang, Patrick Hwu, John V. Heymach, William N. William. Combined immune checkpoint targeting with anti-PD-1 plus anti-CD40 antibodies as the most effective approach to eradicate head and neck squamous cell carcinomas (HNSCCs) in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5648. doi:10.1158/1538-7445.AM2017-5648

Abstract 3903: Targeting oral cancer stem cells in the hypoxic niche by BCG infected mesenchymal stem cells

Abstract Background: Tumor hypoxia is a major contributing factor in cancer therapeutic failure. The microenvironment of hypoxia/oxidative stress may reprogram cancer cells to highly tumorigenic, metastatic, angiogenic and stem cell-like state. We have characterized in vitro and in vivo models of hypoxia-induced cancer cell reprogramming to cancer stem cell (CSCs) like cells including an immunocompetent model of oral cancer. These CSCs exhibit ABCG2 cell surface marker. Numerous approaches including targeted therapy using oncolytic virus and bacteria have been attempted to target cancer cells in their hypoxic niche without major therapeutic success. Major therapeutic challenges include the inaccessibility of hypoxic niche by therapy-agents and the poor replication of viruses or bacteria intracellular to hypoxic cancer cells. Furthermore, the immune privileged microenvironment of tumor hypoxia could pose a challenge to viral/bacterial-induced immunity against the tumor. In this context, here we have tested a novel stem cell-based approach to deliver BCG infected stem cells to the hypoxic core of tumors. Methods: Human SCC-25 cell line-derived xenograft in NOD/SCID mice exhibit hypoxic zones, where ABCG2+ resides (1). SCC-25 tumor-bearing mice were injected i.v. with CD271+ bone marrow mesenchymal stem cells (BM-MSCs). We recently found that CD271+ BM-MSCs could be infected with M. tuberculosis. The BCG infected CD271+ BM-MSCs were injected to SCC-25 xenograft-bearing mice. The mice were sacrificed on day-20 of stem cell injection and then evaluated for the hypoxic CSCs. Next, we have developed a 4-NQO induced oral cancer cell line, which was injected orthotropic to the tongue of congenic mice. The mice developed tumors, which was hypoxic. In these tumor-bearing mice, BCG-infected murine CD271 BM-MSCs were injected i.v., and animals were monitored for four weeks for tumor growth. On day-20 of injection, mice were sacrificed, and the tumors were collected. In a control group, BCG alone was injected. Results: In both the SCC-25 and an immunocompetent mouse model, we found that BCG or CD271+ BM-MSCs injection alone did not result in marked anti-tumor activity and or elimination of ABCG2+ cells from their hypoxic niche. In contrast, the injection of BCG infected CD271+ BM-MSCs led to marked reduction of tumor growth. Importantly, we observed marked replication of BCG intracellular to CSCs. These results indicate that CD271+ BM-MSCs facilitated the transfer of BCG to cancer cells residing in the hypoxic zone. Importantly, the transfer of BCG was seen more in the immunocompetent model, suggesting potential role of immune system. Conclusions: Our findings indicate that hypoxic CSCs may be targeted by a few BCG infected CD271 BM-MSCs. We propose this could be a novel therapeutic approach to target drug resistance cancer stem cells residing in the hypoxic niche of tumors.1. Bhuyan R et al, Cancer Research, 2016; Volume 76, Issue 14 Supplement, pp. 935. Note: This abstract was not presented at the meeting. Citation Format: Bidisha Pal, Seema Bhuyan, Jaishree Garhyan, Hong Li, Rashmi Bhuyan, Herman Yeger, Bikul Das. Targeting oral cancer stem cells in the hypoxic niche by BCG infected mesenchymal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3903. doi:10.1158/1538-7445.AM2017-3903

Abstract LB-180: Targeting galectin-1 in combination with radiation and immune checkpoint therapy in head and neck cancers

Abstract Head and neck cancers (HNC) development shows significant changes in the immune profile, with lower systemic lymphocyte counts and reduced tumor-infiltrating lymphocytes, which inversely correlate with survival. Galectin-1 (Gal-1) is secreted at high level by cancer cells, including HNC, and contributes to tumor-immune escape and disease progression. Previously, we have shown that both hypoxia and radiotherapy (RT) can stimulate Gal-1 secretion. Immune checkpoint therapies, specifically targeting the PD1 pathway, allows T cells to be effective in shrinking tumors and lengthen survival in patients with metastatic HNC. However, these therapies work only in a small number of patients with tumors showing significant T cell infiltration. Targeting Gal-1 in HNC therefore, could be a useful therapeutic approach, as it may synergize with radiation and immunotherapy. Using the CRISPR/Cas9 deletion approach, we developed an orthotopic model of HNC with/without Gal-1 expression. We found that in both subcutaneous and orthotopic model of oral cancer, Gal-1 KO (knock-out) tumors showed marked decrease in tumor growth and nodal metastases. These differences were attributed to enhanced infiltration of CD4+ and CD8+ T cells, in the absence of Gal-1. Using a series of transendothelial migration assays in vitro and tracking adoptively transferred T cells in vivo, we show that presence of Gal-1 in the tumor microenvironment strongly suppresses T cell infiltration into tumors. We found that Gal-1 secretion led to a systemic immune suppressive response not just intra-tumorally but also in the circulating blood compartment, resulting in reduction of both CD4+ and CD8+ T cells. Concurrently, we detected higher levels of IFN-γ secretion in mice with Gal-1 KO tumors. We also observed a higher expression of PD-L1 and Gal-9 on the endothelium of WT-Gal1 compared to Gal-1 KO tumors, leading to T cell exhaustion and ineffective systemic immune response. In addition, there was noteworthy increase in the PD1+ and Tim3+ T cells in Gal-1 WT tumors compared to Gal-1 KO tumors, suggesting a role of Gal-1 in T cell anergy. Finally, we show that knocking out Gal-1 either genetically or by a blocking antibody, we could convert an anti-PD1 Ab non-responsive tumor into a responsive tumor with a significant decrease in tumor burden. In summary, our study shows Gal-1 in the tumor microenvironment could hamper immune response by impeding transendothelial T cell migration into the tumor, blocking pro-inflammatory cytokine secretions and up-regulating inhibitory immune checkpoints ligands in both tumor endothelial and T cells. Our data suggests that Gal-1 may mediate poor-response or resistance to anti-PD1 therapy and combinatorial approaches of galectin-1 inhibition with PD-1 checkpoint inhibitors may enhance therapeutic efficacy in HNC. (Acknowledgements: BMS for Anti-PD1 and Anti-Gal1 Abs. Dr R. Uppaluri, for MOC-2 oral cancer cells). Citation Format: Dhanya K. Nambiar, Todd Aguilera, Joshua Daniel Bloomstein, Hongbin Cao, Albert Koong, Quynh Thu Le. Targeting galectin-1 in combination with radiation and immune checkpoint therapy in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-180. doi:10.1158/1538-7445.AM2017-LB-180

Dose-dependent enhancement of T-lymphocyte priming and CTL lysis following ionizing radiation in an engineered model of oral cancer

ObjectivesDetermine if direct tumor cell cytotoxicity, antigen release, and susceptibility to T-lymphocyte killing following radiation treatment is dose-dependent. Materials and methodsMouse oral cancer cells were engineered to express full-length ovalbumin as a model antigen. Tumor antigen release with uptake and cross presentation of antigen by antigen presenting cells with subsequent priming and expansion of antigen-specific T-lymphocytes following radiation was modeled in vitro and in vivo. T-lymphocyte mediated killing was measured following radiation treatment using a novel impedance-based cytotoxicity assay. ResultsRadiation treatment induced dose-dependent induction of executioner caspase activity and apoptosis in MOC1 cells. In vitro modeling of antigen release and T-lymphocyte priming demonstrated enhanced proliferation of OT-1 T-lymphocytes with 8Gy treatment of MOC1ova cells compared to 2Gy. This was validated in vivo following treatment of established MOC1ova tumors and adoptive transfer of antigen-specific T-lymphocytes. Using a novel impedance–based cytotoxicity assay, 8Gy enhanced tumor cell susceptibility to T-lymphocyte killing to a greater degree than 2Gy. ConclusionIn the context of using clinically-relevant doses of radiation treatment as an adjuvant for immunotherapy, 8Gy is superior to 2Gy for induction of antigen-specific immune responses and enhancing tumor cell susceptibility to T-lymphocyte killing. These findings have significant implications for the design of trials combining radiation and immunotherapy.

Effects of High Fat Diet on 4‐NQO‐Induced Changes in a Variety of Tissues in a Mouse Model of Oral Cancer

IntroductionA variety of factors, including use of tobacco, diet, gender, and genetics impact cancer risk. Smoking is a risk factor for about 1/3 of all human cancers. It is well established that administration of 4‐nitroquinonline 1‐oxide (4NQO) in drinking water mimics the effects of tobacco and leads to development of oral carcinoma in mice. The purpose of this study was to compare the effects of high and low in saturated fat diets (HF and LF, respectively) on the incidence and severity of 4NQO‐induced cancers. We hypothesized that 4NQO's carcinogenic effects extend beyond oral tissues and will result in pathological changes in organs beyond the oral cavity. We also hypothesized that a HF diet would increase inflammation and tissue changes when compared to the LF diet.Materials and methodsMale and female C57Bl/6 mice (Jackson Labs, 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 weeks prior to euthanasia. Animals were weighed weekly and upon sacrifice. Salivary glands, lungs, liver, kidney, spleen, and portions of the gastrointestinal tract were harvested, formalin fixed, microtome sectioned, and stained with hematoxylin and eosin (H&E) for histological evaluation. Histopathology criteria used for tissue assessment were based on the presence and degree of cellular atypia, tissue hyperplasia and dysplasia, and lymphoid cells infiltration.ResultsHistopathological changes in 4NQO‐treated animals were identified in esophagus (epithelial hyperplasia, dysplasia, inflammation, and development of carcinoma), lungs (significant peribronchial lymphoid cell infiltrates and – characteristic of emphysema – increased in size air spaces), and liver (lymphoid cell infiltrates in portal tracts and liver parenchyma and periportal fibrosis). Kidney, spleen, salivary glands, and pancreas demonstrated little changes based on H&E‐stained tissue analysis. Gender had no significant effect on a degree of 4NQO‐induced tissue pathology. However, HF diet exacerbated pathological changes compared to LF diet. This was particularly evident in liver, with males but not females demonstrating severe steatosis and liver enlargement (3× higher liver index in HF males versus all other treatment groups).ConclusionThis mouse model of oral cancer, initially established to mimic the effects of smoking, may be extended to serve as a model for the effects of tobacco and diet on extra‐oral tissues as well.Support or Funding InformationWe recognize the Midwestern University Deans of the College of Dental Medicine and the Basic Science Division for their support of this research. Student research was supported by the Office of Research and Sponsored Programs and the Biomedical Sciences Division.

Low dietary n-6/n-3 polyunsaturated fatty acid ratio prevents induced oral carcinoma in a hamster pouch model

Dietary fatty acid patterns have been linked to the prevalence of certain cancers, however in oral carcinoma is limited. Thus, we investigated the chemopreventive effects of various dietary n-6 and n-3 fatty acids in a 9,10-dimethyl-1,2-benz[a]-anthracene (DMBA)- and betel quid extract (BQE) -induced hamster oral cancer model. Thirty 6-week-old adult male hamsters were housed and divided into normal, low, and high dietary n-6 and n-3 fatty acid groups under DMBA + BQE treatment for 16 weeks. The right buccal pouch of all hamsters were evaluated by tumor number, volume, burden and selected inflammatory parameters. The results indicate that the low dietary n-6/n-3 fatty acid group exhibited a significantly lower tumor number, volume, and burden than those of the other groups. Furthermore, this group had significantly lower nuclear factor-κB, proliferating cell nuclear antigen, and cyclin D1 expression in the right buccal pouch tissue. In conclusion, the lower dietary n-6/n-3 fatty acid ratio exerted chemopreventive effects in the DMBA- and BQE-induced hamster oral cancer model.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Preclinical uPAR-targeted multimodal imaging of locoregional oral cancer

ObjectivesEstablishing adequate resection margins and lymphatic mapping are crucial for the prognosis of oral cancer patients. Novel targeted imaging modalities are needed, enabling pre- and intraoperative detection of tumour cells, in combination with improved post-surgical examination by the pathologist. The urokinase-receptor (uPAR) is overexpressed in head and neck cancer, where it is associated with tumour progression and metastasis. Material and methodsTo determine suitability of uPAR for molecular imaging of oral cancer surgery, human head and neck tumours were sectioned and stained for uPAR to evaluate the expression pattern compared to normal mucosa. Furthermore, metastatic oral squamous carcinoma cell line OSC-19 was used for targeting uPAR in in vivo mouse models. Using anti-uPAR antibody ATN-658, equipped with a multimodal label, the in vivo specificity was investigated and the optimal dose and time-window were evaluated. ResultsAll human oral cancer tissues expressed uPAR in epithelial and stromal cells. Hybrid ATN-658 clearly visualized tongue tumours in mice using either NIRF or SPECT imaging. Mean fluorescent TBRs over time were 4.3±0.7 with the specific tracer versus 1.7±0.1 with a control antibody. A significant difference in TBRs could be seen between 1nmol (150μg) and 0.34nmol (50μg) dose groups (n=4, p<0.05). Co-expression between BLI, GFP and the NIR fluorescent signals were seen in the tongue tumour, whereas human cytokeratin staining confirmed presence of malignant cells in the positive cervical lymph nodes. ConclusionThis study shows the applicability of an uPAR specific multimodal tracer in an oral cancer model, combining SPECT with intraoperative guidance.

Targeting metastasis-initiating cells through the fatty acid receptor CD36.

The fact that the identity of the cells that initiate metastasis in most human cancers is unknown hampers the development of antimetastatic therapies. Here we describe a subpopulation of CD44bright cells in human oral carcinomas that do not overexpress mesenchymal genes, are slow-cycling, express high levels of the fatty acid receptor CD36 and lipid metabolism genes, and are unique in their ability to initiate metastasis. Palmitic acid or a high-fat diet specifically boosts the metastatic potential of CD36+ metastasis-initiating cells in a CD36-dependent manner. The use of neutralizing antibodies to block CD36 causes almost complete inhibition of metastasis in immunodeficient or immunocompetent orthotopic mouse models of human oral cancer, with no side effects. Clinically, the presence of CD36+ metastasis-initiating cells correlates with a poor prognosis for numerous types of carcinomas, and inhibition of CD36 also impairs metastasis, at least in human melanoma- and breast cancer-derived tumours. Together, our results indicate that metastasis-initiating cells particularly rely on dietary lipids to promote metastasis.

TM1-IR680 peptide for assessment of surgical margin and lymph node metastasis in murine orthotopic model of oral cancer.

Treatment outcome after surgical removal in oral carcinoma is poor due to inadequate methodologies available for marking surgical margins. Even though some methodologies for intraoperative margin assessment are under clinical and preclinical trials for other solid tumours, a promising modality for oral cancer surgery is not developed. Fluorescent-based optical imaging using Near Infrared (NIR) dyes tagged to tumour specific target will be an optimal tool for this purpose. One such target, Gastrin Releasing Peptide Receptor (GRPR) was selected for the study, and its binding peptide, TM1-IR680, was tested for its efficacy for surgical margin prediction in murine orthotopic model of oral cancer, derived from primary samples. Here, for the first time in a preclinical analysis, we show that the size and margin of oral cancer can be predicted, as revealed by 3D-imaging. Interestingly, the peptide was sensitive enough to detect lymph nodes that harboured dispersed tumour cells before colonization, which was impossible to identify by conventional histopathology. We recommend the use of TM1-NIR dyes alone or in combination with other technologies to improve the clinical outcome of oral cancer surgery.

Increased survival rate by local release of diclofenac in a murine model of recurrent oral carcinoma.

Despite aggressive treatment with radiation and combination chemotherapy following tumor resection, the 5-year survival rate for patients with head and neck cancer is at best only 50%. In this study, we examined the therapeutic potential of localized release of diclofenac from electrospun nanofibers generated from poly(D,L-lactide-co-glycolide) polymer. Diclofenac was chosen since anti-inflammatory agents that inhibit cyclooxygenase have shown great potential in their ability to directly inhibit tumor growth as well as suppress inflammation-mediated tumor growth. A mouse resection model of oral carcinoma was developed by establishing tumor growth in the oral cavity by ultrasound-guided injection of 1 million SCC-9 cells in the floor of the mouth. Following resection, mice were allocated into four groups with the following treatment: 1) no treatment, 2) implanted scaffolds without diclofenac, 3) implanted scaffolds loaded with diclofenac, and 4) diclofenac given orally. Small animal ultrasound and magnetic resonance imaging were utilized for longitudinal determination of tumor recurrence. At the end of 7 weeks following tumor resection, 33% of mice with diclofenac-loaded scaffolds had a recurrent tumor, in comparison to 90%–100% of the mice in the other three groups. At this time point, mice with diclofenac-releasing scaffolds showed 89% survival rate, while the other groups showed survival rates of 10%–25%. Immunohistochemical staining of recurrent tumors revealed a near 10-fold decrease in the proliferation marker Ki-67 in the tumors derived from mice with diclofenac-releasing scaffolds. In summary, the local application of diclofenac in an orthotopic mouse tumor resection model of oral cancer reduced tumor recurrence with significant improvement in survival over a 7-week study period following tumor resection. Local drug release of anti-inflammatory agents should be investigated as a therapeutic option in the prevention of tumor recurrence in oral squamous carcinoma.

CD24 blunts oral squamous cancer development and dampens the functional expansion of myeloid-derived suppressor cells

ABSTRACTCD24 expression has been implicated in the oncogenesis of multiple types of cancer and high tumor expression is considered a poor prognosis factor; however, the role of CD24 in oral cancer progression is unknown. Unlike other cancer types, we found that higher CD24 levels in human oral cancers are correlated to lower clinical stage and better overall survival. We then dissected the role of CD24 and mechanisms in oral cancer pathogenesis in mice using a genetic strategy and demonstrated that CD24 deficiency increased the oral cavity tumor burden in response to the carcinogen 4-nitroquioline 1-oxide (4-NQO). Immune profile analysis showed a significant expansion as well as increased suppressive function of myeloid-derived suppressor cells (MDSCs) in CD24−/− mice, but no apparent impairment in T cells, B cells, or dendritic cells. Further, studies with an orthotopically transplanted syngeneic squamous carcinoma model in the tongue of CD24−/− and CD24+/− mice confirmed the protective roles of CD24 against cancer. Moreover, the difference in tumor growth between CD24−/− and CD24+/− mice was blunted by immunodepletion of MDSCs. We conclude that CD24 expression impedes MDSC expansion and function, and thus slows oral cancer oncogenesis. This study is the first to examine the role of CD24 in a de novo oral cancer model, and it highlights the need to consider the immune regulatory roles of CD24 in the development of CD24-targeted therapy for cancer.

Abstract 1448: MDSC depletion delays primary tumor growth in syngeneic models of oral cavity cancer

Abstract Carcinogen-associated oral cavity cancers are a heterogeneous group of aggressive cancers with a high recurrence rate after definitive treatment and a poor 5-year survival. The genetic alteration rate of these cancers tends to be high, and many oral cancers express immune checkpoint molecules in the tumor microenvironment with potential to respond to checkpoint blocking immunotherapy. Local immunosuppression mediated by both the tumor cells and other infiltrating immune cells are likely a major mechanism of resistance to immunotherapy. Here, we investigated the role of myeloid derived suppressor cells (MDSCs) in a highly aggressive but poorly immunogenic syngeneic model of carcinogen-induced oral cavity cancer (MOC2). Performing a time course analysis of immune infiltrates, MOC2 tumors demonstrated robust recruitment of CXCR2+CSFR1+CCR2− MDSCs that peaked at 15 days following tumor transplantation. This intratumoral accumulation of MDSCs preceded draining lymph node and splenic MDSC accumulation by 6 and 9 days respectively. The accumulation of MDSCs corresponded to a sharp decrease in the presence of tumor infiltrating CD8+ T-lymphocytes (TIL), CD4+ TIL, FoxP3+CD4+ Tregs and CD3−NK1.1+ NK cells. As CD8+ TIL numbers decreased, cell surface expression of the lymphocyte activation markers CD69, CD44 and ICOS decreased with the checkpoint molecule CTLA-4 increased. Phenotypically, the great majority of tumor-infiltrating MDSCs were granulocytic MDSCs (Ly6GhiLy6Cint), while few were monocytic MDSCs (Ly6GloLy6Chi). In a CFSE-based functional T-lymphocyte suppression assay, sorted peripheral and tumor-infiltrating MDSCs strongly suppressed T-lymphocyte proliferation at MDSC:T-lymphocyte ratios as low as 1:32. Hypothesizing that decreased tumor MDSCs would impact primary tumor growth, MOC2 tumors were allowed to engraft to 100mm3 and depletion of granulocytic MDSCs was performed with a rat anti-mouse Ly6G depleting antibody (clone RB6-8C5). This depletion resulted in a statistically significant delay in MOC2 primary tumor growth. Flow cytometric analysis of tumor tissues revealed that MDSC depletion was transient with rapid rebound of MDSC tumor infiltration within days of depleting antibody administration. This data highlights the critical role that MDSCs play in local immunosuppression and suggest that the syngeneic MOC model represents a powerful tool to study MDSC pathobiology. Functional inhibition or elimination of MDSCs from the tumor microenvironment represents an exciting adjuvant therapy that may enhance the response to checkpoint inhibition in patients with oral cavity cancer. Citation Format: Paul E. Clavijo, Ruth Davis, Zhong Chen, Carter Van Waes, Clint T. Allen. MDSC depletion delays primary tumor growth in syngeneic models of oral cavity cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1448.

Abstract 3234: Genetic deletion of macrophage migration inhibitory factor reduces oral carcinogenesis

Abstract Oral cancer kills about 1 person every hour, each day in the United States and is the 6th most prevalent cancer worldwide. The pro-inflammatory cytokine ‘macrophage migration inhibitory factor’ (MIF) has been shown to be expressed in oral cancer patients, yet its precise role in oral carcinogenesis is not clear. In this study, we examined the impact of global Mif deletion on the cellular and molecular process occurring during oral carcinogenesis using a well-established mouse model of oral cancer with the carcinogen 4-nitroquinoline-1-oxide (4NQO). C57BL/6 Wild-type (WT) and Mif knock-out mice were administered with 4NQO in drinking water for 16 weeks, then regular drinking water for 8 weeks. Mif knock-out mice displayed fewer oral tumor incidence and multiplicity, accompanied by a significant reduction in the expression of pro-inflammatory cytokines Il-1β, Tnf-α, chemokines Cxcl1, Cxcl6, Cxcl8 and Ccl3 and other molecular biomarkers of oral carcinogenesis Mmp1 and Ptgs2. Further, recruitment of myeloid-derived tumor promoting immune cells was inhibited in Mif knock-out mice. Our results demonstrate that genetic Mif deletion reduces the incidence and severity of oral carcinogenesis, by inhibiting the expression inflammatory cytokines and infiltration of tumor promoting immune cells. Thus, targeting MIF is a promising strategy for the prevention or therapy of oral cancer. Citation Format: Steve Oghumu, Thomas Knobloch, Cesar Terrazas, Sanjay Varikuti, Claire Bollinger, Hans Iwenofu, Christopher Weghorst, Abhay Satoskar. Genetic deletion of macrophage migration inhibitory factor reduces oral carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3234.

Enhanced Tumor Control with Combination mTOR and PD-L1 Inhibition in Syngeneic Oral Cavity Cancers.

Significant subsets of patients with oral cancer fail to respond to single-agent programmed death (PD) blockade. Syngeneic models of oral cancer were used to determine if blocking oncogenic signaling improved in vivo responses to PD-L1 monoclonal antibody (mAb). Anti-PD-L1 enhanced durable primary tumor control and survival when combined with mTOR (rapamycin), but not in combination with MEK inhibition (PD901) in immunogenic MOC1 tumors. Conversely, PD-L1 mAb did not enhance tumor control in poorly immunogenic MOC2 tumors. Rapamycin enhanced expansion of peripheral antigen-specific CD8 T cells and IFNγ production following ex vivo antigen stimulation. More CD8 T cells infiltrated and were activated after PD-L1 mAb treatment in mice with immunogenic MOC1 tumors, which were stable or increased by the addition of rapamycin, but suppressed when PD901 was added. Rapamycin increased IFNγ production capacity in peripheral and tumor-infiltrating CD8 T cells. In vivo antibody depletion revealed a CD8 T-cell-dependent, and not NK cell-dependent mechanism of tumor growth inhibition after treatment with rapamycin and PD-L1 mAb, ruling out significant effects from NK cell-mediated antibody-dependent cellular cytotoxicity. Rapamycin also enhanced IFNγ or PD-L1 mAb treatment-associated induction of MHC class I expression on MOC1 tumor cells, an effect abrogated by depleting infiltrating CD8 T cells from the tumor microenvironment. These data conflict with traditional views of rapamycin as a universal immunosuppressant, and when combined with evidence of enhanced antitumor activity with the combination of rapamycin and PD-L1 mAb, suggest that this treatment combination deserves careful evaluation in the clinical setting. Cancer Immunol Res; 4(7); 611-20. ©2016 AACR.

Black raspberries in cancer clinical trials: Past, present and future.

BACKGROUNDBlack raspberries (BRB) inhibit a broad range of cancers in preclinical models, including in vivo models of oral, esophageal, colon, breast and skin cancer. Promising preclinical results have led to clinical evaluations in cancer patients or patients at increased risk for cancer development.OBJECTIVETo summarize clinical investigations targeting cancer or precancerous lesions with BRB and discuss future directions.METHODSA thorough literature search was conducted through December 1, 2015 to identify all published studies evaluating BRB in cancer focused clinical trials.RESULTSResearch investigating BRB in clinical settings report positive effects on preneoplastic lesions or cancers of the oral cavity, esophagus and colon. BRB treatment resulted in: histologic regression of oral intraepithelial neoplasia associated with improved histologic grade and significantly reduced loss of heterozygosity at tumor suppressor gene loci, modulated genes linked to RNA processing and growth factor recycling; in the colon, BRB inhibited FAP-associated polyp progression, demethylated tumor suppressor genes and improved plasma cytokine profiles; in Barrett’s patients, BRB consumption increased tissue levels of GST-pi and decreased 8-isoprostane, a marker of lipid peroxidation/oxidative stress.CONCLUSIONSThe precise dose, duration and optimum mode of BRB delivery for cancer inhibition remains to be fully elucidated. Common themes across studies support that BRB are anti-proliferative, anti- inflammatory, reduce oxidative stress and restore tumor suppressive activity. Future directions are included in the conclusions section.

Deletion of macrophage migration inhibitory factor inhibits murine oral carcinogenesis: Potential role for chronic pro-inflammatory immune mediators.

Oral cancer kills about 1 person every hour each day in the United States and is the sixth most prevalent cancer worldwide. The pro-inflammatory cytokine 'macrophage migration inhibitory factor' (MIF) has been shown to be expressed in oral cancer patients, yet its precise role in oral carcinogenesis is not clear. In this study, we examined the impact of global Mif deletion on the cellular and molecular process occurring during oral carcinogenesis using a well-established mouse model of oral cancer with the carcinogen 4-nitroquinoline-1-oxide (4NQO). C57BL/6 Wild-type (WT) and Mif knock-out mice were administered with 4NQO in drinking water for 16 weeks, then regular drinking water for 8 weeks. Mif knock-out mice displayed fewer oral tumor incidence and multiplicity, accompanied by a significant reduction in the expression of pro-inflammatory cytokines Il-1β, Tnf-α, chemokines Cxcl1, Cxcl6 and Ccl3 and other molecular biomarkers of oral carcinogenesis Mmp1 and Ptgs2. Further, systemic accumulation of myeloid-derived tumor promoting immune cells was inhibited in Mif knock-out mice. Our results demonstrate that genetic Mif deletion reduces the incidence and severity of oral carcinogenesis, by inhibiting the expression of chronic pro-inflammatory immune mediators. Thus, targeting MIF is a promising strategy for the prevention or therapy of oral cancer.

Overexpression of Bcl‐2, SOCS 1, 3 and Cdh 1, 2 are associated with the early neoplasic changes in modified 4‐nitroquinoline 1‐oxide‐induced murine oral cancer model

The objective was to assess histopathological changes and the expression of proliferating cell nuclear antigen (PCNA), Bcl-2, suppressor of cytokine signaling (SOCS) 1 and 3, Vimentin, TWIST1, and Cdh 1 and 2 in early stages of experimental oral carcinogenesis process using a shorter period of exposure to 4-nitroquinoline oxide (4-NQO) model. In this study, 20 rats were divided into control group (n = 10), sacrificed on the first day of the experiment, and experimental group (n = 10) treated with 50 ppm of 4-NQO solution dissolved in drinking water for 8 and 12 weeks. The histological sections were stained with H&E or subjected to immunohistochemistry for detecting PCNA, Bcl-2, SOCS 1 and 3, and STAT 3. Some specimens were used for verification of Vimentin expression, Cdh 1, Cdh 2, and TWIST1 by RT-qPCR. At both 8 and 12 weeks, morphological changes occurred mainly in the posterior portion of the tongue and were limited to the epithelial tissue, including moderate to severe dysplasia at 8 weeks, and severe dysplasia with exacerbation of atypical cells at 12 weeks. Expression of SOCS 1 and 3 increased from 8 to 12 weeks (P < 0.05), whereas STAT 3 expression was reduced mainly at 12 weeks (P < 0.05) in comparison with the control group. The expression of all epithelial-mesenchymal transition markers (EMT) was increased after 12 weeks, reaching statistical significance (P < 0.05) for Cdh 1 and 2. Together, the results suggested that overexpression of Bcl-2, SOCS 1 and 3, and Cdh 1 and 2 is associated with the early neoplasic changes in modified 4-nitroquinoline 1-oxide-induced murine oral cancer model.

MTOR and MEK1/2 inhibition differentially modulate tumor growth and the immune microenvironment in syngeneic models of oral cavity cancer.

We investigated the effects of mTOR and MEK1/2 inhibition on tumor growth and the tumor microenvironment in immunogenic and poorly immunogenic models of murine oral cancer. In vitro, rapamycin and PD901 inhibited signaling through expected downstream targets, but only PD901 reduced viability and altered function of MOC cells. Following transplantation of MOC cells into immune-competent mice, effects on both cancer and infiltrating immune cells were characterized following rapamycin and/or PD901 treatment for 21 days. In vivo, both rapamycin and PD901 inhibition reduced primary growth of established MOC tumors on treatment. Following withdrawal of PD901, rapid rebound of tumor growth limited survival, whereas durable tumor control was observed following rapamycin treatment in immunogenic MOC1 tumors despite more robust inhibition of oncogenic signaling by PD901. Characterization of the immune microenvironment revealed diminished infiltration and activation of antigen-specific CD8+ T-cells and other immune cells following PD901 but not rapamycin in immunogenic tumors. Subsequent in vitro T-cell assays validated robust inhibition of T-cell expansion and activation following MEK inhibition compared to mTOR inhibition. CD8 cell depletion abrogated rapamycin-induced primary tumor growth inhibition in MOC1 mice. These data have critical implications in the design of combination targeted and immune therapies in oral cancer.