Orthotopic Model Of Oral Cancer Research Articles

Using Gold-Nanorod-Filled Mesoporous Silica Nanobeads for Enhanced Radiotherapy of Oral Squamous Carcinoma.

Radiotherapy (RT), in combination with surgery, is an essential treatment strategy for oral cancer. Although irradiation provides effective control over tumor growth, the surrounding normal tissues are almost inevitably affected. With further understanding of the molecular mechanisms involved in radiation response and recent advances in nanotechnology, using gold nanoparticles as a radiosensitizer provides the preferential sensitization of tumor cells to radiation and minimizes normal tissue damage. Herein, we developed gold nano-sesame-beads (GNSbs), a gold-nanorod-seeded mesoporous silica nanoparticle, as a novel radioenhancer to achieve radiotherapy with a higher therapeutic index. GNSbs in combination with 2 Gy irradiation effectively enhanced the cytotoxic activity CAL-27 cells. The well-designed structure of GNSbs showed preferential cellular uptake by CAL-27 cells at 24 h after incubation. Gold nanorods with high density modified on mesoporous silica nanoparticles resulted in significant reactive oxygen species (ROS) formation after irradiation exposure compared with irradiation alone. Furthermore, GNSbs and irradiation induced more prominent DNA double-strand breaks and G2/M phase arrest in CAL-27 than those in L929. In animal studies, radiotherapy using GNSbs as a radiosensitizer showed significant suppression of tumor growth in an orthotopic model of oral cancer. These results demonstrate that using GNSbs as a radiosensitizer could possess clinical potential for the treatment of oral squamous carcinoma.

Neutrophil Elastase Facilitates Tumor Cell Intravasation and Early Metastatic Events.

SummaryFunctional roles of neutrophil elastase (NE) have not been examined in distinct steps of the metastatic cascade. NE, delivered to primary tumors as a purified enzyme or within intact neutrophils or neutrophil granule content, enhanced human tumor cell intravasation and subsequent dissemination via NE-mediated formation of dilated intratumoral vasculature. These effects depended on picomole range of NE activity, sensitive to its natural inhibitor, α1PI. In Elane-negative mice, the lack of NE decreased lung retention of human tumor cells in experimental metastasis. Furthermore, NE was essential for spontaneous metastasis of murine carcinoma cells in a syngeneic orthotopic model of oral cancer. NE also induced tumor cell survival and migration via Src/PI3K-dependent activation of Akt signaling, vital for tumor cell dissemination in vivo. Together, our findings implicate NE, a potent host enzyme specific for first-responding innate immune cells, as directly involved in early metastatic events and a potential target for therapeutic intervention.

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

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.

Abstract 3934: Restoration of NOTCH signaling in head and neck squamous cell carcinoma inhibits tumor growth and cell migratory abilities

Abstract Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common malignancy worldwide, with roughly 300,000 cancer related deaths occurring globally each year. The survival of patients with HNSCC hasn't changed significantly over the past decade leading investigators to search for promising molecular targets. To identify new treatment targets and biomarkers that could better guide therapy, we previously characterized the genomic alterations from primary HNSCC patient samples. We were among the first to discover that NOTCH1 is one of the most frequently mutated genes in this cancer type. The spectrum of inactivating NOTCH1 mutations in HNSCC suggested a tumor suppressive role for this protein, however, the mechanism of its function is currently unknown. Procedure: We used Sanger sequencing and immunoblotting to characterize 50 well- established HNSCC cell lines as being wild-type or mutant for NOTCH1. We cloned the full length NOTCH1 receptor to restore the NOTCH signaling function and activated this pathway by culturing cells on immobilized NOTCH ligand, Jagged1, coated plates. Clonogenic assays and competitive cell proliferation assays was used to evaluate cell growth and proliferation. Transwell Boyden chambers was used to investigate migration/invasion. Tumorigenicty was evaluated in vivo in a mice orthotopic model of oral cancer. An unbiased gene expression analysis was performed to identify potential downstream targets of the NOTCH pathway. Results: We show here that restoration of NOTCH signaling in mutant HNSCC cell lines significantly decreases cell growth and migratory abilities. Activation of NOTCH1 led to decreased tumorigenicity in a mice orthotopic oral cancer model. Conversely, abrogation of the NOTCH pathway using the dominant negative form of the NOTCH co-activator, MAML1, enhanced tumor growth in a weakly tumorigenic HNSCC cell line harboring wild-type NOTCH1. To evaluate the mechanism of NOTCH induced inhibition of growth and migration, we performed an unbiased microarray analysis and observed suppression of two proto-oncogenes, AXL and CTNNAL1 in the presence of NOTCH1, but not in its absence. Restoration of NOTCH1 significantly decreases protein expression levels of AXL and CTNNAL1 which then results in decreased cell growth and migratory abilities. Furthermore, we show here a novel link between NOTCH1, AXL and CTNNAL1 via HES5, one of the canonical NOTCH downstream targets. We suggest that NOTCH1 activates HES5 which then downregulates AXL and CTNNAL1, thus inhibiting cell growth and migratory abilities. Conclusions: We have data linking NOTCH1 reactivation to suppression of proto-oncogenes, AXL and CTNNAL1 along with decreased growth and migration of HNSCC cells. This underscores the need to understand the clinical significance of the NOTCH pathway and its downstream targets which can then be exploited for potential therapeutic strategies. Citation Format: Shhyam Moorthy, Rami Saade, Curtis Pickering, David Neskey, Noriaki Tanaka, Patrick Zweidler-McKay, Mitchell Frederick, Jeffrey Myers. Restoration of NOTCH signaling in head and neck squamous cell carcinoma inhibits tumor growth and cell migratory abilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3934. doi:10.1158/1538-7445.AM2015-3934

Disruptive TP53 Mutation Is Associated with Aggressive Disease Characteristics in an Orthotopic Murine Model of Oral Tongue Cancer

To characterize tumor growth and metastatic potential in head and neck squamous cell carcinoma (HNSCC) cell lines in an orthotopic murine model of oral tongue cancer and to correlate TP53 mutation status with these findings. Cells from each of 48 HNSCC cell lines were orthotopically injected into the oral tongues of nude mice. Tumor volume, cervical lymph node metastasis, and mouse survival were recorded. Direct sequencing of the TP53 gene and Western blot analysis for the p53 protein after induction with 5-fluorouracil was conducted. Cell lines were categorized as either mutant TP53 or wild-type TP53, and lines with TP53 mutation were further categorized on the basis of type of mutation (disruptive or nondisruptive) and level of p53 protein expression. The behavior of tumors in these different groups was compared. These 48 HNSCC cell lines showed a wide range of behavior from highly aggressive and metastatic to no tumor formation. Mice injected with cells harboring disruptive TP53 mutations had faster tumor growth, greater incidence of cervical lymph node metastasis, and shorter survival than mice injected with cells lacking these mutations. HNSCC cell lines display a wide spectrum of behavior in an orthotopic model of oral cancer. Cell lines with disruptive TP53 mutations are more aggressive in this system, corroborating clinical reports that have linked these mutations to poor patient outcome.

Abstract 1504: Characterization of the murine oral squamous cell carcinoma cell line AT84 stably overexpressing uPAR

Abstract Cancers of the oral cavity are associated with poor prognosis, and almost half of the patients diagnosed with oral squamous cell carcinoma (SCC) will die from the disease. Compared to its counterpart in skin, one main differing feature is the ability of oral SCC to metastasize. A prerequisite for metastasis is the capability to invade the surrounding tissue and penetrate into the nearest blood- or lymphatic vessel. This initial step in metastasis requires several extracellular matrix degrading enzymes. One such enzyme is uPA (urokinase-type plasminogen activator), involved in pericellular proteolysis, where high levels are correlated with poor prognosis in several cancers. Upon binding of pro-uPA to its extracellular receptor uPAR, the enzyme is cleaved and activated. uPA further activates matrix metalloproteases (MMP's), also needed for matrix degradation. In addition, ample evidence shows that binding of uPA to uPAR triggers receptor-mediated cellular signaling. uPAR lacks a membrane spanning domain hence signal transduction has to be mediated through one or several membrane partners. uPARs role in the regulation of cell adhesion, migration and proliferation has been linked to its interplay with membrane partners such as integrins, EGFR, caveolin and G-protein coupled receptors (e.g. FPRL1). To better understand the role of uPAR in the invasive growth and metastasis of oral SCCs, the mouse oral SCC cell line, AT84 [1, 2], was stably transfected with uPAR. Overexpression of uPAR resulted in down regulation of epithelial marker E-cadherin and the cells display a more mesenchymal appearance, indicating epithelial-mesenchymal transition (EMT). In addition, elevation of uPAR expression increased migration in a wound healing assay. In contrast to previous reports, an increase in MMP-9 activity was not observed in these cells, although an increase in overall proteolytic activity could be found using gelatin as substrate. The cells were reintroduced into the C3H mouse to observe the role of uPAR in cancer progression in vivo. Results from the analysis of the host-tumor interactions within this syngenous mouse model will be presented. 1. Lou, E., et al., Clinical and pathological features of the murine AT-84 orthotopic model of oral cancer. Oral Dis, 2003. 9(6): p. 305-12. 2. Schultz-Hector, S. and S. Haghayegh, Beta-fibroblast growth factor expression in human and murine squamous cell carcinomas and its relationship to regional endothelial cell proliferation. Cancer Res, 1993. 53(6): p. 1444-9. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1504. doi:10.1158/1538-7445.AM2011-1504

Abstract 1046: Differential interactions between c-Src and c-Met mediate resistance to c-Src inhibitors

Abstract Cancers of the head and neck are difficult to treat because both the therapy and the tumor impact essential functions such as speech and swallowing and they can also significantly alter facial appearance. One promising molecular target for which new agents have been developed is the Src family kinases (SFKs). SFK inhibition in cancer cells leads to an universal abrogation of invasion but a variable and weak effects on apoptosis and proliferation. The pathways downstream of c-Src promoting survival are not well-characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal for head and neck cancers, we sought to characterize the mechanisms of resistance to SFK inhibition in Head and Neck squamous cell carcinoma (HNSCC). SFKs were inhibited in a panel of oral cancer cell lines and the effects on subsequent survival and signaling were measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and their intrinsic kinase activity by in vitro kinase assay. Cytotoxicity was measured using an MTT assay and the Chou-Talalay combination index calculated. The in vivo effects of c-Met and SFK inhibitors were assessed using an orthotopic model of oral cancer. SFK inhibition resulted in c-Met inhibition in cell lines that were sensitive to SFK inhibitors, but not in resistant cell lines. Isolated c-Met act as a c-Src substrate in both sensitive and resistant cells, whereas distinct difference exists in c-Src and c-Met interaction in intact sensitive and resistant cells. The epidermal growth factor receptor contributed to c-Met activation in resistant cells whereas in sensitive cells c-Met acts as a direct downstream target for c-Src. We demonstrated the biological consequences of this mechanism in vitro with synergistic cytotoxicity and enhanced apoptosis when SFK and c-Met inhibitors were combined. Likewise, the combination resulted in decreased tumor size in vivo. In conclusion, we demonstrate that sustained c-Met activation can mediate resistance to SFK inhibition. The differences between c-Src and c-Met signaling in sensitive and resistant cells are not due to intrinsic structural changes in c-Src or c-Met, but rather to distinct interactions within the intact cells. The synergistic cytotoxic effects of SFK and c-Met inhibition may be important for the treatment of head and neck cancers and should be tested in future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1046. doi:10.1158/1538-7445.AM2011-1046

Distinct Interactions Between c-Src and c-Met in Mediating Resistance to c-Src Inhibition in Head and Neck Cancer

c-Src inhibition in cancer cells leads to an abrogation of invasion but a variable effect on apoptosis. The pathways downstream of c-Src promoting survival are not well characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal, we sought to characterize the mechanisms of resistance to c-Src inhibition. c-Src was inhibited in a panel of oral cancer cell lines and subsequent survival and signaling measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and an in vitro kinase assay. Cytotoxicity was measured and the Chou-Talalay combination index calculated. An orthotopic model of oral cancer was used to assess the effects of c-Met and c-Src inhibitors. Inhibition of c-Src resulted in c-Met inhibition in sensitive cells lines, but not in resistant cell lines. Isolated c-Met was a c-Src substrate in both sensitive and resistant cells, but there was no interaction of c-Src and c-Met in intact resistant cells. To examine the biological consequences of this mechanism, we demonstrated synergistic cytotoxicity, enhanced apoptosis, and decreased tumor size with the combination of c-Src and c-Met inhibitors. Sustained c-Met activation can mediate resistance to c-Src inhibition. These data suggest that the differences between c-Met and c-Src signaling in sensitive and resistant cells are due to distinct factors promoting or inhibiting interactions, respectively, rather than to intrinsic structural changes in c-Src or c-Met. The synergistic cytotoxic effects of c-Src and c-Met inhibition may be important for the treatment of head and neck cancers.

Abstract 2343: Overexpression of uPAR in the murine oral squamous cell carcinoma cell line AT84

Abstract Cancers of the oral cavity are associated with bad prognosis and have been so for the last 20-30 years. Almost half of the patients diagnosed with oral squamous cell carcinoma (SCC) will die from the disease. When comparing oral SCC to its counterpart in skin, one of the main features that differ between the two is the ability of the oral SCC to metastasize. A prerequisite for metastasis is the capability to invade the surrounding tissue and penetrate into the nearest blood- or lymphatic vessel. This initial step in metastasis requires several extracellular matrix degrading enzymes. One such enzyme is uPA (urokinase-type plasminogen activator), involved in pericellular proteolysis, where high levels are known to be correlated to bad prognosis in several cancers. Pro-uPA binds to its extracellularly located receptor uPAR. Upon binding, pro-uPA is cleaved and activated, and ample evidence shows that binding of uPA triggers uPAR-mediated cellular signalling. Being that uPAR has no membrane spanning domain, the signal transduction has to be mediated through one or several membrane partners. uPARs role in the regulation of cell adhesion, migration and proliferation have been dedicated to its interplay with membrane partners such as integrins, chemokine receptors, EGFR, G-protein coupled receptors and caveolin. To better understand the role of uPAR in the metastasis of oral SCCs, a mouse oral SCC cell line, AT84, was stably transfected with uPAR. AT84 was initially obtained from a spontaneously originating oral SCC of the C3H mouse (Lou et al., 2003; Schultz-Hector and Haghayegh, 1993). Characterization of these cell lines with respect to adhesion, migration, invasion, and effects on gene expression and activation of other proteolytic enzymes will be presented. Lou, E., R.M. Kellman, R. Hutchison, and E.J. Shillitoe. 2003. Clinical and pathological features of the murine AT-84 orthotopic model of oral cancer. Oral Dis. 9:305-12. Schultz-Hector, S., and S. Haghayegh. 1993. Beta-fibroblast growth factor expression in human and murine squamous cell carcinomas and its relationship to regional endothelial cell proliferation. Cancer Res. 53:1444-9. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2343.

302. Herpes Simplex Virus Thymidine Kinase/Ganciclovir Therapy Mediated by Transferrin-Associated Lipoplexes in a Murine Model for Oral Cancer

Current treatments of oral cancer are largely unsatisfactory, and the five-year survival rate has not improved over the last two decades. In fact, despite recent advances, the present therapeutic approaches such as surgery, radiation and chemotherapy have significant limitations both in terms of efficacy and ability to prolong survival in patients with advanced cancer disease. Although viral vectors are generally efficient in transducing cells, the drawbacks associated with their use, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery. Cationic lipid-based systems (lipoplexes) have been extensively used, both in vitro and in vivo, despite their limited efficiency of delivery and gene expression. Our previous studies have shown that transferrin-associated lipoplexes composed of DOTAP:cholesterol constitute a promising tool for delivering Herpes Simplex Virus thymidine kinase (HSV-tk) gene to the squamous cell carcinoma cells, including human squamous cells (HSC-3) and murine squamous carcinoma cells (SCC VII). High cytotoxicity (95 %) was achieved for both types of cells upon treatment with 100 |[mu]|M ganciclovir (GCV) following transfection with 1 |[mu]|g pCMVtk. In this study, we evaluated the in vivo efficiency of the transferrin-associated lipoplexes to deliver a reporter gene in an orthotopic model of oral cancer established in C3H/HeJ immunocompetent mice (upon injection of 1 |[times]| 105 of SCC VII cells into the floor of the mouth). Our results show that DOTAP:cholesterol/transferrin-complexes, when prepared at 3/2 lipid/DNA (+/-) charge ratio (40 |[mu]|g pCMVlacZ), are able to efficiently transfect the tumor cells in vivo, as assessed by scoring the percentage of cells expressing |[beta]|-galactosidase, using both immunohistochemistry and X-gal staining. In addition, we will investigate the efficacy of this formulation to mediate tumor cell killing and tumor size reduction, upon delivery of the HSV-tk gene followed by GCV treatment. Since our in vitro studies have shown that SCC-VII cells are very sensitive to this therapy and significantly transfected in vivo, a regression of the tumor is expected to be achieved. Our results suggest that DOTAP:cholesterol/transferrin-complexes can be promising for the gene therapy of oral cancer in animal models.