Neck Squamous Cell Carcinoma Models Research Articles

Down-regulation of HLA-B-associated transcript 3 impairs the tumoricidal effect of natural killer cells through promoting the T cell immunoglobulin and mucin domain-containing-3 signaling in a mouse head and neck squamous cell carcinoma model

Head and neck squamous cell carcinoma (HNSCC) arises from the malignant mucosal epithelium of the oral cavity, pharynx, and larynx. Natural killer (NK) cells are fundamental immune cells shaping the anti-HNSCC response. Elucidation of the regulatory mechanisms of NK cell activity is crucial for understanding anti-HNSCC immunity. In this study, we characterized the expression and function of HLA-B-associated transcript 3 (Bat3) in NK cells in a mouse HNSCC model. We found that Bat3 expression was down-regulated in HNSCC-infiltrating NK cells. SCC VII, the mouse HNSCC cell line used in this model, induced Bat3 downregulation through direct cell-to-cell contact. By applying lentivirus-mediated silencing of Bat3, we discovered that Bat3 knockdown impaired the tumoricidal effect of NK cells on SCC VII cells and Hepa1-6RAE1, a genetically modified liver cancer cell line. Furthermore, Bat3 knockdown resulted in a significant decrease in perforin, granzyme B, interferon-γ, and tumor necrosis factor-α in NK cells upon co-culture with SCC VII cells. Further investigations revealed that Bat3 knockdown promoted the binding of T cell immunoglobulin and mucin domain-containing-3 (Tim-3) to Fyn and thus activated the Tim-3 signaling. Blockade of Tim-3 with a neutralizing Tim-3 antibody counteracted the effect of Bat3 knockdown on NK cell cytotoxicity. Taken together, our data suggest that HNSCC might down-regulate Bat3 expression to augment Tim-3 signaling and ultimately suppress the tumoricidal activity of NK cells. This study unveils a novel mechanism by which HNSCC evades NK cell killing, and sheds light on designing novel anti-HNSCC immunotherapy targeting Bat3 and Tim-3 signaling.

Explant Modeling of the Immune Environment of Head and Neck Cancer.

Patients exhibit distinct responses to immunotherapies that are thought to be linked to their tumor immune environment. However, wide variations in outcomes are also observed in patients with matched baseline tumor environments, indicating that the biological response to treatment is not currently predictable using a snapshot analysis. To investigate the relationship between the immune environment of tumors and the biological response to immunotherapies, we characterized four murine head and neck squamous cell carcinoma (HNSCC) models on two genetic backgrounds. Using tumor explants from those models, we identified correlations between the composition of infiltrating immune cells and baseline cytokine profiles prior to treatment. Following treatment with PD-1 blockade, CTLA-4 blockade, or OX40 stimulation, we observed inter-individual variability in the response to therapy between genetically identical animals bearing the same tumor. These distinct biological responses to treatment were not linked to the initial tumor immune environment, meaning that outcome would not be predictable from a baseline analysis of the tumor infiltrates. We similarly performed the explant assay on patient HNSCC tumors and found significant variability between the baseline environment of the tumors and their response to therapy. We propose that tumor explants provide a rapid biological assay to assess response to candidate immunotherapies that may allow matching therapies to individual patient tumors. Further development of explant approaches may allow screening and monitoring of treatment responses in HNSCC.

Abstract PO-094: Glutaminase inhibition increases ionizing radiation induced oxidative stress and DNA damage in head and neck squamous cell carcinoma models

Abstract Ionizing radiation (IR) is a pillar of head and neck cancer treatment, the 6th most common cancer worldwide. However, its efficacy is limited by radioresistance, resulting in locoregional failure rates of ~50%. The mechanisms that tumor cells use to evade IR-induced cellular damage are poorly understood. Our initial proteomics analysis indicated that IR induced metabolic pathways, including those involving the Krebs cycle. Glutamate is a key substrate of the Krebs cycle. Glutaminase is the enzyme that converts glutamine to glutamate. Analysis of The Cancer Genome Atlas’s transcriptome database revealed that glutaminase overexpression (upper 25%) in patients with HNSCC was associated with reduced patient survival (p≤0.03). In this study, we therefore aimed to identify whether glutaminase inhibition with CB-839 (currently in Phase 1/2 trials) could enhance cellular response to IR. To do this, we used three representative human-derived HNSCC cell lines: CAL-27, FaDu, and HN5. First, we established that proliferation of all 3 HNSCC cell lines was glutamine dependent (20-50% increase, n=3 per line, p≤0.01). Second, a synergistic decrease in cell survival was observed in clonogenic assays with HNSCC cell lines treated with IR and CB-839 relative to either treatment alone (n=3 per line, p≤0.05). Thirdly, in 3D cell culture, which better represents the host microenvironment, combinatorial treatment significantly reduced HNSCC spheroid size (11-26% decrease, n=3 per line, p≤0.0001). The functional effect of CB-839 on cellular metabolism was examined using Seahorse MitoStress test assays. CB-839 significantly reduced spare respiratory capacity and OCR/ECAR ratio in HNSCC cell lines examined (p≤0.001, p≤2.0 × 10−6, n>3). To understand the mechanism behind this reduced cell growth and survival, we examined whether CB-839 enhanced oxidative stress and DNA damage when combined with IR using immunofluorescence. Oxidative stress marker, 8-oxoguanine, was ~2 fold higher with combined treatment as compared to CB-839 or IR alone in CAL-27 cells (p≤0.01, p≤0.05; respectively). Additionally, increased levels of y-H2AX were observed in CAL-27 cells treated with both CB-839 and IR relative to independent treatment. Combination of CB-839 with IR significantly inhibited tumor growth in CAL-27 xenograft mice relative to vehicle (p≤0.01), CB-839 (p≤0.05) or IR (p≤0.01) (p values, day 19). To summarize, successful treatment of HNSCC is impeded by adaptive resistance to IR and locoregional failure. Overall, these data suggest that the rational combination of IR and glutaminase inhibitor (CB-839) enhances cellular response and tumor control relative to independent treatment and provides pre-clinical data in support of additional investigation in patients with HNSCC. Citation Format: Christina A. Wicker, Brian G. Hunt, Sarah Palackdharry, William R. Elaban, Trisha M. Wise-Draper, Gordon B. Mills, Susan E. Waltz, Vinita Takiar. Glutaminase inhibition increases ionizing radiation induced oxidative stress and DNA damage in head and neck squamous cell carcinoma models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-094.

Sample-size calculation for preclinical dose–response experiments using heterogeneous tumour models

BackgroundIn preclinical radio-oncological research, local tumour control is considered the most relevant endpoint as it reflects the inactivation of cancer stem cells. Preclinical tumour-control assays may compare dose–response curves between different radiotherapy strategies, e.g., assessing additional targeted drugs and immunotherapeutic interventions, or between different radiation modalities. To mimic the biological heterogeneity of human tumour populations and to accommodate for approaches of personalized oncology, preclinical studies are increasingly performed combining larger panels of tumour models. For designing the study protocols and to obtain reliable results, prospective sample-size planning has to be developed that accounts for such heterogeneous cohorts. MethodsA Monte-Carlo-based method was developed to estimate the sample size of a comparative 1:1 two-arm prospective tumour-control assay. Based on repeated logistic regression analysis, pre-defined dose levels, assumptions on the dose–response curves of the included tumour models and on the dose-modifying factors (DMF), the power is calculated for a given number of animals per dose group. ResultsTwo applications are presented: (i) For a simple tumour-control assay with the head and neck squamous cell carcinoma (HNSCC) model FaDu, 10 animals would be required for each of 7 dose levels per arm to reveal a DMF of 1.25 with a power of 0.82 without drop out (total: 140 animals). (ii) In a more complex experiment combining six different lung tumour models to a heterogeneous population, 21 animals would be required for each of 11 dose levels per arm to reveal a DMF of 1.25 with a power of 0.81 without drop out (total: 462 animals). Analyzing the heterogeneous cohort reduces the required animal number by more than 50% compared to six individual tumour-control assays. ConclusionAn approach for estimating the required animal number for comparative tumour-control assays in a heterogeneous population is presented, allowing also the inclusion of different treatments as a personalized approach in the experimental arm. The software is publicly available and can be applied to plan comparisons of sigmoidal dose–response curves based on logistic regression.

Glutaminase inhibition with telaglenastat (CB-839) improves treatment response in combination with ionizing radiation in head and neck squamous cell carcinoma models.

The efficacy of ionizing radiation (IR) for head and neck cancer squamous cell carcinoma (HNSCC) is limited by poorly understood mechanisms of adaptive radioresistance. Elevated glutaminase gene expression is linked to significantly reduced survival (p<0.03). The glutaminase inhibitor, telaglenastat (CB-839), has been tested in Phase I/II cancer trials and is well tolerated by patients. This study investigated if telaglenastat enhances the cellular response to IR in HNSCC models. Using three human HNSCC cell lines and two xenograft mouse models, we examined telaglenastat's effects on radiation sensitivity. IR and telaglenastat combinatorial treatment reduced cell survival (p≤0.05), spheroid size (p≤0.0001) and tumor growth in CAL-27 xenograft bearing mice relative to vehicle (p≤0.01), telaglenastat (p≤0.05) or IR (p≤0.01) monotherapy. Telaglenastat significantly reduced the Oxygen Consumption Rate/Extracellular Acidification Rate ratio in CAL-27 and HN5 cells in the presence of glucose and glutamine (p≤0.0001). Telaglenastat increased oxidative stress and DNA damage in irradiated CAL-27cells. These data suggest that combination treatment with IR and telaglenastat leads to an enhanced anti-tumor response. This pre-clinical data, combined with the established safety of telaglenastat justifies further investigation for the combination in HNSCC patients.

Combined EGFR1 and PARP1 Inhibition Enhances the Effect of Radiation in Head and Neck Squamous Cell Carcinoma Models

Head and neck squamous cell carcinoma (HNSCC) is a challenging cancer with little change in five-year overall survival rate of 50-60% over the last two decades. Radiation with or without platinum-based drugs remains the standard of care despite limited benefit and high toxicity. HNSCCs often overexpress epidermal growth factor receptor (EGFR) and inhibition of EGFR signaling enhances radiation sensitivity by interfering with repair of radiation-induced DNA breaks. Poly (adenosine diphosphate-ribose) polymerase-1 (PARP1) also participates in DNA damage repair, but its inhibition provides benefit in cancers that lack DNA repair by homologous recombination (HR) such as BRCA-mutant breast cancer. HNSCCs in contrast are typically BRCA wild-type and proficient in HR repair, making it challenging to apply anti-PARP1 therapy in this model. A recently published study showed that a combination of EGFR and PARP1 inhibition induced more DNA damage and greater growth control than each single agent in HNSCC cells. This led us to hypothesize that a combination of EGFR and PARP1 inhibition would enhance the efficacy of radiation to a greater extent than each single agent, providing a rationale for paradigm-shifting combinatorial approaches to improve the standard of care in HNSCC. Here, we report a proof-of-concept study using Detroit562 HNSCC cells, which are proficient for DNA repair by both HR and non-homologous end joining (NHEJ) mechanisms. We tested the effect of adding cetuximab and/or olaparib (inhibitors of EGFR and PARP1, respectively) to radiation and compared it to that of cisplatin and radiation combination, which is the standard of care. Our results demonstrate that the combination of cetuximab and olaparib with radiation was superior to the combination of any single drug with radiation in terms of induction of unrepaired DNA damage, induction of senescence, apoptosis and clonogenic death, and tumor growth control in mouse xenografts. Combined with our recently published phase I safety data on cetuximab/olaparib/radiation triple combination, the data reported here demonstrate a potential for combining biologically-based therapies that might optimize radiosensitization in HNSCC.

Valproic Acid Synergizes With Cisplatin and Cetuximab in vitro and in vivo in Head and Neck Cancer by Targeting the Mechanisms of Resistance.

Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. The combination of cisplatin (CDDP) plus cetuximab (CX) is one of the standard first-line treatments in this disease. However, this therapeutic regimen is often associated with high toxicity and resistance, suggesting that new combinatorial strategies are needed to improve its therapeutic index. In our study, we evaluated the antitumor effects of valproic acid (VPA), a well-known antiepileptic agent with histone deacetylase inhibitory activity, in combination with CDDP/CX doublet in head and neck squamous cell carcinoma (HNSCC) models. We demonstrated, in HNSCC cell lines, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX resulted in a clear synergistic antiproliferative and pro-apoptotic effects. The synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, suggesting the ability of the combined approach to affect also the cancer stem cells compartment. Mechanistically, VPA enhanced DNA damage in combination treatment by reducing the mRNA expression of ERCC Excision Repair 1, a critical player in DNA repair, and by increasing CDDP intracellular concentration via upregulation at transcriptional level of CDDP influx channel copper transporter 1 and downregulation of the ATPAse ATP7B involved in CDDP-export. Valproic acid also induced a dose-dependent downregulation of epidermal growth factor receptor (EGFR) expression and of MAPK and AKT downstream signaling pathways and prevent CDDP- and/or CX-induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of genes induced by non-canonical activity of nuclear EGFR, such as cyclin D1 and thymidylate synthase. Finally, we confirmed the synergistic antitumor effect also in vivo in both heterotopic and orthotopic models, demonstrating that the combined treatment completely blocked HNSCC xenograft tumors growth in nude mice. Overall, the introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. A phase II clinical trial exploring the combination of VPA and CDDP/CX in R/M HNSCC patients is currently ongoing in our institute.

Abstract 6278: Glutaminase inhibition in combination with ionizing radiation improves treatment response in head and neck squamous cell carcinoma models

Abstract Head and neck cancer is the 6th most common cancer worldwide. Ionizing radiation (IR) is an important aspect of cancer treatment. However, its efficacy is limited by radioresistance, resulting in locoregional failure rates as high as 50%. The pathways used by cells to evade the cellular damage caused by IR are poorly understood. Initial Reverse Phase Protein Array analyses in our laboratory demonstrated that IR induced metabolic pathways, including those involving the Krebs cycle. Glutamate is a key substrate of the Krebs cycle, and essential for the production of biomolecules necessary for tumor proliferation. Glutaminase is the enzyme that converts glutamine to glutamate. Analysis of The Cancer Genome Atlas's transcriptome database revealed that glutaminase overexpression (upper 25%) in patients with HNSCC was associated with significantly reduced patient survival (p&amp;lt;0.03). In this study, we aimed to identify whether glutaminase inhibition with CB-839 could enhance cellular response to IR. To do this, we used three representative human-derived HNSCC cell lines: CAL-27, FaDu, and HN5. We first established that proliferation of all 3 HNSCC cell lines was glutamine dependent (20-50% increase, n=3 per line, p&amp;lt;0.01). Moreover, a synergistic decrease in cell survival was observed in clonogenic assays with HNSCC cell lines treated with IR and CB-839 relative to either treatment alone (n=3 per line, p&amp;lt;0.05). In 3D cell culture, which better represents the host microenvironment, combinatorial treatment significantly reduced HNSCC spheroid size (11-26% decrease, n=3 per line, p&amp;lt;0.0001). To understand the mechanism behind this reduced cell growth and survival, we examined CB-839's effect on cellular metabolism using Seahorse MitoStress test assays. CB-839 significantly reduced spare respiratory capacity and OCR/ECAR ratio in HNSCC cell lines examined (p&amp;lt;0.001, p&amp;lt;2.0 × 10−6, n&amp;gt;3). Combination of CB-839 with IR significantly inhibited tumor growth in Cal-27 xenograft mice relative to vehicle (p&amp;lt;0.01), CB-839 (p&amp;lt;0.05) or IR (p&amp;lt;0.01) (p values representative of day 20). In summary, adaptive resistance to IR and locoregional failure is a major hurdle in the successful treatment of HNSCC. Overall, these data suggest that combinatorial treatment with IR and the glutaminase inhibitor, CB-839, lead to an enhanced response over either treatment alone and provide pre-clinical data to justify further investigation for clinical use in patients with HNSCC. Citation Format: Christina A. Wicker, Brian G. Hunt, Sarah Palackdharry, William R. Elaban, Gordon B. Mills, Trisha Wise-Draper, Susan Waltz, Vinita Takiar. Glutaminase inhibition in combination with ionizing radiation improves treatment response in head and neck squamous cell carcinoma models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6278.

CD244 represents a new therapeutic target in head and neck squamous cell carcinoma

BackgroundDeveloping novel strategies to overcome the immunosuppressive tumor microenvironment is a critically important area of cancer therapy research. Here, we assess the therapeutic potential of CD244 (2B4/signaling lymphocyte activation molecule...

E6 Oncoproteins from High-Risk Human Papillomavirus Induce Mitochondrial Metabolism in a Head and Neck Squamous Cell Carcinoma Model

Head and neck squamous cell carcinoma (HNSCC) cells that are positive for human papillomavirus (HPV+) favor mitochondrial metabolism rather than glucose metabolism. However, the involvement of mitochondrial metabolism in HNSCC HPV+ cells is still unknown. The aim of this work was to evaluate the role of E6 oncoproteins from HPV16 and HPV18 in the mitochondrial metabolism in an HNSCC model. We found that E6 from both viral types abates the phosphorylation of protein kinase B-serine 473 (pAkt), which is associated with a shift in mitochondrial metabolism. E6 oncoproteins increased the levels of protein subunits of mitochondrial complexes (I to IV), as well as the ATP synthase and the protein levels of the voltage dependent anion channel (VDAC). Although E6 proteins increased the basal and leak respiration, the ATP-linked respiration was not affected, which resulted in mitochondrial decoupling. This increase in leak respiration was associated to the induction of oxidative stress (OS) in cells expressing E6, as it was observed by the fall in the glutathione/glutathione disulfide (GSH/GSSG) rate and the increase in reactive oxygen species (ROS), carbonylated proteins, and DNA damage. Taken together, our results suggest that E6 oncoproteins from HPV16 and HPV18 are inducers of mitochondrial metabolism.

Inhibition of EphB4-Ephrin-B2 Signaling Reprograms the Tumor Immune Microenvironment in Head and Neck Cancers.

Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study, we identified EphB4-ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell-cell contact, resulting in bidirectional signaling. We found that EphB4-ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8+ and CD4+Foxp3- T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4-ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression, particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4-ephrin-B2 inhibition with RT. In vitro, EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall, our study is the first to implicate the role of EphB4-ephrin-B2 in tumor immune response. Moreover, our findings suggest that EphB4-ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy. SIGNIFICANCE: These findings present EphB4-ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC.

C6 ceramide motivates the anticancer sensibility induced by PKC412 inpreclinical head and neck squamous cell carcinoma models.

The purpose of this study was to evaluate the anti-head and neck squamous cell carcinoma (anti-HNSCC) cell activity by C6 ceramide and multikinase inhibitor PKC412. Experiments were performed on HNSCC cell lines (SQ20B and SCC-9) and primary human oral carcinoma cells. Results showed that PKC412 inhibited HNSCC cell proliferation without provoking apoptosis activation. Cotreatment of C6 ceramide significantly augmented PKC412-induced lethality in HNSCC cells. PKC412 decreased Akt-mammalian target of rapamycin (mTOR) activation in HNSCC cells, facilitated with cotreatment of C6 ceramide. In contrast, exogenous expression of a constitutively active Aktrestored Akt-mTOR activation and attenuated lethality by the cotreatment. We propose that Mcl-1 is a primary resistance factor of PKC412. The cytotoxicity of PKC412 in HNSCC cells was potentiated with Mcl-1 short hairpin RNA knockdown, but was attenuated with Mcl-1 overexpression. Intriguingly, C6 ceramide downregulated Mcl-1 in HNSCC cells. In vivo, PKC412 oral administration inhibited SQ20B xenograft tumor growth in severe combined immunodeficient mice. The antitumor activity of PKC412 was further sensitized with coadministration of liposomal C6 ceramide. Together, we suggest that PKC412 could be further studied as a promising anti-HNSCC strategy, alone or in combination with C6 ceramide.

Optimising intratumoral treatment of head and neck squamous cell carcinoma models with the diterpene ester Tigilanol tiglate

SummaryThe five-year survival rate for patients with head and neck squamous cell carcinoma (HNSCC) has remained at ~50% for the past 30 years despite advances in treatment. Tigilanol tiglate (TT, also known as EBC-46) is a novel diterpene ester that induces cell death in HNSCC in vitro and in mouse models, and has recently completed Phase I human clinical trials. The aim of this study was to optimise efficacy of TT treatment by altering different administration parameters. The tongue SCC cell line (SCC-15) was identified as the line with the lowest efficacy to treatment. Subcutaneous xenografts of SCC-15 cells were grown in BALB/c Foxn1nu and NOD/SCID mice and treated with intratumoral injection of 30 μg TT or a vehicle only control (40% propylene glycol (PG)). Greater efficacy of TT treatment was found in the BALB/c Foxn1nu mice compared to NOD/SCID mice. Immunohistochemical analysis indicated a potential role of the host’s innate immune system in this difference, specifically neutrophil infiltration. Neither fractionated doses of TT nor the use of a different excipiant led to significantly increased efficacy. This study confirmed that TT in 40% PG given intratumorally as a single bolus dose was the most efficacious treatment for a tongue SCC mouse model.

Metformin Effects on Metabolic Coupling and Tumor Growth in Oral Cavity Squamous Cell Carcinoma Coinjection Xenografts.

Objective Many aggressive head and neck cancers contain 2 metabolically coupled tumor compartments: a glycolytic stromal compartment with low caveolin-1 (CAV1) and high monocarboxylate transporter 4 (MCT4) expression and a highly proliferative carcinoma cell compartment with high MCT1. Metabolites are shuttled by MCTs from stroma to carcinoma to fuel tumor growth. We studied the effect of carcinoma-fibroblast coinjection and metformin administration on a mouse model of head and neck squamous cell carcinoma. Study Design Xenograft head and neck squamous cell carcinoma model. Setting Basic science laboratory. Subjects and Methods Oral cavity carcinoma cells were injected alone or as coinjection with human fibroblasts into nude mice to generate xenograft tumors. Tumors were excised and stained with immunohistochemistry for markers of metabolic coupling and apoptosis, including MCT1, MCT4, CAV1, and TUNEL assay (terminal deoxynucleotidyl transferase nick end labeling). Strength of staining was assessed by a pathologist or computer-assisted pathology software. Metformin was administered orally to mice to test effects on immunohistochemical markers in xenografts. Results Coinjection tumors were 2.8-fold larger ( P = .048) and had 1.4-fold stronger MCT1 staining ( P = .016) than tumors from homotypic carcinoma cell injection. Metformin decreased the size of coinjection xenograft tumors by 45% ( P = .025). Metformin reduced MCT1 staining by 28% ( P = .05) and increased carcinoma cell apoptosis 1.8-fold as marked by TUNEL assay ( P = .005). Metformin did not have a significant effect on tumor size when CAV1 knockdown fibroblasts were used in coinjection. Conclusion Coinjection with fibroblasts increases tumor growth and metabolic coupling in oral cavity cancer xenografts. Fibroblast CAV1 expression is required for metformin to disrupt metabolic coupling and decrease xenograft size.

Galectin-1 Inhibitor OTX008 Induces Tumor Vessel Normalization and Tumor Growth Inhibition in Human Head and Neck Squamous Cell Carcinoma Models.

Galectin-1 is a hypoxia-regulated protein and a prognostic marker in head and neck squamous cell carcinomas (HNSCC). Here we assessed the ability of non-peptidic galectin-1 inhibitor OTX008 to improve tumor oxygenation levels via tumor vessel normalization as well as tumor growth inhibition in two human HNSCC tumor models, the human laryngeal squamous carcinoma SQ20B and the human epithelial type 2 HEp-2. Tumor-bearing mice were treated with OTX008, Anginex, or Avastin and oxygen levels were determined by fiber-optics and molecular marker pimonidazole binding. Immuno-fluorescence was used to determine vessel normalization status. Continued OTX008 treatment caused a transient reoxygenation in SQ20B tumors peaking on day 14, while a steady increase in tumor oxygenation was observed over 21 days in the HEp-2 model. A >50% decrease in immunohistochemical staining for tumor hypoxia verified the oxygenation data measured using a partial pressure of oxygen (pO2) probe. Additionally, OTX008 induced tumor vessel normalization as tumor pericyte coverage increased by approximately 40% without inducing any toxicity. Moreover, OTX008 inhibited tumor growth as effectively as Anginex and Avastin, except in the HEp-2 model where Avastin was found to suspend tumor growth. Galectin-1 inhibitor OTX008 transiently increased overall tumor oxygenation via vessel normalization to various degrees in both HNSCC models. These findings suggest that targeting galectin-1—e.g., by OTX008—may be an effective approach to treat cancer patients as stand-alone therapy or in combination with other standards of care.

Immuno-PET imaging based radioimmunotherapy in head and neck squamous cell carcinoma model.

The epidermal growth factor receptor (EGFR) is one of the most comprehensively studied molecular targets in head and neck squamous cell carcinoma (HNSCC). However, inherent and acquired resistance are serious problems and are responsible for limited clinical efficacy and tumor recurrence. In this study, we evaluated the feasibility of immuno-positron emission tomography (PET) imaging and radioimmunotherapy (RIT) with 64Cu-/177Lu-PCTA-cetuximab in cetuximab-resistant SNU-1066 HNSCC xenografted model. The cellular uptake of 64Cu/177Lu-3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid (PCTA)-cetuximab showed good correlation with western blot and flow cytometry analysis in EGFR expression level of various HNSCC cells. 177Lu-PCTA-cetuximab selectively killed cetuximab-resistant SNU-1066 cells in vitro. 64Cu-/177Lu-PCTA-cetuximab specifically accumulated in SNU-1066 tumor and those uptakes were peaked at 48 h and 7 day, respectively in biodistribution, PET and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging. RIT with single dose of 177Lu-PCTA-cetuximab exhibited significant tumor regression and markedly reduced 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) uptake, compared to other groups. Proliferation index were dramatically decreased and apoptotic index increased in RIT group. These results suggest that a diagnostic and therapeutic convergence radiopharmaceutical, 64Cu-/177Lu-PCTA-cetuximab has the potential of target selection using immuno-PET imaging and targeted therapy by RIT in EGFR expressing cetuximab-resistant HNSCC tumors.

Tumor heterogeneity determined with a γH2AX foci assay: A study in human head and neck squamous cell carcinoma (hHNSCC) models

PurposeThis study aimed to analyze the intra-tumoral heterogeneity of γH2AX foci in tumor specimens following ex vivo radiation to evaluate the potential of γH2AX foci as predictors for radiosensitivity. Material and methodsγH2AX foci were quantified in tumor specimens of 3hHNSCC tumor models with known differences in radiosensitivity after reoxygenation in culture medium (10h, 24h), single dose exposure (0Gy, 4Gy), and fixation 24h post-irradiation. Multiple, equally treated samples of the same tumor were analyzed for foci, normalized and fitted in a linear mixed-effects model. ResultsThe ex vivo reoxygenation time had no significant effect on γH2AX foci counts. A significant intra model heterogeneity could be shown for FaDu (p=0.033) but not for SKX (p=0.167) and UT-SCC-5 (p=0.082) tumors, respectively. All tumor models showed a significant intra-tumoral heterogeneity between specimens of the same tumor (p<0.01) or among microscopic fields of a particular tumor specimen (p<0.0001). ConclusionSimilar results for ex vivo γH2AX foci between 10h and 24h reoxygenation time support the applicability of the assay in a clinical setting. The high intra-tumoral heterogeneity underlines the necessity of multiple analyzable samples per patient and therewith the need for an automated foci analysis.

Abstract 1065: MMP sensitive liposomes followed by radiotherapy improves tumor control in head and neck cancer mice models

Abstract Many cancers at advanced stages require a combination of chemotherapy and radiotherapy. A big challenge with this approach is accumulated toxicity because both of these treatment modalities are non-selective and inevitably damage non-target tissues. Nanoparticles such as liposomes provide the ability to differentiate between healthy and cancerous tissues and thus offer efficient delivery of chemotherapy to cancerous tissues. Recently, we developed a matrix metalloproteinase (MMP)-sensitive liposomal platform to carry anticancer agents to the tumor. High local expression of MMPs within the tumor microenvironment ensures bioavailability of the carried agent by converting a polyethylene glycol (PEG)ylated anionic liposome into a dePEGylated cationic liposome that is readily internalized by cells. Using oxaliplatin-loaded MMP-sensitive liposomes, we previously demonstrated great in-vivo efficacy in mice bearing CT26 tumors. In the present study, we assessed the combinatorial effect of MMP-sensitive liposomes and radiotherapy in head and neck squamous cell carcinoma models. Using a clonogenic cell survival assay, we evaluated MMP-sensitive liposomes combined with radiotherapy. A slightly sensitizing effect was produced by oxaliplatin-loaded MMP-sensitive liposomes while free oxaliplatin produced an additive effect. Similarly, levels of platinum intercalated into DNA were three times higher in cells treated with oxaliplatin-loaded MMP-sensitive liposomes than in those treated with free oxaliplatin. The in-vivo efficacy of oxaliplatin-loaded MMP-sensitive liposomes combined with radiotherapy was studied in the same tumor models, FaDu and HN5, where zymogram gels confirmed an MMP-positive tumor microenvironment. MMP-sensitive liposomes and radiation had a much greater anti-tumor effect than any of the monotherapies or free oxaliplatin combined with radiation. In FaDu, the mean tumor size between liposomes + radiation and liposomes only were significantly different (p&amp;lt;0.05). Thus, our preliminary results suggest that MMP-sensitive liposomes in combination with radiation hold a great potential for clinical translation. The MMP-sensitive liposome offers a valuable tool to deliver the payload in a controlled fashion into the tumor microenvironment, where radiation can potentially amplify the therapeutic index. Note: This abstract was not presented at the meeting. Citation Format: Rikke Y. Brogaard, Allison Khoo, Rasmus Eliasen, Fredrik Melander, Awalpreet Chadha, Kathryn Aziz, Sunil Krishnan, Thomas L. Andresen. MMP sensitive liposomes followed by radiotherapy improves tumor control in head and neck cancer mice 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 1065. doi:10.1158/1538-7445.AM2017-1065

Abstract 2644: Antibody-drug conjugates (ADCs) with a novel DNA-alkylating agent, DGN462, are highly potent in vitro and in vivo against human cancer models

Abstract Most ADCs in the clinic utilize a tubulin-binding small molecule as the cytotoxic payload. Although many cancers are sensitive to tubulin agents, some are more responsive to DNA-interacting agents. We have developed a new class of highly potent cytotoxic agents, IGNs, for use in ADCs that consists of indolino-benzodiazepine dimers with a novel DNA-alkylating mechanism of action. We found that IGNs containing a di-imine moiety act via DNA-crosslinking and -alkylating, whereas IGNs with a mono-imine induce DNA alkylation only. Although ADCs with either form of IGN were highly active, ADCs containing di-imine IGNs caused delayed toxicity in mice, leading to mortality. The mono-imine IGN, DGN462, was selected as our lead IGN as, when conjugated to an antibody via a cleavable linkage, the resultant ADC had favorable tolerability in mice (maximally tolerated dose of 40 mg/kg; 700 µg/kg DGN462 dose) without delayed toxicity. The ability of DGN462 to alkylate DNA was demonstrated by measurement of DNA adduct formation with double-stranded DNA and in cells. DGN462 formed covalent DNA adducts through alkylation of the C2- amino group of guanine. Consistent with the activity of other DNA alkylating agents, DGN462-treated cells progressed slowly through S-phase and were arrested in G2/M phase of the cell cycle in a dose dependent manner. ADCs consisting of ∼3 molecules of DGN462 per antibody, attached via a cleavable linker, had desirable biochemical characteristics, including high monomer content (&amp;gt; 97%), low free drug (&amp;lt; 0.1%) and symmetrical mass distribution profiles. These DGN462 ADCs were highly potent and antigen-specific in vitro against various cell types, including PgP-expressing cells, with IC50 values ranging from 2 pM to 3 nM depending on the target, cell type and antigen density. DGN462 ADCs display bystander activity in vitro, indicating that intracellular processing of conjugate by target cancer cells releases metabolites that can kill antigen-negative cells in direct proximity, which may provide an advantage in solid tumors expressing antigen heterogeneously. A CD33-targeting DGN462 ADC was highly active against AML xenografts, with a minimal efficacious dose (MED) of 0.6 mg/kg (conjugate dose), while a non-targeting control was inactive. A DGN462 ADC targeting epidermal growth factor receptor was highly active and antigen-specific against a head and neck squamous cell carcinoma model, with a 1.6 mg/kg MED. The intact conjugate half-life was approximately 90 hours in mice, and bioactivity of conjugate was maintained at 72 hours post dosing, indicating that intact conjugate remains active in vivo. DGN462 ADCs have an acceptable safety profile and potent antigen-specific DNA-alkylating mechanism of action with the potential for activity in tumors with low sensitivity to tubulin agents, heterogeneous antigen expression and/or PgP-mediated drug resistance. Citation Format: Kathleen Whiteman, Charlene Audette, Andre Dandeneau, Megan Ellis, Nathan Fishkin, Lauren Harvey, Holly Johnson, Yelena Kovtun, Erin Maloney, Michael Miller, Alan Wilhelm, Ravi Chari. Antibody-drug conjugates (ADCs) with a novel DNA-alkylating agent, DGN462, are highly potent in vitro and in vivo against human cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2644. doi:10.1158/1538-7445.AM2014-2644

Carfilzomib and oprozomib synergize with histone deacetylase inhibitors in head and neck squamous cell carcinoma models of acquired resistance to proteasome inhibitors

Acquired resistance to proteasome inhibitors represents a considerable impediment to their effective clinical application. Carfilzomib and its orally bioavailable structural analog oprozomib are second-generation, highly-selective, proteasome inhibitors. However, the mechanisms of acquired resistance to carfilzomib and oprozomib are incompletely understood, and effective strategies for overcoming this resistance are needed. Here, we developed models of acquired resistance to carfilzomib in two head and neck squamous cell carcinoma cell lines, UMSCC-1 and Cal33, through gradual exposure to increasing drug concentrations. The resistant lines R-UMSCC-1 and R-Cal33 demonstrated 205- and 64-fold resistance, respectively, relative to the parental lines. Similarly, a high level of cross-resistance to oprozomib, as well as paclitaxel, was observed, whereas only moderate resistance to bortezomib (8- to 29-fold), and low level resistance to cisplatin (1.5- to 5-fold) was seen. Synergistic induction of apoptosis signaling and cell death, and inhibition of colony formation followed co-treatment of acquired resistance models with carfilzomib and the histone deacetylase inhibitor (HDACi) vorinostat. Synergism was also seen with other combinations, including oprozomib plus vorinostat, or carfilzomib plus the HDACi entinostat. Synergism was accompanied by upregulation of proapoptotic Bik, and suppression of Bik attenuated the synergy. The acquired resistance models also exhibited elevated levels of MDR-1/P-gp. Inhibition of MDR-1/P-gp with reversin 121 partially overcame carfilzomib resistance in R-UMSCC-1 and R-Cal33 cells. Collectively, these studies indicate that combining carfilzomib or oprozomib with HDAC or MDR-1/P-gp inhibitors may be a useful strategy for overcoming acquired resistance to these proteasome inhibitors.