Head And Neck Squamous Cell Carcinoma Mouse Model Research Articles

Therapeutic Targeting of the GLS1-c-Myc Positive Feedback Loop Suppresses Glutaminolysis and Inhibits Progression of Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is addicted to glutaminolysis. Targeting this metabolic dependency has emerged as a potential therapeutic approach for HNSCC. Here, we conducted a bioinformatic analysis of the TCGA HNSCC cohort that revealed a robust correlation between expression of c-Myc and GLS1, which catalyzes the first step in glutaminolysis. Intriguingly, disruption of GLS1 signaling in HNSCC cells by genetic depletion or CB-839 treatment resulted in a reduction in c-Myc protein stability via a USP1-dependent ubiquitin-proteasome pathway. On the other hand, c-Myc directly binds to the promoter region of GLS1 and upregulates its transcription. Notably, the GLS1-c-Myc pathway enhanced ACC-dependent SLUG acetylation, prompting cancer cell invasion and metastasis. Thus, the GLS1-c-Myc axis emerged as a positive feedback loop critical for driving the aggressiveness of HNSCC. Therapeutically, combining CB-839 with the c-Myc inhibitor MYCi975 strongly suppressed GLS1-c-Myc signaling, resulting in a superior antitumor effect compared to either single agent in an orthotopic mouse model of HNSCC. These findings hold promise for the development of effective therapies for HNSCC patients, addressing an urgent need arising from the significant incidence and high metastatic rate of the disease.

Lauric acid-based thermosensitive delivery system for the treatment of head and neck squamous cell carcinoma

Traditional treatments for head and neck squamous cell carcinoma (HNSCC) such as surgery, radiation therapy, and chemotherapy, often have severe side effects. Local delivery of chemotherapeutic agents can be a promising approach to minimise systemic toxicity and improve efficacy. Lauric acid (LA), was explored as a novel injectable thermosensitive drug reservoir as a depot for sustained release of anticancer drugs to treat HNSCC. LA was characterised in terms of melting temperature and gelation time. The efficacy of LA-based drug formulations was tested in vitro in a HNSCC cell line and in vivo in a mouse model of HNSCC. LA was modified to have a melting point of 38.5 °C and a gelation time of 40 s at 37.5 °C, rendering it suitable for injection at body temperature. LA- based doxorubicin (DOXO) formulation showed slow release with a maximum of 18% release after 3 days. The in vitro study showed that LA enhanced the cytotoxic effect of DOXO. LA combined with DOXO prevented tumour progression and LA alone significantly reduced the original tumour volume compared to the untreated control group. These findings confirmed that LA can function as practical carrier for the local delivery of chemotherapeutics and provides a safe and simple strategy for the delivery of hydrophobic anticancer drugs and warrant further testing in clinical trials.

Bifunctional Tumor-Targeted Bioprobe for Phothotheranosis.

Background: Near-infrared (NIR) phototheranostics provide promising noninvasive imaging and treatment for head and neck squamous cell carcinoma (HNSCC), capitalizing on its adjacency to skin or mucosal surfaces. Activated by laser irradiation, targeted NIR fluorophores can selectively eradicate cancer cells, harnessing the power of synergistic photodynamic therapy and photothermal therapy. However, there is a paucity of NIR bioprobes showing tumor-specific targeting and effective phototheranosis without hurting surrounding healthy tissues. Methods: We engineered a tumor-specific bifunctional NIR bioprobe designed to precisely target HNSCC and induce phototheranosis using bioconjugation of a cyclic arginine-glycine-aspartic acid (cRGD) motif and zwitterionic polymethine NIR fluorophore. The cytotoxic effects of cRGD-ZW800-PEG were measured by assessing heat and reactive oxygen species (ROS) generation upon an 808-nm laser irradiation. We then determined the invivo efficacy of cRGD-ZW800-PEG in the FaDu xenograft mouse model of HNSCC, as well as its biodistribution and clearance, using a customized portable NIR imaging system. Results: Real-time NIR imaging revealed that intravenously administered cRGD-ZW800-PEG targeted tumors rapidly within 4 h postintravenous injection in tumor-bearing mice. Upon laser irradiation, cRGD-ZW800-PEG produced ROS and heat simultaneously and exhibited synergistic photothermal and photodynamic effects on the tumoral tissue without affecting the neighboring healthy tissues. Importantly, all unbound bioprobes were cleared through renal excretion. Conclusions: By harnessing phototheranosis in combination with tailored tumor selectivity, our targeted bioprobe ushers in a promising paradigm in cancer treatment. It promises safer and more efficacious therapeutic avenues against cancer, marking a substantial advancement in the field.

Efficacy of natural killer cell therapy combined with chemoradiotherapy in murine models of head and neck squamous cell carcinoma

Background aimsNatural killer (NK) cell-based cancer immunotherapy is effective when combined with other treatment modalities such as irradiation and chemotherapy. NK cell's antitumor function to treat solid tumor, including head and neck squamous cell carcinoma (HNSCC), has been targeted recently. This study assessed NK cell recruitment in response to chemoradiation therapy (CRT) in HNSCC. MethodsEx vivo expansion of NK cell, flow cytometry, cell viability assay, cytotoxicity assay, immunohistochemistry, and animal model were performed. ResultsMouse NK cells were recruited to the tumor site by CRT in a nude mouse model. Furthermore, expanded and activated human NK cells (eNKs) were recruited to the tumor site in response to CRT, and CRT enhanced the anti-tumor activity of eNK in an NOD/SCID IL-2Rγnull mouse model. Various HNSCC cancer cell lines exhibited different NK cell ligand activation patterns in response to CRT that correlated with NK cell-mediated cytotoxicity. ConclusionsIdentifying the activation patterns of NK cell ligands during CRT might improve patient selection for adjuvant NK cell immunotherapy combined with CRT. This is the first study to investigate the NK cell's antitumor function and recruitment with CRT in HNSCC mouse model.

Abstract PO-034: Optimizing chemotherapeutic regimen through metabolic interrogation to maximize radiosensitizing effects in HNSCC

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally. Cisplatin (CDDP) has been widely utilized as a chemotherapeutic agent and is often combined with ionizing radiation (IR) to improve overall survival of patients with advanced stage HNSCC and other malignancies. Current treatment algorithms have limited efficacy due to empiric approaches and the lack of analysis to optimize CDDP treatment effectiveness and reduce normal tissue toxicity. We utilized metabolic interrogation as a tool to optimize CDDP treatment regimens in conjunction with IR to maximize radiosensitizing effects in a pre-clinical HNSCC mouse model. Materials/Methods: We performed flow cytometry to optimize the combination regiment of CDDP and IR. The surviving fraction of cells was determined through clonogenic assays. Acute lactate production was monitored as a biomarker for metabolic perturbation induced by CDDP and IR. We compared a metabolism-based CDDP radiosensitization regimen with a standard clinical regimen in a pre-clinical HNSCC mouse model. The metabolism-based CDDP regimen involved administering CDDP (2.5 mg/kg, IV) one hour prior to irradiation (2 Gy) on day 1, followed by another irradiation (2Gy) alone on day 3, while standard clinical regimen involved administering irradiation (2 Gy) alone one day 1 and day 3, followed by CDDP (2.5 mg/kg, IV) treatment on day 4. Both regimens were repeated in the second week. Tumor size was measured using a caliper method twice a week and median survival time was recorded. Results: CDDP caused cell cycle arrest in S and G2/M phases from 24-72 hours. Although there was no significant cell apoptosis in the Sub-G1 phase, CDDP induced senescence, leading to a decreased clone formation on clonogenic survival. Both CDDP and IR triggered a dose and time-dependent, transient decrease in cellular lactate levels, which may serve as sensitive biomarker for predicting treatment response. The metabolism-based CDDP regimen resulted in delayed tumor growth and prolonged median survival time when compared to control standard clinical group. The metabolism-based CDDP regimen outperformed the standard regimen in terms of tumor growth inhibition and prolonged median survival time (p<0.005). Conclusions: Both CDDP and IR triggered acute transient perturbations in cellular lactate levels, which may predict CDDP effects and improve radiosensitization. The real-time metabolic interrogation of acute lactate changes reflects treatment effects on DNA damage, cell death and tumor growth delay. To optimize CDDP regimens prior to clinical translation, we developed an adaptive and robust preclinical platform to deliver the maximum tolerable dose and minimize toxicity. A metabolism-based CDDP regimen can significantly enhance radiosensitization, improve therapeutic effectiveness, and reduce normal tissue toxicity by potentially decreasing the CDDP dose. Minimizing normal tissue toxicity by reducing the CDDP dose and maximizing radiosensitizing effects may have a significant impact on patient survival. Citation Format: Yunyun Chen, Wangjie Yu, Ying Henderson, Vlad Sandulache, Stephen Y. Lai. Optimizing chemotherapeutic regimen through metabolic interrogation to maximize radiosensitizing effects in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-034.

Specific knockout of Notch2 in Treg cells significantly inhibits the growth and proliferation of head and neck squamous cell carcinoma in mice

ObjectiveTo investigate the effect of Notch2 gene knockout in Treg cells on head and neck squamous cell carcinoma (HNSCC) in mice. MethodsA mouse model of HNSCC was constructed. Flow cytometry and immunofluorescence were used to examine the numbers of related immune cells and programmed cell death in tumor cells in the spleen and tumor microenvironment of mice. Western blotting was used to measure the expression of related proteins in tumor tissues. ResultsThe tumor volume of regulatory T (Treg) cell-specific Notch2-knockout mice (experimental group) was significantly smaller than that of control mice (control group) (P < 0.05). Compared with those in the control group, the number of Treg cells and the expression of Ki67 in Treg cells in the spleen and tumor tissue were significantly decreased in the experimental group, while the numbers of CD45+ hematopoietic cells, CD4+ T cells, CD8+ T cells, T helper 1 (Th1) cells, CD11b+ cells (macrophages), and CD11b+CD11c+ cells (dendritic cells) and the expression of Ki67 in CD4+ T cells and CD8+ T cells were significantly increased (P < 0.05). There was no significant difference in the number of Th2 cells between the two groups (P > 0.05). Immunofluorescence analysis showed that the numbers of CD4+ T cells and CD8+ T cells in the tumor tissue in the experimental group were significantly higher than those in the control group (P < 0.05). Compared with that in the control group, programmed cell death in the experimental group was significantly increased (P < 0.05). Moreover, the expression levels of NLRP3, Caspase-1 and GSDMD in the tumor tissues of the experimental group were higher than those in the control group (P < 0.01), while the expression levels of BCL2, Bax, ATG5, LC3 and p62 were not significantly different (P > 0.05). ConclusionsSpecific knockout of the Notch2 gene in Treg cells significantly decreases the function of Treg cells, inhibits the growth of HNSCC and improves the immune microenvironment in mice, thus effectively treating HNSCC.

CCR7 Mediated Mimetic Dendritic Cell Vaccine Homing in Lymph Node for Head and Neck Squamous Cell Carcinoma Therapy.

Immunotherapy has been recognized as one of the most promising treatment strategies for head and neck squamous cell carcinoma (HNSCC). As a pioneering trend of immunotherapy, dendritic cell (DC) vaccines have displayed the ability to prime an immune response, while the insufficient immunogenicity and low lymph node (LN) targeting efficiency, resulted in an unsubstantiated therapeutic efficacy in clinical trials. Herein, a hybrid nanovaccine (Hy-M-Exo) is developed via fusing tumor-derived exosome (TEX) and dendritic cell membrane vesicle (DCMV). The hybrid nanovaccine inherited the key protein for lymphatic homing, CCR7, from DCMV and demonstrated an enhanced efficiency of LN targeting. Meanwhile, the reserved tumor antigens and endogenous danger signals in the hybrid nanovaccine activated antigen presenting cells (APCs) elicited a robust T-cell response. Moreover, the nanovaccine Hy-M-Exo displayed good therapeutic efficacy in a mouse model of HNSCC. These results indicated that Hy-M-Exo is of high clinical value to serve as a feasible strategy for antitumor immunotherapy.

Tertiary Lymphoid Structure Raises Survival and Immunotherapy in HPV− HNSCC

Immune checkpoint blockade (ICB) targeting PD-1/PD-L1 has been used for the treatment of head and neck squamous cell carcinoma (HNSCC). However, the overall response rate to ICB therapy for HNSCC remains less than 20%. It has recently been reported that the appearance of tertiary lymphoid structures (TLSs) in tumor tissue is correlated with better prognosis and response to ICB treatment. Here, we demonstrated an immune classification for the tumor microenvironment (TME) of HNSCC by analyzing The Cancer Genome Atlas (TCGA)–HNSCC data set and found that immunotype D with TLS enrichment had a better prognosis and response to ICB treatment. Furthermore, we observed that TLSs were present in a part of tumor samples of human papillomavirus (HPV) infection negative HNSCC (HPV− HNSCC) and were associated with the densities of dendritic cell (DC)–LAMP+ DCs, CD4+ T cells, CD8+ T cells, and progenitor T cells in TME. We established an HPV− HNSCC mouse model with TLS-enriched TME by overexpressing LIGHT in a mouse HNSCC cell line. We found that the induction of TLS formation enhanced the response to PD-1 blockade treatment in the HPV− HNSCC mouse model, accompanied by increases in DCs and progenitor exhausted CD8+ T cells in the TME. Elimination of CD20+ B cells attenuated the therapeutic effect of PD-1 pathway blockade in TLS+ HPV− HNSCC mouse models. These results indicate that TLSs contribute to the favorable prognosis and antitumor immunity of HPV− HNSCC. Inducing TLS formation in HPV− HNSCC tumors is a potential therapeutic method for improving the ICB response rate in patients with HPV− HNSCC.

Stress keratin 17 as a novel biomarker of response in immune checkpoint blockade–treated head and neck squamous cell carcinoma.

3117 Background: Low response rates in immune checkpoint blockade (ICB) treated head and neck squamous cell carcinoma (HNSCC)drive a critical need for robust clinically validated biomarkers that can predict response to ICB. Stress keratin 17 (K17) is a known prognostic marker in various types of cancer, including HNSCC; however, its predictive value for ICB response has not been investigated. Preclinical studies suggest K17 suppresses macrophage-mediated CXCL9/CXCL10 chemokine signaling involved in attracting activated CD8+ T cells into tumors. Furthermore, knocking out K17 results in restored response to ICB in a HNSCC mouse model. Here, we evaluated if K17 protein expression predicts response to ICB in human HNSCC patients. Methods: We conducted a retrospective analysis of 26 HNSCC patients that received at least one cycle of pembrolizumab at the University of Wisconsin-Madison Carbone Cancer Center. Pretreatment, archival, formalin-fixed, paraffin-embeded samples were stained by immunohistochemistry using a K17 monoclonal antibody. Clinical outcomes were investigator-assessed for all patients with at least one post-treatment scan or evidence of clinical progression after treatment initiation. Based on independent pathology review, cases were categorized into K17 high vs K17 low based on a cut-off of &gt; 5% strong cytoplasmic staining intensity of tumor cells in the invasive carcinoma component. Correlation between K17 expression and clinical outcomes was assesed using Fischer's exact test and log rank test. Results: The 26 patients included in this study were 85% male, median age 60.5 years, 74% ECOG performance status &lt; 2, with 80% having received prior chemotherapy. Primary site included oral cavity (54%), oropharynx (23%), larynx (4%), or other (19%). Seventeen tumors (65%) showed high K17 expression, and 9 tumors (35%) showed low K17 expression. Eleven patients (42%) had programmed death ligand 1 (PD-L1)+ tumors as determined by combined positive score. Six patients (23%, all K17 low) achieved clinical benefit, while 20 patients (77%, 17 K17 high and 2 K17 low) had progressive disease. High K17 expression was associated with lack of clinical benefit (p &lt; 0.001), shorter time to treatment failure (p &lt; 0.001), progression-free (p = 0.004) and overall survival (p = 0.02). PD-L1 expression by immunohistochemistry (clone 22C3) did not correlate with K17 expression or clinical outcome. Conclusions: Our findings suggest that K17 expression may predict clinical benefit from ICB in HNSCC patients, thus supporting further validation studies. [Table: see text]

A Novel CXCR4-Targeted Diphtheria Toxin Nanoparticle Inhibits Invasion and Metastatic Dissemination in a Head and Neck Squamous Cell Carcinoma Mouse Model.

Loco-regional recurrences and metastasis represent the leading causes of death in head and neck squamous cell carcinoma (HNSCC) patients, highlighting the need for novel therapies. Chemokine receptor 4 (CXCR4) has been related to loco-regional and distant recurrence and worse patient prognosis. In this regard, we developed a novel protein nanoparticle, T22-DITOX-H6, aiming to selectively deliver the diphtheria toxin cytotoxic domain to CXCR4+ HNSCC cells. The antimetastatic effect of T22-DITOX-H6 was evaluated in vivo in an orthotopic mouse model. IVIS imaging system was utilized to assess the metastatic dissemination in the mouse model. Immunohistochemistry and histopathological analyses were used to study the CXCR4 expression in the cancer cells, to evaluate the effect of the nanotoxin treatment, and its potential off-target toxicity. In this study, we report that CXCR4+ cancer cells were present in the invasive tumor front in an orthotopic mouse model. Upon repeated T22-DITOX-H6 administration, the number of CXCR4+ cancer cells was significantly reduced. Similarly, nanotoxin treatment effectively blocked regional and distant metastatic dissemination in the absence of systemic toxicity in the metastatic HNSCC mouse model. The repeated administration of T22-DITOX-H6 clearly abrogates tumor invasiveness and metastatic dissemination without inducing any off-target toxicity. Thus, T22-DITOX-H6 holds great promise for the treatment of CXCR4+ HNSCC patients presenting worse prognosis.

CXCR4-targeted nanotoxins induce GSDME-dependent pyroptosis in head and neck squamous cell carcinoma

BackgroundTherapy resistance, which leads to the development of loco-regional relapses and distant metastases after treatment, constitutes one of the major problems that head and neck squamous cell carcinoma (HNSCC) patients currently face. Thus, novel therapeutic strategies are urgently needed. Targeted drug delivery to the chemokine receptor 4 (CXCR4) represents a promising approach for HNSCC management. In this context, we have developed the self-assembling protein nanotoxins T22-PE24-H6 and T22-DITOX-H6, which incorporate the de-immunized catalytic domain of Pseudomonas aeruginosa (PE24) exotoxin A and the diphtheria exotoxin (DITOX) domain, respectively. Both nanotoxins contain the T22 peptide ligand to specifically target CXCR4-overexpressing HNSCC cells. In this study, we evaluate the potential use of T22-PE24-H6 and T22-DITOX-H6 nanotoxins for the treatment of HNSCC.MethodsT22-PE24-H6 and T22-DITOX-H6 CXCR4-dependent cytotoxic effect was evaluated in vitro in two different HNSCC cell lines. Both nanotoxins cell death mechanisms were assessed in HNSCC cell lines by phase-contrast microscopy, AnnexinV/ propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and western blotting. Nanotoxins antitumor effect in vivo was studied in a CXCR4+ HNSCC subcutaneous mouse model. Immunohistochemistry, histopathology, and toxicity analyses were used to evaluate both nanotoxins antitumor effect and possible treatment toxicity. GSMDE and CXCR4 expression in HNSCC patient tumor samples was also assessed by immunohistochemical staining.ResultsFirst, we found that both nanotoxins exhibit a potent CXCR4-dependent cytotoxic effect in vitro. Importantly, nanotoxin treatment triggered caspase-3/Gasdermin E (GSDME)-mediated pyroptosis. The activation of this alternative cell death pathway that differs from traditional apoptosis, becomes a promising strategy to bypass therapy resistance. In addition, T22-PE24-H6 and T22-DITOX-H6 displayed a potent antitumor effect in the absence of systemic toxicity in a CXCR4+ subcutaneous HNSCC mouse model. Lastly, GSDME was found to be overexpressed in tumor tissue from HNSCC patients, highlighting the relevance of this strategy.ConclusionsAltogether, our results show that T22-PE24-H6 and T22-DITOX-H6 represent a promising therapy for HNSCC patients. Remarkably, this is the first study showing that both nanotoxins are capable of activating caspase-3/GSDME-dependent pyroptosis, opening a novel avenue for HNSCC treatment.

Immunomodulation via FGFR inhibition augments FGFR1 targeting T-cell based antitumor immunotherapy for head and neck squamous cell carcinoma

ABSTRACT Fibroblast growth factor receptor 1 (FGFR1) is overexpressed in multiple types of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Being associated with poor prognosis, FGFR1 is a potential therapeutic target for aggressive tumors. T cell-based cancer immunotherapy has played a central role in novel cancer treatments. However, the potential of antitumor immunotherapy targeting FGFR1 has not been investigated. Here, we showed that FGFR-tyrosine kinase inhibitors (TKIs) augmented antitumor effects of immune checkpoint inhibitors in an HNSCC mouse model and upregulated tumoral MHC class I and MHC class II expression in vivo and in vitro. This upregulation was associated with the mitogen-activated protein kinase signaling pathway, which is a crucial pathway for cancer development through FGFR signaling. Moreover, we identified an FGFR1-derived peptide epitope (FGFR1305-319) that could elicit antigen-reactive and multiple HLA-restricted CD4+ T cell responses. These T cells showed direct cytotoxicity against tumor cells that expressed FGFR1. Notably, FGFR-TKIs augmented antitumor effects of FGFR1-reactive T cells against human HNSCC cells. These results indicate that the combination of FGFR-TKIs with immunotherapy, such as an FGFR1-targeting peptide vaccine or immune checkpoint inhibitor, could be a novel and robust immunologic approach for treating patients with FGFR1-expressing cancer cells.

Black raspberry restores the expression of the tumor suppressor p120ctn in the oral cavity of mice treated with the carcinogen dibenzo[a,l]pyrene diol epoxide.

One of the major risk factors for head and neck squamous cell carcinoma (HNSCC) is tobacco smoke exposure, but the mechanisms that can account for disease development remain to be fully defined. Utilizing our HNSCC mouse model, we analyzed oral squamous cell carcinomas (OSCC) induced by the active metabolite of a common smoke constituent, dibenzo[a,l]pyrene diol-epoxide (DBPDE). Analyzing protein expression by either immunofluorescence or immunohistochemistry, we identified biologic processes that are dysregulated in premalignant and invasive cancer lesions induced by DBPDE. Interestingly, p120ctn expression is downregulated in both stages of the disease. In addition to decreased p120ctn expression, there was also increased proliferation (as measured by Ki67), inflammation (as measured by NFkB (p65) expression), neovascularization (as measured by CD31) and recruitment of Ly6G-positive immune cells as well as strong EGFR expression. We also examined the effect of the chemopreventive agent black raspberry (BRB) on p120ctn and EGFR protein expression in DBPDE treated mice. p120ctn, but not EGFR, protein expression increased in mice treated with BRB. Our results suggest that modulation of p120ctn may, in part, account for the mechanism by which BRB inhibits DBPDE induced OSCC in mice.

Genome-Wide Enhancer Analysis Reveals the Role of AP-1 Transcription Factor in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the world, but its epigenomic features have not been determined. Here, we studied the chromatin landscape of active enhancers of HNSCC head tumor tissues by performing H3K27ac and H3K4me1 ChIP-Seq with a Tgfbr1/Pten double conditional knockout HNSCC mouse model. We identified 1,248 gain variant enhancer loci (VELs) and 2,188 lost VELs, as well as 153 gain variant super enhancer loci (VSELs) and 234 lost VSELs. Potentially involved transcription factors were predicted with motif analysis, and we identified AP-1 as one of the critical oncogenic transcription factors in HNSCC and many other types of cancer. Combining transcriptomic and epigenomic data, our analysis also showed that AP-1 and histone modifications coordinately regulate target gene expression in HNSCC. In conclusion, our study provides important epigenomic information for enhancer studies in HNSCC and reveals new mechanism for AP-1 regulating HNSCC.

The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity

Current therapies for treating heterogeneous cancers such as head and neck squamous cell carcinoma (HNSCC) are non-selective and are administered independent of response biomarkers. Therapy resistance subsequently emerges, resulting in increased cellular proliferation that is associated with loss of differentiation. Whether a cancer cell differentiation potential can dictate therapy responsiveness is still currently unknown. A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity was employed in a HNSCC mouse model, primary patients’ data, and human cell lines to assess the potential of functional differentiation in predicting therapy response. Interestingly, a subset of HNSCC with effective GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-Myc, and STAT3 signaling. Furthermore, we identified the GRHL3-differentiation target gene Filaggrin (FLG) as a response biomarker and more importantly, stratified HNSCC subsets as treatment resistant based on their FLG mutational profile. The loss of FLG in sensitive HNSCC resulted in a dramatic resistance to targeted therapies while the GRHL3-FLG signature predicted a favorable patient prognosis. This study provides evidence for a functional GRHL3-FLG tumor-specific differentiation axis that regulates targeted therapy response in HNSCC and establishes a rationale for clinical investigation of differentiation-paired targeted therapy in heterogeneous cancers.

Effects of prexasertib, a CHK1 inhibitor, in the immune microenvironment of head and neck squamous cell carcinoma (HNSCC).

e18541 Background: Despite considerable advancements in the standard therapies including surgery, chemotherapy and radiotherapy, the clinical outcome for head and neck squamous cell carcinoma (HNSCC) remains poor due to tumor recurrence and metastasis. Molecular-targeted therapies have evolved as novel and promising treatment for HNSCC patients. Herein, we identified a CHK1 inhibitor, prexasertib, as a therapeutic target that enhanced the infiltration of innate and adaptive immune markers in the mice tumor immune microenvironment (TIME) and subsequently sensitizes the tumors. Methods: For this study we used syngeneic mouse model of HNSCC developed using mouse tonsil epithelial cells transformed with HRAS expression and PTPN13 knockdown to represent tobacco-induced HNSCC (MTE-Ras). In vitro drug activity of prexasertib was determined using immunoblotting, flow cytometry, cell proliferation and colony formation assays. The effect of prexasertib on TIME was quantified by Quantigene Plex panel and multiplex immunohistochemistry (mIHC). Results: We found that in vitro treatment with prexasertib increased the amount of DNA damage in the cancer cells and eventually lead to their death. Similarly, our in vivo data showed that treatment with prexasertib resulted in significant tumor regression and increased mice survival. At the molecular level, prexasertib treatment resulted in an upregulation of transcripts associated with T-cell activation, cytokines, chemokines and macrophages indicating an upregulation of inflammatory gene signature. This was further supported by our findings from mIHC staining of mice tumors showing increased infiltration of natural killer cells, natural killer T cells and dendritic cells, following prexasertib treatment. Interestingly, we also saw an increase in the Tregs after prexasertib treatment, which is indicative of an immunosuppressive environment, and we speculate this could be as a result of negative feedback following immune activation by prexasertib treatment. Conclusions: Our results uncover a previously unidentified role of prexasertib in regulating the innate and adaptive immune response in HNSCC, and therefore further corroborate CHK1 as a promising therapeutic target in HNSCC.

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...

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

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

Sentinel Lymph Node-Targeted Therapy by Oncolytic Sendai Virus Suppresses Micrometastasis of Head and Neck Squamous Cell Carcinoma in an Orthotopic Nude Mouse Model.

In clinical N0 (cN0) cases with head and neck squamous cell carcinoma (HNSCC), a treatment selection is still controversial: elective neck dissection or watchful waiting. We focused on sentinel lymph node (SLN)-targeted therapy using the urokinase-type plasminogen activator (uPA)-dependent oncolytic Sendai virus "BioKnife." The objectives of this study were to investigate BioKnife migration into SLNs and elucidate its antitumor effect on lymph node metastases (LNM). We established an orthotopic nude mouse model of HNSCC, with LNM being frequently induced. We inoculated HSC-3-M3, human highly metastatic tongue squamous cell carcinoma cells, in the tongue of the nude mice, and after 2 weeks, we injected BioKnife into the primary tumor. We tracked BioKnife migration into the SLNs by immunostaining, RT-PCR, and an in vivo imaging system. We also examined its antitumor effects and mechanisms through serial section analysis of lymph nodes. GFP reporter expression was clearly visible in the lymph nodes of virus groups, which corresponded to SLNs. Relative GFP mRNA was significantly increased in both the tongues and lymph nodes in the virus groups compared with that in the control group (P < 0.05). Serial section analysis showed that BioKnife infected cancer cells and exhibited significant antitumor effect against LNM compared with the control groups (P < 0.05). We detected apoptosis in LNM infected by BioKnife. BioKnife migrated into SLNs after its injection into the primary tumor and effectively suppressed LNM, suggesting that SLN-targeted therapy using BioKnife has great potential to provide a novel and promising alternative to elective neck dissection in cN0 patients with HNSCC.

PDC depletion induced by CD317 blockade drives the antitumor immune response in head and neck squamous cell carcinoma

ObjectivesDysregulation of immune cells in the tumor microenvironment is a hallmark of head and neck squamous cell carcinoma (HNSCC). Increased infiltration of pDCs has been reported in the microenvironment of HNSCC. However, the precise immunological role of pDC and the therapeutic effects of pDC depletion in HNSCC need to be further investigated. Materials and methodsCD317 antibodies were applied for depleting pDCs in an immunocompetent transgenic HNSCC mouse model. Tumor volume was monitored. Flow cytometric analysis was conducted for studying the immune profile changes after pDC depletion. In addition, immunohistochemical staining was carried out in a human HNSCC tissue microarray for detecting the infiltration of pDCs. We also analyzed the survival implication of pDCs and its correlation with other immune related markers in human HNSCC. ResultspDC depletion in the transgenic HNSCC mouse model significantly delayed tumor growth. After pDCs were depleted, T cells were markedly revitalized, and the proportions of regulatory T cells (Tregs) and monocytic myeloid-derived suppressor cells (MDSCs) were decreased. In human HNSCC microenvironment, pDC infiltration was upregulated and its high infiltration conferred a poor prognosis. Moreover, pDC infiltration was closely correlated with the expression of Foxp-3, PD-1, TIM-3, and LAG-3. ConclusionOur findings demonstrated that pDCs play a negative immunomodulatory role in HNSCC and may present as a target for effective immunotherapy.