SCC Cell Lines Research Articles

Shikonin Stimulates Mitochondria-Mediated Apoptosis by Enhancing Intracellular Reactive Oxygen Species Production and DNA Damage in Oral Cancer Cells.

Phytotherapy has rendered a new insight towards the treatment of various cancers, including oral cancer with fewer side effects, over the traditional chemotherapeutic drugs to overcome chemoresistance. Shikonin (Shk) is a natural biologically active alkaloid found in the Lithospermum erythrorhizon plant's root. It has potent cytotoxic activities against various cancers. Our study revealed the release time and anticancer potential of Shk on the SCC9 and H357 oral cancer cell lines. We investigated the antiproliferative, antimigratory, cell cycle arresting and apoptosis promoting activity of Shk in oral cancer cells by performing MTT and morphological assay, colony, and tumor sphere formation assay, AO/EtBr and DAPI staining, Annexin V-FITC/PI staining, assay for reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) measurement, comet assay, qRT-PCR, and western blot analysis. We also checked the interaction of DNA and Shk by docking and CD spectroscopy and EtBr displacement assay. As a result, we found that Shk reduced the viability, proliferation, and tumorigenicity of SCC9 and H357 cells in a time and concentration-dependent manner. We obtained half-maximal inhibitory concentration (IC50) at 0.5 µM for SCC9 and 1.25 µM for H357. It promotes apoptosis via overexpressing proapoptotic Bax and caspase 3 via enhancing ROS that leads to MMP depletion and DNA damage and arrests cells at the G2/M & G2/S phase. The antimigratory activity of Shk was performed by analyzing the expression of markers of epithelial-mesenchymal transition like E-cadherin, ZO-1, N-cadherin, and vimentin. These overall results recommended that Shk shows potent anticancer activity against oral cancer cell lines in both in vitro and ex vivo conditions. So, it could be an excellent agent for the treatment of oral cancer.

CHRDL1 inhibits OSCC metastasis via MAPK signaling-mediated inhibition of MED29

BackgroundCHRDL1 belongs to a novel class of mRNA molecules. Nonetheless, the specific biological functions and underlying mechanisms of CHRDL1 in oral squamous cell carcinoma (OSCC) remain largely unexplored.MethodsRT-qPCR and immunohistochemical staining were employed to assess the mRNA and protein expression levels of the MED29 gene in clinical samples of OSCC. Additionally, RT-qPCR and Western Blot analyses were conducted to investigate the mRNA and protein expression levels of the MED29 gene specifically in OSCC. The impact of MED29 on epithelial-mesenchymal transition (EMT), invasion, and migration of OSCC was evaluated through scratch assay, transwell assay, and immunofluorescence staining. Furthermore, wound healing assay and Transwell assay were utilized to examine whether CHRDL1 influences the malignant behavior of OSCC by modulating MED29 in vitro. The regulatory role of CHRDL1 on MED29 was further elucidated in vivo through a tail vein lung metastasis model in nude mice.ResultsMED29 expression was elevated in tumor tissues of OSCC patients compared with adjacent cancer tissues. Moreover, in CAL27 and SCC25 cell lines, MED29 was upregulated and associated with increased cell migration and invasion abilities. Overexpression of MED29 facilitated EMT in OSCC cell lines, whereas knockdown of MED29 impeded EMT, resulting in diminished cell migration and invasion capacities. CHRDL1 exerted inhibitory effects on the expression of MED29, thereby suppressing EMT progression and consequently restraining the invasion and migration of OSCC cells. Furthermore, CHRDL1 mediated the inhibition of migration of OSCC cell lines to the OSCC through its regulation of MED29.ConclusionsMED29 facilitated the epithelial-mesenchymal transition process in OSCC, thereby promoting migration and invasion. On the other hand, CHRDL1 exerted inhibitory effects on the invasion and metastasis of OSCC by suppressing MED29 through the inhibition of the MAPK signaling pathway.

Pterostilbene suppresses head and neck cancer cell proliferation via induction of apoptosis.

Head and neck cancer (HNC) is one of the most prevalent causes of death worldwide, and so discovering anticancer agents for its treatment is very important. Pterostilbene (PS) is a trans-stilbene reported to be beneficial in managing various cancers. The objective of the study was to evaluate the cytotoxic, antiproliferative, proapoptotic, and antimigrative effect of PS on HEp-2, SCC-90, SCC-9, FaDu, and Detroit-551 cell lines. MTT and live/dead assays were employed to assess cell viability, while a cell migration test was performed to evaluate wound healing capacity. The mRNA, protein, and intracellular expression levels of CASP-3, BAX, and BCL-2 genes were evaluated by real-time PCR, western blotting, and immunofluorescence staining. Annexin V-PI staining was conducted to assess the amounts of viable, apoptotic, and necrotic cells. The results revealed that PS displayed cytotoxic, antiproliferative activity in a dose-dependent manner in HNC cells by upregulating CASP-3 and BCL-2 while downregulating BCL-2 in the apoptotic pathway. The proapoptotic properties were confirmed by the annexin-V-IP results. Moreover, PS displayed a significant suppressive efficacy on the migration capacity of HNC cells. The present study provides proof that PS has the prospective to be improved as an attractive anticancer agent against HNC following advanced studies.

Y27632 induces tongue squamous cell carcinoma cell apoptosis through MAPK-ERK/JNK signal

Abstract Objectives ROCK signaling is considered a therapeutic target for oral squamous cell carcinoma (OSCC). Y27632, a well-established ROCK inhibitor, has previously been reported to block oral squamous cell carcinoma cell growth and has shown cell type dependence in the treatment of other cancers. TP53 is one of the most frequently mutated genes in head and neck cancer. Here, we aim to investigate the role of Y27632 in wild-type and p53 mutant (R175H) SCC9 cells. Methods The p53-mutation (R175H) and p53-null SCC9 cell line were conducted, then, CCK8, colony formation, wound-healing assays, and transwell assay were employed to investigate the role of Y27632 in wtp53 and mutp53 SCC9 cells. The effects of Y27632 in SCC9 cells were also confirmed by the knockdown of ROCK1/2. Additionally, cell cycle and apoptosis were assessed using flow and western blot analysis. The impact of Y27632 on cell senescence was confirmed through the senescence-associated β-gal staining. Furthermore, the inhibition of Y27632 was examined in vivo using tumor-bearing nude mice. Results Our study demonstrates that Y27632 effectively impeded the proliferation of tongue squamous cell carcinoma (TSCC) cells in vitro and in vivo. Additionally, the proliferation and migration of wtp53 and mutp53 SCC9 cells were also significantly suppressed by Y27632 or ROCK siRNA in vitro. Mechanistically, Y27632 induced apoptosis in SCC9 cells via the MAPK-ERK/JNK signaling pathway. Conclusions Our data demonstrated that Y27632 induces apoptosis in SCC9 cells via the MAPK-ERK/JNK signaling pathway, regardless of the presence of p53 mutant variants (R175H). This will provide a potential therapeutic drug for TSCC treatment in the future.

IN VITRO ANTICANCER ACTIVITY OF HISTATIN-1 COMBINATION WITH CISPLATIN IN HEAD AND NECK CANCER CELL LINES.

Chemotherapy of head and neck squamous cell carcinoma (HNSCC) is associated with significant side effects. Antimicrobial peptides (AMPs), which are naturally occurring defense molecules like defensin-1 and LL-37 found in human secretions, have demonstrated potential in prompting tumor cell apoptosis and enhancing the effect of chemotherapeutic agents. However, the anticancer potential of histatin has not yet been thoroughly examined. The aim of the study was to explore the anticancer activity of histatin, an AMP present in human saliva and used alone or in combination with cisplatin in HNSCC cell lines. The gene expression of histatin was evaluated in the HSC4 and SCC25 cell lines by qRT-PCR. Cell proliferation was investigated at different concentrations of histatin peptide (His-1), cisplatin, and their combination using an MTT assay. SCC25 cells expressed both HTN1 (histatin-1) and HTN3 (histatin-3), whereas the HSC4 cell line expressed only HTN1. The combination of exogenous His-1 and cisplatin demonstrated a synergistic anti-proliferative effect against the HNSCC cell lines in a dosedependent manner. The combination of low-dose cisplatin and histatin inhibits HNSCC cell proliferation. His-1 sensitizes tumor cells to the cytotoxic effects of cisplatin potentially allowing for a reduction in its effective concentration.

TROP2 Expression and Therapeutic Implications in Cutaneous Squamous Cell Carcinoma: Insights From Immunohistochemical and Functional Analysis.

Cutaneous squamous cell carcinoma (cSCC) is a common form of skin cancer, but treatments for advanced cases have limited efficacy. Trophoblast cell-surface antigen 2 (TROP2) is a cell-surface protein that is widely expressed in various tumours, where it exerts significant influence over critical processes such as tumour cell growth, apoptosis, migration, invasion and metastasis. Sacituzumab govitecan, an antibody-drug conjugate (ADC) targeting TROP2, is emerging as a promising strategy for anticancer therapy. In this study, we investigated TROP2 expression in cSCC tissues from 51 patients and evaluated its function in the A431 human SCC cell line. Immunohistochemical analysis revealed TROP2 expression on the plasma membrane of cSCC tissues and A431 cells. A431 cells showed sensitivity to sacituzumab govitecan with a significant concentration-dependent decrease in viable cell number. In addition, Knockdown of TROP2 resulted in decreased expression of cyclin D1 and BCL-2, along with reduced cell viability. Knockdown of TROP2 also resulted in decreased expression of vimentin, along with reduced migratory capacity. These findings suggest that TROP2 plays a crucial role in cSCC cell proliferation and migration, and highlight the potential of sacituzumab govitecan as a promising therapeutic option for cSCC.

Okanin Inhibits Cell Growth and Induces Apoptosis and Pyroptosis in Oral Cancer.

Okanin, a flavonoid compound derived from Bidens pilosa L., has garnered attention for its anti-inflammatory properties. Although Bidens pilosa is commonly used in healthcare products and functional foods, the anticancer potential of okanin, particularly in oral cancer, remains underexplored. This study aims to investigate the effects of okanin on oral cancer cell lines and its potential as a therapeutic agent. The study involved assessing the cytotoxic effects of okanin on oral cancer cell lines SAS, SCC25, HSC3, and OEC-M1. The IC50 values were determined using methylene blue assays, and the clonogenic capacity was evaluated through colony formation assays. Flow cytometry was used to analyze cell cycle progression and apoptosis. Caspase-3/7 activity assays and annexin V/7-AAD staining confirmed the induction of apoptosis and pyroptosis. In vivo efficacy was assessed using a SAS xenograft model, and immunohistochemical analysis of xenograft tissue was performed to examine pyroptosis-related markers. Okanin exhibited potent cytotoxic effects with IC50 values of 12.0 ± 0.8, 58.9 ± 18.7, 18.1 ± 5.3, and 43.2 ± 6.2 μM in SAS, SCC25, HSC3, and OEC-M1 cells, respectively. It caused dose- and time-dependent reductions in cell viability and significantly impaired clonogenic capacity. Flow cytometry revealed G2/M cell cycle arrest and increased sub-G1 population, indicating cell cycle disruption and death. Okanin induced both apoptosis and pyroptosis, as confirmed by caspase-3/7 activity and annexin V/7-AAD staining. In vivo, okanin reduced tumor growth and involved pyroptosis-related markers such as CASP1, GSDMC, GSDMD, and GSDME. Okanin demonstrates significant anticancer potential, particularly in oral cancer, by inducing both apoptosis and pyroptosis. Its efficacy in reducing tumor growth in vivo further supports its potential as a novel therapeutic option. Further mechanistic studies are needed to elucidate the pathways involved in okanin-mediated cell death and to explore its clinical applications.

An Integrated Approach of Network Pharmacology, Bioinformatics, Molecular Docking, and Experimental Verification Uncovers Prunellae Spica as the potential Medicine of Prognosis Improvement for Oral Squamous Cell Carcinoma.

Prunellae Spica (PS), the spike from Prunella vulgaris L., is a traditional Chinese medicine that can treat Oral Squamous Cell Carcinoma (OSCC), whereas its molecular mechanisms and effects on the prognosis of patients remain unclear. Our study aimed to identify potential anti-OSCC targets of PS and explore its mechanisms and effects on prognosis through network pharmacology, bioinformatics analysis, molecular docking, and in vitro cell assays. Sixty-two potential targets of 11 active anti-OSCC ingredients of PS were identified, with Quercetin, the core ingredient of PS, exhibiting the most significant number of OSCC-related targets. GO analysis indicated that the primary biological processes involved in OSCC treatment by PS were the cellular response to nitrogen compound, response to xenobiotic stimulus, and cellular response to organonitrogen compound. KEGG analysis revealed that Pathways in cancer were the top highly enriched signaling pathway in the treatment of OSCC by PS. DisGeNET analysis is mainly about Lip and Oral Cavity Carcinoma. More importantly, 6 of the 62 targets were markedly related to prognosis. Molecular docking revealed high affinities between the key component and the prognosis-related target proteins. Treatment of OSCC cell line SCC-25 with Quercetin could inhibit malignant biological behaviors, such as cell proliferation, colony formation, invasion, and migration, as well as affect the targets related to prognosis and promote autophagy. Overall, these results suggest that PS plays a significant role in treating and improving the prognosis of OSCC by directly influencing various processes in OSCC.

STAT3 interactome predicts presence of proteins that regulate immune system in oral squamous cell carcinoma

ObjectivesSignal transducer and activator of transcription 3 (STAT3) is one of the key proliferation mechanism–related proteins that helps in oral squamous cell carcinoma (OSCC) progression. Immune evasion by STAT3 is mediated by the JAK2/STAT3/PDL1 signaling axis. Based on previous findings, we hypothesized that STAT3-binding partners participate in the inhibition of anti-tumor activity in OSCC. MethodsA 3D cancer-immune co-culture model was constructed using oral cancer cell lines SCC4, SCC9, SCC25, and CAL27 and normal oral cell line OKF6. The cells were co-cultured with natural killer (NK-92) and Jurkat cells. The target protein STAT3 was chosen based on SWATH data, and co-immunoprecipitation (Co-IP)-based proteomics was conducted. The Co-IP LC-MS/MS output was analysed to determine the protein interaction network, gene ontology, pathway analysis, and protein cluster annotation. ResultsSTAT3 in oral cancer cell lines interacts with the epidermal growth factor receptor (EGFR) and other proteins that participate in proliferation and immune mechanisms. Proteome analysis showed that some STAT3-binding proteins found in this study are known immune system regulators. ConclusionOverall, STAT3 interactive proteins regulate the immune system in oral squamous cell carcinoma cells.

The Investigation of Nfκb Inhibitors to Block Cell Proliferation in OSCC Cells Lines.

Oral cancers, with oral squamous cell carcinoma (OSCC) as the predominant type, have a significant impact on morbidity and mortality rates. Therefore, targeting the NFκB pathway shows promise in cancer therapy. This study investigated the impact of two NFκB inhibitors, LY2409881 and MLN4924, on cell proliferation, apoptosis susceptibility, and in vivo tumorigenesis in OSCC cell lines CAL27 and SCC15. The results revealed that both LY2409881 and MLN4924 effectively suppressed cell proliferation, induced apoptosis, and arrested the cell cycle at the G2/M phase-a phenomenon likely associated with the NFκB pathway. Furthermore, MLN4924 demonstrated potent inhibitory effects on cell proliferation at low μM concentrations, surpassing the effectiveness of LY2409881 as an inhibitor. (All results: p<0.05) Conclusion: These findings highlight the potential of LY2409881 and MLN4924 as novel therapeutic agents for OSCC, thereby offering new insights for the clinical management of this condition.

Exploring 4-morpholinopyrrolo-[2,3-d]-pyrimidine derivatives as novel anti-cancer agents: Design, synthesis and biological studies

Despite extensive research efforts, cancer continues to pose a significant global health challenge. Continuing the anti-cancer research, twenty-four 4-Morpholinopyrrolo-[2,3-d]-pyrimidine derivatives were synthesized and characterized via HRMS, 1H, 13C NMR, and FTIR. A single crystal for one compound, PGT4, was developed for further confirmation. All synthesized compounds were assessed for their anti-cancer activity in three cancer cell lines, SCC-25 (oral squamous cell carcinoma), U87-MG (glioblastoma) and MCF-7 (breast cancer) and one normal cell line HEK-293T (human embryonic kidney) using WST assay. Fourteen compounds demonstrated IC50 values ranging from 2 to 10 µM against the SCC-25 cell line. PTT3 and PGT7 were the most effective compounds against the SCC-25 cell line, showing an IC50 of 2 µM. Cell cycle analysis was also performed using propidium iodide staining, and apoptosis studies were carried out using caspase 3/7 activity assay and Annexin V-FITC isothiocyanate assay. PTT3 and PGT7 arrested the cell cycle at the G1 phase and enhanced the apoptosis by 9.1 and 13.8-folds in SCC-25 cells, respectively. Treatment with PTT3 and PGT7 also resulted in a dose-dependent decrease in the colony formation of SCC-25 cells. We carried out the molecular docking and dynamic studies to find the insights on binding mechanisms of PTT3 and PGT7 within the active region of the PI3Kγ target protein (PDB ID:3IBE, 2.80Å). Additionally, in-silico ADME properties were predicted for the most active molecules, PTT3 and PGT7, aiding in assessing their pharmacokinetic profiles.

Gambogenic acid induces apoptosis via upregulation of Noxa in oral squamous cell carcinoma

Gambogenic acid (GNA), a bioactive compound derived from the resin of Garcinia hanburyi, has demonstrated significant antitumor properties. However, its mechanisms of action in oral squamous cell carcinoma (OSCC) remain largely unclear. This study aimed to elucidate the apoptotic effects of GNA on OSCC cell lines CAL-27 and SCC-15. Our results indicated that GNA induced apoptosis by upregulating the pro-apoptotic protein Noxa. Mechanistic investigations revealed that GNA treatment led to the generation of reactive oxygen species (ROS), which activated endoplasmic reticulum (ER) stress, culminating in cell apoptosis. Inhibition of ROS production and ER stress pathways significantly mitigated GNA-induced Noxa upregulation and subsequent apoptosis. Furthermore, in vivo studies using a murine xenograft model demonstrated that GNA administration effectively inhibited the growth of CAL-27 tumors. Collectively, these findings underscore GNA’s potential as a therapeutic agent for the treatment of OSCC.

Bioinformatics approach combined with experimental verification reveals OAS3 gene implicated in paclitaxel resistance in head and neck cancer.

This study aimed to identify a candidate gene associated with paclitaxel (PTX) resistance and to evaluate functionally its biological role in the PTX-resistant head and neck squamous cell carcinoma (HNSCC) cell lines and clinical specimens. Microarray data series containing samples of different types of cancers resistant to PTX were analyzed and then a candidate gene associated with PTX resistance was identified using various bioinformatics tools. After the suppression of the target gene expression, changes in cell viability and colony-forming ability were evaluated in PTX-resistant FaDu and SCC-9 cell lines. Bioinformatics analyses of upregulated genes in PTX-resistant cancer cells indicated that OAS3 was associated with PTX resistance. The downregulation of OAS3 expression significantly reduced the viability and colony-forming capacity of PTX-resistant SCC-9 cells by inducing apoptosis and cell cycle arrest at G0/G1 phase. The therapeutic targeting of OAS3 may resensitize PTX-resistant HNSCC cells with high OAS3 expression to PTX treatment.

Macrophorins H and L, two new HMG-conjugate macrophorins from rihzospheric Penicillium sp. NX-05-G-3

Macrophorins H (4) and L (5), two rare HMG-conjugate macrophorins along with three known macrophorins (1-3), three DMOA-derived meroterpenoids (6-8) and two ergosterol derivates (9-10) were isolated from sterilized rice medium cultured Penicillium sp. NX-05-G-3. Their structures were elucidated by 1D and 2D NMR. The cytotoxicities of all compounds were evaluated, and compounds 1 and 2 showed extensive cytotoxicity against human cancer cell lines Hela, SCC15, MDA-MB-453 and A549, with IC50 values ranging from 17.6 to 32.8 µM.

Verbascoside inhibits oral squamous cell carcinoma cell proliferation, migration, and invasion by the methyltransferase 3-mediated microRNA-31-5p/homeodomain interacting protein kinase 2 axis

ObjectiveThe study aimed to investigate the effects of verbascoside on oral squamous cell carcinoma (OSCC) cellular behaviors and underlying molecular mechanisms. DesignFor this purpose, SCC9 and UM1 cell lines were treated with verbascoside, and their biological behaviors, including proliferation, migration, and invasion, were evaluated using cell counting kit-8, 5-Ethynyl-2′-deoxyuridine, and Transwell assays. The expression of methyltransferase-3 (METTL3), microRNA (miR)− 31–5p, and homeodomain interacting protein kinase-2 (HIPK2) were examined using quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between METTL3 and miR-31–5p was evaluated by RNA immunoprecipitation and methylated RNA immunoprecipitation, while the interaction between miR-31–5p and HIPK2 was evaluated by dual-luciferase reporter analysis. ResultsThe results indicated inhibition of OSCC cell proliferation, migration, and invasion post verbascoside treatment. Similarly, METTL3 was upregulated in OSCC cells and was inhibited post-verbascoside treatment. Overexpressing METTL3 promoted the cellular processes. Moreover, miR-31–5p was upregulated in OSCC cells, where METTL3 facilitated the processing of miR-31–5p in an N6-methyladenosine (m6A)-dependent manner. The HIPK2 served as miR-31–5p target, where overexpressing miR-31–5p or HIPK2 knockdown reversed the suppression of verbascoside-induced biological behaviors. ConclusionsVerbascoside inhibited the progression of OSCC by inhibiting the METTL3-regulated miR-31–5p/HIPK2 axis. These findings suggested that verbascoside might be an effective drug for OSCC therapy.

Macrophage membrane-camouflaged pH-sensitive nanoparticles for targeted therapy of oral squamous cell carcinoma

BackgroundOral cancer is the most common malignant tumor of the head and neck, and 90% of cases are oral squamous cell carcinoma (OSCC). Chemotherapy is an important component of comprehensive treatment for OSCC. However, the clinical treatment effect of chemotherapy drugs, such as doxorubicin (DOX), is limited due to the lack of tumor targeting and rapid clearance by the immune system. Thus, based on the tumor-targeting and immune evasion abilities of macrophages, macrophage membrane-encapsulated poly(methyl vinyl ether alt maleic anhydride)-phenylboronic acid-doxorubicin nanoparticles (MM@PMVEMA-PBA-DOX NPs), briefly as MM@DOX NPs, were designed to target OSCC. The boronate ester bonds between PBA and DOX responded to the low pH value in the tumor microenvironment, selectively releasing the loaded DOX.ResultsThe results showed that MM@DOX NPs exhibited uniform particle size and typical core-shell structure. As the pH decreased from 7.4 to 5.5, drug release increased from 14 to 21%. The in vitro targeting ability, immune evasion ability, and cytotoxicity of MM@DOX NPs were verified in HN6 and SCC15 cell lines. Compared to free DOX, flow cytometry and fluorescence images demonstrated higher uptake of MM@DOX NPs by tumor cells and lower uptake by macrophages. Cell toxicity and live/dead staining experiments showed that MM@DOX NPs exhibited stronger in vitro antitumor effects than free DOX. The targeting and therapeutic effects were further confirmed in vivo. Based on in vivo biodistribution of the nanoparticles, the accumulation of MM@DOX NPs at the tumor site was increased. The pharmacokinetic results demonstrated a longer half-life of 9.26 h for MM@DOX NPs compared to 1.94 h for free DOX. Moreover, MM@DOX NPs exhibited stronger tumor suppression effects in HN6 tumor-bearing mice and good biocompatibility.ConclusionsTherefore, MM@DOX NPs is a safe and efficient therapeutic platform for OSCC.

Induction of Immunological Antitumor Effects by the Combination of Adenovirus-Mediated Gene Transfer of B7-1 and Anti-Programmed Cell Death-1 Antibody in a Murine Squamous Cell Carcinoma Model.

The goal of this study was to evaluate the antitumor immune effects of B7-1 gene expression in addition to immune checkpoint inhibitor against squamous cell carcinoma. A murine SCC cell line, KLN205, was infected with adenoviral vector carrying B7-1 (AdB7). Infected cells were injected subcutaneously in the flanks of DBA/2 mice. Three weeks after implantation, anti-mouse PD-1 antibody (antiPD1) was intraperitonially administrated twice a week for a total of six times. CD80 was significantly overexpressed in the AdB7-infected tumors. IFN-gamma in the T cells in the spleen was significantly increased and tumor size was significantly reduced in the mice treated with both AdB7 and antiPD1. Targeted tumors treated with both AdB7 and antiPD1 exhibited significantly increased cell densities of total immune cells as well as Ki-67+ CD8+ T cells and decreased regulatory T cells. These results suggest that the B7-1 gene transfer may enhance the antitumor effect of anti-PD1 antibody against SCC.

Chemerin promotes invasion of oral squamous cell carcinoma by stimulating IL-6 and TNF-α production via STAT3 activation.

Elevated levels of adipokine chemerin have been identified in oral squamous cell carcinoma (OSCC) and found to be associated with metastasis to the cervical lymph nodes. The underlying mechanism through which chemerin affects OSCC progression is unclear. The aims of this study were firstly to determine chemerin levels and cytokine concentrations in serum from patients with OSCC and in OSCC cell cultures, and secondly to observe chemerin effects on OSCC cell cytokine secretion, migration, and invasion in vitro. Serum samples were collected from 20 patients diagnosed with OSCC, including groups with (LN+) and without (LN-) cervical lymph node metastasis. A Luminex liquid suspension assay was used to quantify serum concentrations of 27 types of cytokines. Correlations between chemerin and cytokines (i.e., IL-6, IL-15, GM-CSF, RANTES, TNF-α, and VEGF) were analyzed. ELISAs (enzyme-linked immunosorbent assays) were used to determine concentrations of chemerin and selected cytokines in serum and in supernatants of OSCC cell cultures (SCC9 and SCC25 cell lines). OSCC cells were stimulated with human recombinant chemerin, STAT3 inhibitor, or IL-6 together with TNF-α neutralizing antibodies. Phosphorylated STAT3 protein levels were measured with western blot analysis. OSCC cell migration and invasion were investigated with Transwell assays. Compared to the LN- group, OSCC patients with cervical lymph node metastasis had higher levels of IL-6 (P = 0.006), IL-15 (P = 0.020), GM-CSF (P = 0.036), RANTES (P = 0.032), TNF-α (P = 0.005), VEGF (P = 0.006), and chemerin (P = 0.001). Patients' serum chemerin levels correlated directly with IL-6, GM-CSF, TNF-α, and VEGF levels in OSCC patients. Exogenous recombinant chemerin treatment promoted secretion of IL-6 and TNF-α via activation of STAT3 in OSCC cells. Chemerin induced OSCC-cell migration and invasion, and these effects were reduced by IL-6 and TNF-α neutralizing antibodies. Our findings indicate that chemerin may play a role in advancing OSCC progression by increasing production of IL-6 and TNF-α, perhaps via a mechanism involving STAT3 signaling.

Abstract 5438: Circulating tumor cells exhibit a tumor stemness defense phenotype in Head and Neck cancer subjects undergoing cisplatin based chemotherapy

Abstract Introduction: We have proposed that tumor has its own defense mechanism similar to an organism having its own defense at the population level. We also proposed that the center of this tumor defense is the hypoxic niche of cancer stem cells (CSCs). Our lab has previously characterized the hypoxic niche of CSCs, and demonstrated that these CSCs reprogram to a highly inflammatory, aggressive and embryonic stemness phenotype during oxidative stress (1). These reprogrammed CSCs, the tumor stemness defense (TSD) phenotype is transient, and exert altruistic defense against bacterial invasion in SCC-25 cell line (2). Further, VEGF/VEGFR1 autocrine signaling was found in cisplatin induced TSD phenotype (4) and Myc/Hif2alpha stemness pathway was found in TSD phenotype of a leukemia stem cell model (3). We hypothesize that the platinum-induced stress may also activate TSD phenotype in the dormant CSC fraction of head and neck squamous cell carcinoma (HNSCC), which may mobilize to the distant tissue via circulation as circulating tumor cells (CTCs). Methods: To test the hypothesis, we isolated EpCAM+/ABCG2+ CTCs (blood, n=14) and EpCAM+/ABCG2+ CSCs (tumor tissue, n=6/14) of HNSCC subjects under cisplatin treatment. CTCs were collected from peripheral blood mononuclear cells (PBMNC) by the immunomagnetic sorting of EpCAM+ cells, and then cells were expanded in a defined serum free media that we used to maintain the hypoxic CSCs (1). We evaluated the EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs for TSD phenotype by performing qPCR gene expression, Boyden chamber assay of invasion, clonogenic assay and serial transplantation assay in NOD/SCID mice. Data was compared with the EpCAM+/ABCG2- cell population. We also developed a pre-clinical model of cisplatin-induced reprogramming of HNSCC derived CSCs to understand the molecular mechanisms. Results: In 8/14 patient derived EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs showed TSD phenotype associated gene expression, high migration/invasion, self sufficiency (ability to form spheroid when grown in serum free media without growth factors), enhanced secretion of protumorigenic growth factors; VEGF, SDF-1, PIGF, and HMGB1. These CTCs and CSCs have shown tumor formation with significant CSC frequency, when injected to NOD/SCID mice. Finally, using a SCC-25 cell line derived pre-clinical model, we demonstrated that cisplatin therapy reprogram CSCs to TSD phenotype. Conclusion: Our work indicates that CTCs with TSD phenotype may indicate the interaction between cisplatin and CSC niche defense. Using the pre-clinical model, the TSD phenotype based CSC niche defense mechanism may be further explored to develop markers for therapy response, metastasis or recurrence in HNSCC.

Abstract 6646: Investigating the combination of radiation and subsequent immune checkpoint inhibitor treatment in head and neck squamous cell carcinoma models

Abstract Purpose: Head and neck squamous cell carcinoma (HNSCC) is often marked by an immunosuppressive tumor microenvironment, which contributes to the dismal 10-20% response rate to immune checkpoint inhibitor (ICI) therapy in HNSCC patients. Radiotherapy (RT) is a currently standard of care and frontline therapy for HNSCC, yet resistance is common. We sought to examine the combination therapy of RT followed by ICI therapy to test if RT induces an in situ vaccination which enhances the efficacy of subsequent ICI. Methods: We utilized SCC cell lines derived from K15.KrasG12D/Smad4−/− SCCs, which mimic the progression of HPV-unrelated HNSCC, and transplanted SCC cells into immune-competent C57BL/6J mouse recipients. Mice were subjected to RT followed by anti-PD-L1/anti-TGFb therapy. CD8+ splenic T cells of responder or non-responder mice were examined by paired single-cell VDJ sequencing and single cell RNA sequencing (TCR/sc-RNAseq) to look for adaptations of the cytotoxic T cell compartment following combination therapy. Responder and non-responder tumor lines or tumors were treated with RT alone in vitro and in vivo, respectively, to examine major histocompatibility complex I (MHC-I) levels on tumor cells. Results: Responders to this combination therapy exhibited complete tumor eradication while non-responders displayed rapid tumor progression. When re-challenged with same HNSCCs cells, responders rejected implanted tumor cells while naïve recipients rapidly developed HNSCCs. Single-cell TCR sequencing of splenic CD8+ T cells revealed that responders, but not naïve recipients exhibited multiclonal CD8+ T cell expansion specifically of memory cell populations. Responder tumor cells exhibited greater levels of MHC-I protein level presentation than non-responders when stimulated with RT. Conclusions: Our data suggest that 1) elevated MHC-I by SCC tumor cells presentation driven by RT and ICI combination therapy facilitate a systemic expansion of memory CD8+ T cell clonotypes which drive anti-tumor immunity and 2) MHC-I expression at baseline or when induced by RT may serve as a predictive marker for therapeutic response to RT and ICI combination therapy in HNSCCs. Citation Format: Hanne Lind, Alexander Strait, Spencer Hall, Jack B. Goon, John D. Aleman, Samantha Chen, Philip Owens, Jing H. Wang, Christian Young, Xiao-Jing Wang. Investigating the combination of radiation and subsequent immune checkpoint inhibitor treatment in head and neck squamous cell carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6646.