The effect of matrices on the gene expression profile of patient-derived head and neck carcinoma cells for in vitro therapy testing.

OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive tumor with a 5-year mortality rate of ~ 50%. New in vitro methods are needed for testing patients' cancer cell response to anti-cancer treatments. We aimed to investigate how the gene expression of fresh carcinoma tissue samples and freshly digested single cancer cells change after short-term cell culturing on plastic, Matrigel or Myogel. Additionally, we studied the effect of these changes on the cancer cells' response to anti-cancer treatments. MATERIALS/METHODS: Fresh tissue samples from HNSCC patients were obtained perioperatively and single cells were enzymatically isolated and cultured on either plastic, Matrigel or Myogel. We treated the cultured cells with cisplatin, cetuximab, and irradiation; and performed cell viability measurement. RNA was isolated from fresh tissue samples, freshly isolated single cells and cultured cells, and RNA sequencing transcriptome profiling and gene set enrichment analysis were performed. RESULTS: Cancer cells obtained from fresh tissue samples changed their gene expression regardless of the culturing conditions, which may be due to the enzymatic digestion of the tissue. Myogel was more effective than Matrigel at supporting the upregulation of pathways related to cancer cell proliferation and invasion. The impacts of anti-cancer treatments varied between culturing conditions. CONCLUSIONS: Our study showed the challenge of in vitro cancer drug testing using enzymatic cell digestion. The upregulation of many targeted pathways in the cultured cells may partially explain the common clinical failure of the targeted cancer drugs that pass the in vitro testing.

The effect of fascin 1 inhibition on head and neck squamous cell carcinoma cells.

Fascin 1 plays important pro-metastatic roles in head and neck carcinoma (HNSCC) migration, invasion, and metastasis. However, limited advancement in targeting metastasis remains a major obstacle in improving HNSCC patients' survival. Therefore, we assessed the therapeutic potential of fascin 1 targeted inhibition and its potential prognostic value in HNSCC patients. Using in vitro and in vivo approaches, we investigated the effect of compound G2, a novel fascin 1 inhibitor, on HNSCC cells migration, invasion, and metastasis. High-throughput screening (HTS) was used to assess cytotoxic activity of compound G2 alone or combined with irradiation. We also evaluated the prognostic potential of fascin 1 in HNSCC patients. Interestingly, compound G2 reduced carcinoma cells migration and invasion in vitro and inhibited metastasis in vivo. Moreover, HTS revealed a modest cytotoxic activity of the compound G2 on HNSCC cell lines. Irradiation did not synergistically enhance the compound G2-mediated cytotoxic activity. Survival analyses showed that high fascin 1 immunoexpression, at the tumor invasive front, was associated with cancer-specific mortality in the advanced stages of HNSCC. Collectively, our findings suggest that fascin 1 represents a promising anti-metastatic therapeutic target and a useful prognostic marker in patients with HNSCC. Novel anti-metastatic agents could provide a valuable addition to cancer therapy.

In vitro humanized 3D microfluidic chip for testing personalized immunotherapeutics for head and neck cancer patients.

OBJECTIVES: Immunotherapy and personalized medicine therapeutics are emerging as promising approaches in the management of head and neck squamous cell carcinoma (HNSCC). In spite of that, there is yet no assay that could predict individual response to immunotherapy. METHODS: We manufactured an in vitro 3D microfluidic chip to test the efficacy of immunotherapy. The assay was first tested using a tongue cancer cell line (HSC-3) embedded in a human tumour-derived matrix "Myogel/fibrin" and immune cells from three healthy donors. Next, the chips were used with freshly isolated cancer cells, patients' serum and immune cells. Chips were loaded with different immune checkpoint inhibitors, PD-L1 antibody and IDO 1 inhibitor. Migration of immune cells towards cancer cells and the cancer cell proliferation rate were evaluated. RESULTS: Immune cell migration towards HSC-3 cells was cancer cell density dependent. IDO 1 inhibitor induced immune cells to migrate towards cancer cells both in HSC-3 and in two HNSCC patient samples. Efficacy of PD-L1 antibody and IDO 1 inhibitor was patient dependent. CONCLUSION: We introduced the first humanized in vitro microfluidic chip assay to test immunotherapeutic drugs against HNSCC patient samples. This assay could be used to predict the efficacy of immunotherapeutic drugs for individual patients.

PCR-based zebrafish model for personalised medicine in head and neck cancer.

BACKGROUND: Currently, in vivo model for personalised cancer drug testing is challenging. A zebrafish larvae xenograft model has been applied in recent years to cancer research, particularly for drug testing purposes, showing promising results in drug testing against patient-derived tumour xenografts. Currently, these xenograft models apply imaging techniques to measure drug efficacy. However, this method carries several limitations, including timely imaging, thereby reducing the available number of tested fish and drugs. Here, we propose a PCR-based fast assay to evaluate drug efficacy in a zebrafish larvae xenograft model. METHODS: We tested two primary and corresponding metastatic head and neck squamous cell carcinoma (HNSCC) cell lines and patient-derived tongue cancer sample applying zebrafish larvae xenograft model. Cisplatin efficacy was tested using imaging technique and compared the results with PCR-based methods. Drug screening of eight compounds was applied on both cell lines and patient sample using PCR. RESULTS: In a head-to-head comparison, all the three techniques (imaging, quantitative PCR, and droplet digital PCR) showed similar reduction of the cancer cells growth after cisplatin treatment. Using the quantitative PCR assay, we demonstrated a dose-dependent response of HNSCC cells to cisplatin. Drug screening results of four HNSCC cell lines and patient sample revealed different drug efficacy between tested cancer cells. CONCLUSION: We introduce a novel, easy, fast and cost-effective PCR-based in vivo zebrafish larvae assay to test the response of cell lines and clinical tumour samples to anti-cancer drugs. This method goes hand-by-hand with the commonly used imaging assay.

IDO1 correlates with the immune landscape of head and neck squamous cell carcinoma: a study based on bioinformatics analyses.

Background: Head and neck squamous cell carcinoma (HNSCCs) is a common cancer type with a high mortality rate and poor prognosis. Recent studies have focused on the role of immune checkpoints in HNSCC progression and in their potential use as prognostic markers and immunotherapeutic candidates. Some immune checkpoints, such as PD-1 and PD-L1, have been studied thoroughly in HNSCC. Other molecules, such as indoleamine 2,3-dioxygenase 1 (IDO1), have been investigated minimally. Methods: IDO1 expression, prognostic potential, and association with the immune profile of HNSCC were explored using online databases, including GEPIA, UALCAN, TIMER2.0, cBioPortal, and LinkedOmics, which utilize TCGA datasets and are freely available for use. For validation purposes, seven pairs of primary and metastatic HNSCC were immunostained for IDO1. Results: Our analysis revealed significantly higher expression of IDO1 in HNSCC, especially in HPV+ SCCs compared with healthy control tissue. However, IDO1 expression showed weak to no prognostic potential for overall and disease-free survival in HNSCC. IDO1 expression in HNSCC was positively correlated with several immune-related molecules, including most of the immune checkpoints. Additionally, GO enrichment analysis revealed that several immune-related pathways are positively correlated with IDO1 expression in HNSCC, such as response to type I interferon and lymphocyte-mediated immunity pathways. Finally, IDO1 expression positively correlated with infiltration of most of the immune cells in HNSCC, such as CD4+ T cells, CD8+ T cells, M1 and M2 macrophages, dendritic cells, and B cells. Conclusion: IDO1 expression is closely correlated with the immune profile of the HNSCC. This observation should be explored further to elucidate the potential of targeting IDO1 as a novel immunotherapeutic approach for HNSCC.

Prognostic histological markers in oral tongue squamous cell carcinoma patients treated with (chemo)radiotherapy.

Treatment of oral tongue squamous cell carcinoma (OTSCC) frequently includes surgery with postoperative radiotherapy (RT) or chemoradiotherapy (CRT). Resistance to RT or CRT remains a major clinical challenge and highlights the need to identify predictive markers for it. We included 71 OTSCC patients treated with surgery combined with RT or CRT. We evaluated the association between tumor budding, tumor-stroma ratio (TSR), depth of invasion (DOI), tumor-infiltrating lymphocytes (TILs), hypoxia-inducible factor-1alpha (HIF-1alpha) expression, octamer-binding transcription factor 4 (OCT4) expression, high-endothelial venules (HEVs), and disease-free survival (DFS) using uni- and multivariate analyses. No significant association was observed between the different histological and molecular markers (TSR, DOI, TILs, HEV, HIF-1alph, OCT4) and DFS. However, an associative trend between DOI, budding, and DFS was noted. Further studies with larger cohorts are needed to explore the prognostic value of DOI and budding for OTSCC patients treated with postoperative RT or CRT.

Angiogenesis Inhibitors for Head and Neck Squamous Cell Carcinoma Treatment: Is There Still Hope?

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) carries poor survival outcomes despite recent progress in cancer treatment in general. Angiogenesis is crucial for tumour survival and progression. Therefore, several agents targeting the pathways that mediate angiogenesis have been developed. We conducted a systematic review to summarise the current clinical trial data examining angiogenesis inhibitors in HNSCC. METHODS: We carried out a literature search on three angiogenesis inhibitor categories-bevacizumab, tyrosine kinase inhibitors and endostatin-from Ovid MEDLINE, Cochrane Library, Scopus and ClinicalTrials.gov database. RESULTS: Here, we analysed 38 clinical trials, total of 1670 patients, investigating 12 angiogenesis inhibitors. All trials were in phase I or II, except one study in phase III on bevacizumab. Angiogenesis inhibitors were used as mono- and combination therapies together with radio-, chemo-, targeted- or immunotherapy. Among 12 angiogenesis inhibitors, bevacizumab was the most studied drug, included in 13 trials. Although bevacizumab appeared effective in various combinations, it associated with high toxicity levels. Endostatin and lenvatinib were well-tolerated and their anticancer effects appeared promising. CONCLUSIONS: Most studies did not show benefit of angiogenesis inhibitors in HNSCC treatment. Additionally, angiogenesis inhibitors were associated with considerable toxicity. However, some results appear encouraging, suggesting that further investigations of angiogenesis inhibitors, particularly in combination therapies, for HNSCC patients are warranted. SYSTEMATIC REVIEW REGISTRATION: PROSPERO (https://www.crd.york.ac.uk/prospero/), identifier CRD42020157144.

Evaluation Challenges in the Validation of B7-H3 as Oral Tongue Cancer Prognosticator.

B7-H3 was the only molecule identified with prognostic potential from a recent systematic review of the prognostic value of immune checkpoints in oral cancer. We aimed to validate this finding in a multicenter international cohort. We retrospectively retrieved 323 oral tongue squamous cell carcinoma (OTSCC) samples from three different countries (Brazil, Finland, and Norway) for immunostaining and scoring for B7-H3. We evaluated tumor immunogenicity by analyzing the amount of tumor-infiltrating lymphocytes and divided the tumors into immune hot and cold. To increase the reliability of the results, both digital and manual visual scoring were used. Survival curves were constructed based on the Kaplan-Meier method, and the Cox proportional hazard model was utilized for univariate and multivariate survival analysis. B7-H3 expression was not significantly associated with overall or disease-specific survival in the whole OTSCC cohort. When divided into immune hot and cold tumors, high B7-H3 expression was significantly associated with poor disease-specific and overall survival in the immune hot group, depending on the scoring method and the country of the cohort. This was achieved only in the univariate analysis. In conclusion, B7-H3 was a negative prognosticator for OTSCC patient survival in the subgroup of immune hot tumors, and was not validated as a prognosticator in the full cohort. Our findings suggest that the immune activity of the tumor should be considered when testing immune checkpoints as biomarkers.

High-throughput compound screening identifies navitoclax combined with irradiation as a candidate therapy for HPV-negative head and neck squamous cell carcinoma.

Conventional chemotherapeutic agents are nonselective, often resulting in severe side effects and the development of resistance. Therefore, new molecular-targeted therapies are urgently needed to be integrated into existing treatment regimens. Here, we performed a high-throughput compound screen to identify a synergistic interaction between ionizing radiation and 396 anticancer compounds. The assay was run using five human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) cell lines cultured on the human tumor-derived matrix Myogel. Our screen identified several compounds with strong synergistic and antagonistic effects, which we further investigated using multiple irradiation doses. Navitoclax, which emerged as the most promising radiosensitizer, exhibited synergy with irradiation regardless of the p53 mutation status in all 13 HNSCC cell lines. We performed a live cell apoptosis assay for two representative HNSCC cell lines to examine the effects of navitoclax and irradiation. As a single agent, navitoclax reduced proliferation and induced apoptosis in a dose-dependent manner, whereas the navitoclax-irradiation combination arrested cell cycle progression and resulted in substantially elevated apoptosis. Overall, we demonstrated that combining navitoclax with irradiation resulted in synergistic in vitro antitumor effects in HNSCC cell lines, possibly indicating the therapeutic potential for HNSCC patients.