Stroma modulation of radiation response in head and neck squamous cell carcinoma: Insights from zebrafish larvae xenografts.

BACKGROUND: The tumour microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC) consists of different subtypes of cells that interact with the tumour or with each other. This study investigates the possibility of co-culturing HNSCC cells with different stroma cells in a zebrafish xenograft model, focusing on the effect of stroma cells on HNSCC growth and response to irradiation. MATERIAL AND METHOD: HNSCC metastatic cell line HSC-3 was used along with five types of stroma cells: normal gingival fibroblasts (NOF), cancer associated fibroblasts (CAF), macrophages, CD4+ T cells, and human umbilical vein endothelial cells (HUVEC). The mixture of HSC-3 cells and each-stroma cell type-was injected into 2-day post-fertilization zebrafish embryos, and the effect of stroma cells on tumour growth was tested. The study also aimed to mimic the HNSCC tumour by injecting a mixture of HSC-3 cells, CAFs, macrophages, and HUVECs into zebrafish embryos and testing the effect of these stroma cells on the cancer cells' response to irradiation compared to HSC-3-only tumours. RESULTS: CAFs had a significant inducement effect on tumour size, while HUVECs showed the opposite effect. The irradiated group of HSC-3-only tumour had a significantly smaller tumor cell area compared to the control, while the group with stroma cells and HSC-3 cells showed cancer cells being resistant to irradiation. CONCLUSION: This is the first report of co-culturing cancer cells with several types of stroma cells using a zebrafish xenograft model. This study also highlighted the role of stroma cells in turning the cancer cells from radioresponsive to radioresistant.

Novel human lymph node-derived matrix supports the adhesion of metastatic oral carcinoma cells.

BACKGROUND: 3D culture is increasingly used in cancer research, as it allows the growth of cells in an environment that mimics in vivo conditions. Metastases are the primary cause of morbidity and mortality in cancer patients, and solid tumour metastases are mostly located in lymph nodes. Currently, there are no techniques that model the pre-metastatic lymph node microenvironment in vitro. In this study, we prepared a novel extracellular matrix, Lymphogel, which is derived from lymph nodes, mimicking the tumour microenvironment (TME) of metastatic carcinoma cells. We tested the suitability of the new matrix in various functional experiments and compared the results with those obtained using existing matrices. METHODS: We used both commercial and patient-derived primary and metastatic oral tongue squamous cell carcinoma (OTSCC) cell lines. We characterized the functional differences of these cells using three different matrices (human uterine leiomyoma-derived Myogel, human pre-metastatic neck lymph node-derived Lymphogel (h-LG), porcine normal neck lymph node-derived Lymphogel (p-LG) in proliferation, adhesion, migration and invasion assays. We also performed proteomic analyses to compare the different matrices in relation to their functional properties. RESULTS: OTSCC cells exhibited different adhesion and invasion patterns depending on the matrix. Metastatic cell lines showed improved ability to adhere to h-LG, but the effects of the matrices on cell invasion fluctuated non-significantly between the cell lines. Proteomic analyses showed that the protein composition between matrices was highly variable; Myogel contained 618, p-LG 1823 and h-LG 1520 different proteins. The comparison of all three matrices revealed only 120 common proteins. Analysis of cellular pathways and processes associated with proteomes of each matrix revealed similarities of Myogel with h-LG but less with p-LG. Similarly, p-LG contained the least adhesion-related proteins compared with Myogel and h-LG. The highest number of unique adhesion-related proteins was present in h-LG. CONCLUSIONS: We demonstrated that human pre-metastatic neck lymph node-derived matrix is suitable for studying metastatic OTSCC cells. As a whole-protein extract, h-LG provides new opportunities for in vitro carcinoma cell culture experiments.

The critical effects of matrices on cultured carcinoma cells: Human tumor-derived matrix promotes cell invasive properties.

The interaction between squamous cell carcinoma (SCC) cells and the tumor microenvironment (TME) plays a major role in cancer progression. Therefore, understanding the TME is essential for the development of cancer therapies. We used four (primary and metastatic) head and neck (HN) SCC cell lines and cultured them on top of or within 5 matrices (mouse sarcoma-derived Matrigel®, rat collagen, human leiomyoma-derived Myogel, human fibronectin and human fibrin). We performed several assays to study the effects of these matrices on the HNSCC behavior, such as proliferation, migration, and invasion, as well as cell morphology, and molecular gene profile. Carcinoma cells exhibited different growth patterns depending on the matrix. While fibrin enhanced the proliferation of all the cell lines, collagen did not. The effects of the matrices on cancer cell migration were cell line dependent. Carcinoma cells in Myogel-collagen invaded faster in scratch wound invasion assay. On the other hand, in the spheroid invasion assay, three out of four cell lines invaded faster in Myogel-fibrin. These matrices significantly affected hundreds of genes and a number of pathways, but the effects were cell line dependent. The matrix type played a major role in HNSCC cell phenotype. The effects of the ECMs were either constant, or cell line dependent. Based on these results, we suggest to select the most suitable matrix, which provides the closest condition to the in vivo TME, in order to get reliable results in in vitro experiments.

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.

Evaluation of BH3 mimetics as a combination therapy with irradiation in head and neck squamous cell carcinoma.

INTRODUCTION: Head and neck squamous cell carcinoma (HNSCC) is a common cancer with a five-year survival rate around 60%, indicating a need for new treatments. BH3 mimetics are small molecules that inhibit anti-apoptotic Bcl-2 family proteins, resulting in apoptosis induction. METHODS: We performed a high-throughput screen using a Myogel matrix to identify the synergy between irradiation and the novel BH3 mimetics A-1155463, A-1331852, and navitoclax in 12 HNSCC cell lines, normal (NOF) and cancer-associated fibroblasts (CAF), and dysplastic keratinocytes (ODA). Next, we examined synergy in an apoptosis assay, followed by a clonogenic assay and a Myogel spheroid on selected HNSCC cell lines. Finally, we applied zebrafish larvae xenograft to validate the effects of navitoclax and A-1331852. RESULTS: All three BH3 mimetics exhibited a strong synergy with irradiation in eight HNSCC cell lines and ODAs, but not in NOFs and CAFs. A-1155463 and A-1331852 induced apoptosis and reduced proliferation, and together with irradiation, significantly increased apoptosis and arrested proliferation. A-1331852 and navitoclax significantly decreased the clonogenicity compared with the control, and combination treatment led to a decreased clonogenicity compared with monotherapy or irradiation. However, unlike navitoclax or A-1155463, only A-1331852 significantly reduced cancer cell invasion. Furthermore, in spheroid and zebrafish, irradiation appeared ineffective and failed to significantly increase the drug effect. In the zebrafish, A-1331852 and navitoclax significantly reduced the tumor area and metastasis. CONCLUSIONS: Our findings encourage the further preclinical investigation of BH3 mimetics, particularly A-1331852, as a single agent or combined with irradiation as a treatment for HNSCC.

Evaluation of in vitro and in vivo personalized cancer treatment assays for oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common cancer with a high heterogeneity and few approved treatments. OSCC is one of the least explored areas for precision oncology. In this study, we aimed to test the reliability of our three established rapid cancer systemic treatment-testing assays: human tumour-derived matrix (Myogel)-coated well-plates, zebrafish xenografts, and 3D microfluidic chips. METHODS: Chemo-, radio- and targeted-therapy testing in Myogel-coated wells and zebrafish xenografts was conducted nine times using five samples; two primary and three metastatic lymph node samples from three OSCC patients. Peripheral blood mononuclear cells (PBMNCs) were isolated from the patients' blood. The response of the tumour cells to radio-, chemo-, and targeted therapy was tested using Myogel-coated wells and zebrafish larvae xenografts. The tumour cells' response to immunotherapy was tested using 3D microfluidic chips. The cells' sensitivity to the treatments was compared with the patients' clinical response. Primary and metastatic lymph node tissue-derived DNA samples from two patients underwent whole exome sequencing to compare the mutational profiles of the samples. RESULTS: Test results were in line with patients' responses in 7/9 (77%) zebrafish xenograft assays and 5/9 (55%) Myogel-coated wells assays. Immunotherapy testing was done using one metastatic patient sample which matched the patients' response. Differences in responses to treatments between primary and metastatic samples of the same patient were detected in 50% of the zebrafish larvae assays. CONCLUSIONS: Our results show the potential of using personalized cancer treatment testing assays - specifically zebrafish xenografts that revealed promising results - in OSCC patient samples.

Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix.

Oxygen tension varies during placental and fetal development. Although hypoxia drives early trophoblast invasion, low placental oxygen levels during pregnancy show association with pregnancy complications including fetal growth restriction and preeclampsia. JEG-3 cells are often used as a trophoblast model. We studied transcriptional changes of JEG-3 cells on a uterine leiomyoma derived matrix Myogel. This might be the closest condition to the real uterine environment that we can get for an in vitro model. We observed that culturing JEG-3 cells on the leiomyoma matrix leads to strong stimulation of ribosomal pathways, energy metabolism, and ATP production. Furthermore, Myogel improved JEG-3 cell adherence in comparison to tissue culture treated plastic. We also included PDMS microchip hypoxia creation, and observed changes in oxidative phosphorylation, oxygen related genes and several hypoxia genes. Our study highlights the effects of Myogel matrix on growing JEG-3 cells, especially on mitochondria, energy metabolism, and protein synthesis.

Stress induced phosphoprotein 1 overexpression controls proliferation, migration and invasion and is associated with poor survival in oral squamous cell carcinoma.

Objective: Although there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs. Methods: STIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection. Results: STIP1 was highly expressed in OSCCs and significantly associated with shortened survival and higher risk of recurrence. STIP1 down-regulation decreased proliferation, migration and invasion of tumor cells, and reduced the number of metastases in the Zebrafish model. STIP1 and miR-218-5p were inversely expressed, and the transfection of miR-218-5p mimics into OSCC cells decreased STIP1 levels as well as proliferation, migration and invasion. Conclusion: Our findings show that STIP1 overexpression, which is inversely associated with miR-218-5p levels, contributes to OSCC aggressiveness by controlling proliferation, migration and invasion and is a determinant of poor prognosis.

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.

Tumour cells express functional lymphatic endothelium-specific hyaluronan receptor in vitro and in vivo: Lymphatic mimicry promotes oral oncogenesis?

Lymphatic metastasis represents the main route of tumour cell dissemination in oral squamous cell carcinoma (OSCC). Yet, there are no FDA-approved therapeutics targeting cancer-related lymphangiogenesis to date. The lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1), a specific lymphatic marker, is associated with poor survival in OSCC patients. In this study, we present a potential novel mechanism of lymphatic metastasis in OSCC-lymphatic mimicry (LM), a process whereby tumour cells form cytokeratin+/LYVE-1+, but podoplanin-negative, mosaic endothelial-like vessels. LM was detected in one-third (20/57; 35.08%) of randomly selected OSCC patients. The LM-positive patients had shorter overall survival (OS) compared to LM-negative group albeit not statistically significant. Highly-metastatic tumour cells formed distinct LM structures in vitro and in vivo. Importantly, the siRNA-mediated knockdown of LYVE-1 not only impaired tumour cell migration but also blunted their capacity to form LM-vessels in vitro and reduced tumour metastasis in vivo. Together, our findings uncovered, to our knowledge, a previously unknown expression and function of LYVE-1 in OSCC, whereby tumour cells could induce LM formation and promote lymphatic metastasis. Finally, more detailed studies on LM are warranted to better define this phenomenon in the future. These studies could benefit the development of targeted therapeutics for blocking tumour-related lymphangiogenesis.

Bilberry (Vaccinium myrtillus L.) Powder Has Anticarcinogenic Effects on Oral Carcinoma In Vitro and In Vivo.

Previous studies indicate that bilberry with high amounts of phenolic compounds can inhibit carcinogenic processes of colorectal cancer in vitro and in vivo. However, no studies have focused on the effects of bilberry on oral cancer. In this study, we aimed to examine the effects of bilberry powder on oral squamous cell carcinoma (OSCC) cells using both in vitro and in vivo assays. The effects of 0, 1, 10, and 25 mg/mL of whole bilberry powder on the viability, proliferation, migration, and invasion of OSCC (HSC-3) cells were examined and compared with 0.01 mg/mL of cetuximab. Two oral keratinocyte cell lines served as controls. Tumor area was analyzed in zebrafish microinjected with HSC-3 cells and treated with 2.5, 10, or 25 µg/mL of bilberry powder. Metastases in the head or tail areas were counted. Bilberry powder inhibited the viability, proliferation, migration, and invasion of HSC-3 cells (p < 0.05), which was more pronounced with higher concentrations. Cetuximab had no effect on HSC-3 cell migration or invasion. Compared to controls, the tumor area in zebrafish treated with bilberry powder (10 and 25 µg/mL) was reduced significantly (p = 0.038 and p = 0.021, respectively), but the number of fish with metastases did not differ between groups. Based on our in vitro and in vivo experiments, we conclude that whole bilberry powder has anti-tumor effects on OSCC cells.

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.