Advancing preclinical evaluation : 3D and Zebrafish models for head and neck cancer treatment testing

Head and neck squamous cell cancer (HNSCC) ranks as the eighth most prevalent cancer globally, often diagnosed at a locally advanced stage in approximately 60% of HNSCC patients. Despite strides in cancer diagnosis and treatment, therapy failure leading to local recurrence and the development of metastatic tumors remains a significant challenge. Additionally, HNSCC exhibits considerable heterogeneity, resulting in varied treatment responses within the patient population. Given this diversity, there is a pressing need for an individualized treatment approach, and better preclinical models to test different therapies in the most convenient and in vivo like conditions, in order to predict treatment responses. The development of better preclinical models represents a promising and innovative avenue in cancer therapy, aiming to enhance the efficacy of anti-cancer treatments and alleviate the burden on individuals diagnosed with HNSCC. This study encompasses three interconnected investigations, shedding light on environment-dependent cell behaviors, personalized treatment testing assays, and the impact of stroma cells on HNSCC growth and response to treatment. The objective of this study is to facilitate effective drug testing for HNSCC through the utilization of diverse in vitro and in vivo models. The primary goals encompass elucidating the impact of various culture matrices on the behavioral patterns of HNSCC cells, envisioning personalized cancer treatment assays tailored for HNSCC, exploring the predictive capabilities of novel models in evaluating the responses of HNSCC patients to diverse therapeutic interventions, and ultimately emphasizing the critical role of employing the appropriate tumour microenvironment (TME) to influence the responsiveness of HNSCC cells to treatment modalities. Extracellular matrix (ECM) is a major component of the TME that interacts closely with HNSCC cells. It also regulates tumour behaviour, progression, and responsiveness to therapy. The ECM, which is made up of proteins, glycoproteins, and polysaccharides, gives surrounding cells structural and functional support. Changes in ECM composition contribute to tumour invasion, metastasis, and treatment resistance in HNSCC. Considering the previous, we studied the effect of different ECM components in form of 3D matrices, on the cell behavior. We cultured four primary and metastatic HNSCC cell lines on five matrices (Matrigel, collagen, Myogel, fibronectin, and fibrin). Matrix effects were evaluated through assays evaluating proliferation, migration, invasion, cell morphology, and molecular gene profiles. The matrix type significantly influenced HNSCC cell phenotype, exhibiting both constant and cell line-dependent effects. The study indicates the importance of selecting matrices that closely mimic the in vivo TME for reliable in vitro experiments. In the quest for precision oncology in oral squamous cell carcinoma (OSCC), three rapid systemic treatment-testing assays—Myogel-coated wells, zebrafish xenografts, and 3D microfluidic chips—were assessed for their reliability. These assays were applied to test chemo-, radio-, targeted-therapy, and immunotherapy using samples from OSCC patients. Results demonstrated a correlation between assay outcomes and patients' clinical responses, with promising potential identified in zebrafish xenografts. The study highlights the feasibility and benefit of personalized cancer treatment testing assays in OSCC patient samples. The third study investigated co-culturing HNSCC cells with various stroma cells in a zebrafish xenograft model. The effects of normal gingival fibroblasts (NOF), cancer-associated fibroblasts (CAF), macrophages, CD4+ T cells, and human umbilical vein endothelial cells (HUVEC) on HNSCC growth and response to irradiation were explored. CAFs induced significant tumor growth, while HUVECs exhibited an opposite effect. Additionally, the study revealed stroma cells' role in rendering cancer cells radioresistant. This pioneering investigation presents a novel approach to study stroma cells interactions in HNSCC using a zebrafish xenograft model.