Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiologically representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qualitative analysis including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiological relevance of these models continues to improve.

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents.

Cancer causes major patient morbidity and mortality and is a critical health concern worldwide. The recent GLOBOCAN 2019 factsheet recorded nearly 19.2 million new cancer cases, 9.9 million cancer deaths and 50.55 million people suffering from different kinds of cancer globally within 5 years after diagnosis. Growth factors (GF) are a group of proteins that can affect cellular processes, including differentiation, division, intravasation, extravasation and dissemination. The circulating tumor cells in the bloodstream can populate distant tissues and organs and believe to be the primary cause of metastasis. Extravasation is a crucial phase in the metastasis process, in which tumor cells leave the bloodstream and enter the host tissue. The progress of metastasis is triggered by the tendency of cancer cells to disseminate to target organs from the site of the primary tumor. Despite extensive basic scientific and clinical investigations, cancer is still a major clinical and public health problem. The development of cancer can be influenced by genetics, environmental factors, gene-environment interaction, lifestyle, age and a number of other factors. The harnessing and enhancement of the body’s own cytotoxic cells to prevent basement membrane rupture and the intervening dissemination processes can provide useful insight into the development of cancer. The mutation in oncogenes and tumour suppressor genes, and chromosomal aberration is a cornerstones of the molecular basis of cancer. The basement Membrane (BM) acts as a cell invasion shield, thus identification of processes that underlie in breaching of BM can contribute to understanding the disease pathogenesis. TGF-β is known for its dual function; it requires inhibition in the advanced stage however, the growth inhibitory properties are displayed in the early stages of tumorigenesis. Therefore, inhibition of TGF-β signalling in the CD8+ T cell compartment may be necessary for tumor immunity to be restored. Quantitation of tumour cell dissemination is important and plays significant role in elucidating mechanisms of cancer and strategies for therapeutic intervention.

Immunotherapy is one of the important modalities in the treatment of cancer since it can directly target the tumor and its microenvironment with lesser side effects and cytotoxicity. The main goal of immunotherapy in the treatment of cancer is the reactivation of the immune system against cancer cells. In this way, the body fights against cancer using its immune system rather than relying on external agents which might be harmful to other healthy parts of the body. The development of monoclonal antibodies (Mabs) has delivered a significant therapeutic effect. Mab therapy is one of the most evolving techniques in cancer immunotherapy and has shown efficacy in controlling several types of malignancies. There are several other methods by which the activation of the immune system can be achieved, such as by using small molecules or by targeting ligands. Interestingly, studies have demonstrated that cancer stem cells have also been found as a target for effective immunotherapy. Additionally, the complete elimination of the cancer cells requires longer sustainability of tumor-specific T cells. Primitive results suggest that these T cells can be localized to tumor cells, mediating highly effective immunotherapy. However, despite these huge successes, several problems still persist and must be overcome. This chapter discusses the current and cutting-edge immunotherapeutic approaches to fight against cancer cells.