title: Quantitative and Qualitative Characterisation of Head and Neck Tumoroid Cultures With and Without Extracellular Matrix
Creators: name: Wong, Michael
Publisher: name: Griffith University
Year: 2024
Subjects: subject: organoids
subject: oncology
subject: hydrogels
subject: squamous cell carcinoma (SCC)

description: Head and neck malignancy, and in particular squamous cell carcinoma (SCC), is responsible for a significant disease burden globally. Head and neck SCC (HNSCC) epidemiology has changed over the past 40 years, with human papillomavirus infection now demonstrated to be a major causative factor in rising rates of oral cavity and oropharyngeal mucosal SCC. The lack of an optimal in vitro model system to accurately recapitulate in vivo responses to therapy in HNSCC remains a challenge. The development of tumour-derived three-dimensional cultures known as tumour organoids, or tumoroids, has provided for improved modelling of the tumour microenvironment through simulation of important characteristics such as tumour hypoxia, cell-cell interactions, and nutrient diffusion characteristics. In turn, this has revealed broad promise in translational research towards the characterisation of cancer therapy responses within the domain of precision medicine. Current methods of tumoroid development utilise Matrigel® and CultrexTM Basement Membrane Extract 2 - two common proprietary murine-derived hydrogels containing extracellular matrix proteins. While useful for facilitating cell-matrix interactions and providing a scaffold for three-dimensional cell growth and organisation, these have notable limitations. Hydrogels are temperature sensitive and liable to solidification which can impede the addition and removal from supernatant of chemotherapeutic agents, secreted vesicles, and growth factors. Their murine origin and absence of human immune cells are also inherently suboptimal in creating the correct substrate for the culture and modelling of human cancers. There exists a need for further optimisation of these organotypic models as a platform for novel drug discovery and drug testing. A protocol that reliably and reproducibly facilitates the propagation of tumoroids from a heterogeneous patient-derived tumour specimens will be invaluable in achieving this aim. A novel hydrogel-free method of establishing in vitro tumoroid cultures has been subject to experimentation in colorectal malignancy but not in head and neck malignancy. The absence of a hydrogel provides for the de novo synthesis of extracellular matrix native to the tumour and self-organisation of cells within this scaffold through the use of ultralow attachment plates. This model demonstrates similar structural and physiological properties to native tissue, whilst enabling more accurate biomimicry of the tumour microenvironment for drug testing. We applied a novel hydrogel-free method to establish an end-to-end protocol for the development of HNSCC tumoroids and compared them quantitatively and qualitatively to existing hydrogel-based methods using CultrexTM and Matrigel®. A total of 26 successful tumoroid lines were created using the three culture methods from 27 patient-derived tumour samples, representing the highest rate of success in the literature. These patient-derived tissues encompassed the breadth of the upper aerodigestive tract from a broad, heterogeneous demographic. Tumoroids developed without hydrogels demonstrated significantly higher logarithmic growth over a 14-day incubation period compared to their hydrogel-based counterparts. Importantly, increased cell proliferation was observed in proportion to lower initial cell counts in the hydrogel-free model, further highlighting its ability to replicate in vivo cancers, which typically start with small numbers of cells. Further characterisation of the tumoroids with static interval imaging and timelapse imaging provided opportunities for qualitative assessment. Dominant tumoroid morphologies of heterogenous size and density were viewed under low magnification and observed to increase in size exponentially with static imaging. However, the timelapse imaging experiment was not successful secondary to a number of logistical, hardware and so\ware issues experienced. Immunofluorescence of key cell elements including pancytokeratin and cell-surface vimentin confirmed the presence of tumoroid organisation, diseased squamous epithelium, and de novo synthesis of cytoskeletal elements. The presence of tumour hypoxia is a key phenomenon of the tumour microenvironment and was demonstrated in one of the hydrogel-free tumoroids. An assay of metabolic activity demonstrated dose-dependent responses to chemotherapeuGc agents used in treatment of HNSCC. These were independent of the culture methods, indicating the utility of the tumoroids in in vitro drug testing. This study iterates our understanding of tumour organoids in HNSCC and at large, by introducing hydrogel-free culture as a novel method achieving greater cell yield and closer recapitulation of the tumour microenvironment. The results are promising for groups looking to replicate the method at scale and serve as a gateway to furthering the application of this work to precision medicine.