Abstract. Nasopharyngeal carcinoma (NPC) is a type of cancer endemic in Asia, including Malaysia, Southern China, Hong Kong and Taiwan. Treatment resistance, particularly in recurring cases, remains a challenge. Thus, studies to develop novel therapeutic agents are important. Potential therapeutic compounds may be effectively examined using two-dimensional (2D) cell culture models, three-dimensional (3D) spheroid models or in vivo animal models. The majority of drug assessments for cancers, including for NPC, are currently performed with 2D cell culture models. This model offers economical and high-throughput screening advantages. However, 2D cell culture models cannot recapitulate the architecture and the microenvironment of a tumor. In vivo models may recapitulate certain architectural and microenvironmental conditions of a tumor, however, these are not feasible for the screening of large numbers of compounds. By contrast, 3D spheroid models may be able to recapitulate a physiological microenvironment not observed in 2D cell culture models, in addition to avoiding the impediments of in vivo animal models. Thus, the 3D spheroid model offers a more representative model for the study of NPC growth, invasion and drug response, which may be cost-effective without forgoing quality. Contents1. Introduction 2. 2D cell culture models 3. In vivo animal models 4. 3D spheroid models 5. 3D spheroid models for high-throughput drug screening 6. NPC 3D spheroids 7. Conclusion IntroductionNasopharyngeal carcinoma (NPC) is a type of cancer that affects the nasopharynx, commonly at the posterior and superior region in the fossa of Rosenmüller (1). It is a geographically distinct cancer, which is prevalent in south-east Asia, southern China and southern Africa (2). Viral, dietary, hereditary and lifestyle factors have been identified as risk factors for NPC (2). Current NPC treatment consists of radiotherapy, chemotherapy or chemo-radiotherapy; treatment resistance, particularly in advanced and recurrent cases of NPC, remains a challenge (2). NPC is staged according to the TNM system, whereby T describes the primary tumor invasion to the tissue or organs near the nasopharynx, N describes the spread to the lymph nodes and M indicates the metastasis of the tumor. NPC is usually detected at a late stage (III or IV) (2). Early-stage NPC has unspecific and ambiguous clinical symptoms such as neck lumps, bloodstained sputum, mild hearing loss and a unilateral headache which may be ignored by its sufferer or misdiagnosed by a doctor (3). Ultimately, this leads to a late disease presentation as well as detection. The pathogenesis of NPC involves genetic and epigenetic changes in the nasopharyngeal epithelium (4). Previous studies have improved current understanding of the potential molecular targets and signaling pathways involved in NPC pathogenesis, which has assisted the development of targeted therapies for the treatment of NPC, including cetuximab (Erbitux

The BCL-2 anti-apoptotic proteins are over-expressed in many cancers and hence are attractive therapeutic targets. In this study, we tested the sensitivity of two Nasopharyngeal Carcinoma (NPC) cell lines HK1 and C666-1 to Maritoclax, which is reported to repress anti-apoptotic protein MCL-1 and BH3 mimetic ABT-263, which selectively inhibits anti-apoptotic proteins BCL-2, BCL-XL and BCL-w. We investigated the sensitisation of the NPC cell lines to these drugs using the SYBR Green I assay and 3D NPC spheroids. We report that Maritoclax repressed anti-apoptotic proteins MCL-1, BCL-2, and BCL-XL in a dose- and time-dependent manner and displayed a single agent activity in inhibiting cell proliferation of the NPC cell lines. Moreover, combination of Maritoclax and ABT-263 exhibited synergistic antiproliferative effect in the HK1 cells. Similar results were obtained in the 3D spheroids generated from the HK1 cells. More notably, 3D HK1 spheroids either treated with single agent Maritoclax or combination with ABT-263, over 10 days, did not develop resistance to the treatment rapidly. Collectively, the findings illustrate that Maritoclax as a single agent or combination with BH3 mimetics could be potentially useful as treatment strategies for the management of NPC.

Spheroids have been shown to recapitulate the tumour in vivo with properties such as the tumour microenvironment, concentration gradients, and tumour phenotype. As such, it can serve as a platform for determining the growth and invasion behaviour pattern of the cancer cells as well as be utilised for drug sensitivity assays; capable of exhibiting results that are closer to what is observed in vivo compared to two-dimensional (2D) cell culture assays. This study focused on establishing a three-dimensional (3D) cell culture model using the Nasopharyngeal Carcinoma (NPC) cell line, HK1 and analysing its growth and invasion phenotypes. The spheroids will also serve as a model to elucidate their sensitivity to the chemotherapeutic drug, Flavopiridol. The liquid overlay method was employed to generate the spheroids which was embedded in bovine collagen I matrix for growth and invasion phenotypes observation. The HK1 cells formed compact spheroids within 72 hours. Our observation from the 3 days experiments revealed that the spheroids gradually grew and invaded into the collagen matrix, showing that the HK1 spheroids are capable of growth and invasion. Progressing from these experiments, the HK1 spheroids were employed to perform a drug sensitivity assay using the chemotherapeutic drug, Flavopiridol. The drug had a dose-dependent inhibition on spheroid growth and invasion.