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

Background and Objectives: Pfu DNA polymerase is an enzyme that exhibits the lowest error rate in the 3´ to 5´ exonucle- ase (proofreading) activity during DNA synthesis in Polymerase Chain Reactions (PCRs). This study was aimed to express and purify Pfu DNA polymerase in a bacterial expression system under a simple purification method. Materials and Methods: Pfu polymerase gene sequence, derived from Pyrocuccus furiosus (Pfu) genomic DNA, was cloned and overexpressed in E. coli BL21 (DE3) pLysS. Upon overexpression, bacterial lysate containing the Pfu DNA polymerase was heated at 94°C for 5 minutes. Pfu DNA polymerase having high thermal stability was retained while the other bacterial proteins were denatured. The resulting thermo stable Pfu DNA polymerase was separated from the other debris of the dena- tured proteins by simple centrifugation. Results: The enzymatic activity of the resulting Pfu DNA polymerase was estimated by comparing with the commer- cial Pfu DNA Polymerases. An estimated 50000 units of functional Pfu DNA polymerase was produced from a 400 ml culture. Conclusion: The in-house produced Pfu DNA Polymerase could be used for routine amplification that requires high-fidelity such as cloning and DNA sequencing.

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.