RAPID MICROWELL PROTOTYPING, GENERATION OF 3D MULTICEL- LULAR CANCER AGGREGATES, AND EMT DRUG SCREENING Ting-Yuan Tu 1,3 , Wei Sun 1 , Weng Kung Peng 1 , Zhe Wang 1 , Ruby Y.J. Huang 4 , Paul T. Matsudaira 3 , Jean-Paul Thiery 2 , Roger D. Kamm 1* 1 Singapore-MIT Alliance for Research and Technology (SMART) Center, Singapore 2 Institute of Molecular Cell Biology (IMCB), A-STAR, Singapore 3 Mechanobiology Institute (MBI), National University of Singapore, Singapore 4 Cancer Science Institute (CSI), National University of Singapore, Singapore ABSTRACT This work presents a microwell prototyping technique for generating multicellular cancer aggregates in suspension cul- ture for epithelial-mesenchymal-transition (EMT) drug screening. Microwell and aggregate size and geometry were ex- amined in PMMA, PDMS, polystyrene materials materials. Retrieved aggregates were further studied in terms of cell dis- persion through drug screening, to identify the effective drug dosage for the inhibition of EMT. Two distinct cell phenotypic behaviors were discovered from Low-attachment-dish- and microwell-generated aggregates under different conditions. Fu- ture studies of the two types of aggregates may contribute to the understanding of cancer metastasis. KEYWORDS: Microwell, Epithelial-mesenchymal-transition, Drug screening, CO 2 laser INTRODUCTION EMT has been known to play an important role in cancer metastasis[1]. Studying EMT using 3D multicellular aggre- gates presumably provides a better in vivo tumor simulation. There exist various approaches for the generation of aggregates[2-4], e.g. conventional low attachment dish (LAD)[2], hanging drop methods[3], or microstructure confinement[4]. However, these methods either failed to achieve precise control of aggregates size[2, 3] or required a rela- tively sophisticated MEMs fabrication process[4]. Moreover, by judging the chemo-toxicity on viability of cancer aggre- gates[3, 4] may overlook the EMT-induced populations shown to exhibit increased resistance to drug treatment[5]. Therefore, we propose a simple CO 2 laser prototyping technique featuring rapid and ease of fabrication for generating cancer aggregates. Retrieved aggregates are further studied in terms of cell dispersion through drug screening, to identify the effective drug dos- age and signaling pathways for the inhibition of EMT. EXPERIMENTAL We use a 10W CO 2 laser system to ablate the substrate in single hole drilling mode. Substrate removal through ablation causes a Gaussian-profile of hole feature suitable for cells to form aggregates. After four to five days, we retrieved aggregates from microwell and re-plate them in 3D a polymerized collagen-type I environment for dispersion assay. Due to phenotypic change through EMT, cells gain migratory capacity and capable of disseminating from a cell cluster within short period of time. We observe aggregates using microscopy to determine the effective anti-EMT drug dosage for inhibiting cell disper- sion. RESULTS AND DISCUSSION Polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), and polystyrene (PS) materials were quantitatively and qualitatively investigated under laser power from 1W-5W (Fig. 1). Representative hole features (Fig. 1(a)) show marked differences both between materials and as a function of substrate material. Diameter, depth, and aspect ratio of different ma- terials of microwell were quantified, (Fig. 1(b)). In order to more closely examine microwell structure, isometric-view SEM images were obtained for three materials (Fig. 2). Surface profiles are presented in overview (left), single microwell (middle), and inner view (right). PMMA microwells exhibited an uneven surface at the periphery, with micro-size pored distributed over much of the internal surface. PDMS mi- crowells, in contrast, possessed a smooth surface, but optical resolution was impeded by the sharp geometry of the bottom. In order to test these microwells for aggregate formation, A549 lung cancer cells were seeded in the PS microwell and were observed to gradually aggregate with a blurred cell-cell interface, (Fig. 3(a)). There was a weak correlation in size be- tween aggregates and microwell (Fig 3(b) (left)). Cell seeding concentration was varied over 1, 5 & 10 x 10 5 cells/ml, alt- hough this had surprisingly little effect on the probability of forming a aggregate or the resulting aggregate size. 978-0-9798064-4-5/μTAS 2011/$20©11CBMS-0001 82 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA