Abstract A72: Prostate cancer spheroid formation in a 384 hanging drop array plate.
Authors: Camacho Daniel F. , Hsiao Amy Y. , Takayama Shuichi , Pienta Kenneth J. 
Published: June 2012

Abstract
Background: Bridging the gap between in vitro and in vivo studies is critical to therapeutic advancement. Achieving more physiological tumor models in vitro will attenuate dependence on preclinical animal testing and facilitate more accurate prediction of a compound's antitumor behavior in vivo. Multicellular tumor spheroids are 3-dimensional, self-assembled clusters of cancer cells formed in an environment where cell-cell interactions dominate in the absence of cell-surface interactions. A spheroid composed of prostate cancer cells, or prostatosphere, not only provides a 3-dimensional tumor model, but also allows the formation of extracellular matrix as would occur in vivo. Thus, drug screening performed on spheroids should yield results of greater physiological significance than screening performed on 2-dimensional tissue cultures. Furthermore, the use of a 384 hanging drop array plate has previously been shown to enable the convenient formation of spheroids from COS7, mES, and A431.H9 cell lines[1]. Here we chronicle the compatibility of the 384 hanging drop array plate with four prostate cancer cell lines for prostatosphere formation.
Methods: Prostate cancer cell suspensions were deposited into staggered access holes on the 384 hanging drop array plate, creating 15 to 20 L hanging drops with cell concentrations ranging from 500 to 5000 cells per drop. In order to arrest drop evaporation, the hanging drop array plate was sandwiched between a lid and 96-well plate filled with distilled water and was wrapped in Parafilm. Drops were observed using bright field microscopy and imaged daily (Olympus IX71). The image processing software ImageJ was used to obtain spheroid dimensions, and mean spheroid diameter was calculated using the following equation: d = (a × b)∘(1/2), where a and b are orthogonal diameters of the spheroid. Average size was reported as mean diameter ± standard deviation and was measured on the second day after spheroid formation was observed. Results: Spheroid formation was observed within 24 hours for LNCaP and LAPC4 cells, and within 48 hours for VCaP and DU145 cells. LNCaP, LAPC4, and VCaP spheroids characteristically formed one single spheroid per drop, with larger spheroids obtained at higher cell concentrations. These three cell lines exhibited round and oblong spheroid morphologies at low cell concentration, with spheroid shape becoming more irregular, branched, or partially hollow at higher cell concentrations. Hanging drops containing DU145 cells tended to contain multiple spheroids of smaller diameter. Thus, DU145 spheroids were usually limited to round or oblong morphologies. DU145 spheroids attained an average size of 72.1 ± 43.7 microns.
Conclusion: These prostate cancer cell lines are shown to form prostatospheres when cultured in the 384 hanging drop array plate. Preliminary evidence suggests that additional prostate cancer cell lines are also capable of doing so. Our group intends to screen antitumor therapeutics on prostatospheres in comparison to prostate cancer cells under traditional 2-dimensional tissue culture conditions. Co-culture spheroid formation incorporating prostate cancer cells, endothelial cells, and osteoblasts to simulate the tumor microenvironment is expected, as is drug screening on such spheroids.
References:
1. Y. Tung, A. Y. Hsiao, S. G. Allen, Y. Torisawa, M. Ho, and S. Takayama, Analyst, 2011, 136, 473–478.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A72.