Carrie J. Lovitt

Advanced Cell Culture Techniques for Cancer Drug Discovery

Human cancer cell lines are an integral part of drug discovery practices. However, modeling the complexity of cancer utilizing these cell lines on standard plastic substrata, does not accurately represent the tumor microenvironment. Research into developing advanced tumor cell culture models in a three-dimensional (3D) architecture that more prescisely characterizes the disease state have been undertaken by a number of laboratories around the world. These 3D cell culture models are particularly beneficial for investigating mechanistic processes and drug resistance in tumor cells. In addition, a range of molecular mechanisms deconstructed by studying cancer cells in 3D models suggest that tumor cells cultured in two-dimensional monolayer conditions do not respond to cancer therapeutics/compounds in a similar manner. Recent studies have demonstrated the potential of utilizing 3D cell culture models in drug discovery programs; however, it is evident that further research is required for the development of more complex models that incorporate the majority of the cellular and physical properties of a tumor.

Cancer drug discovery: recent innovative approaches to tumor modeling

ABSTRACT Introduction: Cell culture models have been at the heart of anti-cancer drug discovery programs for over half a century. Advancements in cell culture techniques have seen the rapid evolution of more complex in vitro cell culture models investigated for use in drug discovery. Three-dimensional (3D) cell culture research has become a strong focal point, as this technique permits the recapitulation of the tumor microenvironment. Biologically relevant 3D cellular models have demonstrated significant promise in advancing cancer drug discovery, and will continue to play an increasing role in the future. Areas covered: In this review, recent advances in 3D cell culture techniques and their application in tumor modeling and anti-cancer drug discovery programs are discussed. The topics include selection of cancer cells, 3D cell culture assays (associated endpoint measurements and analysis), 3D microfluidic systems and 3D bio-printing. Expert opinion: Although advanced cancer cell culture models and techniques are becoming commonplace in many research groups, the use of these approaches has yet to be fully embraced in anti-cancer drug applications. Furthermore, limitations associated with analyzing information-rich biological data remain unaddressed.

Doxorubicin resistance in breast cancer cells is mediated by extracellular matrix proteins

BackgroundCancer cell resistance to therapeutics can result from acquired or de novo-mediated factors. Here, we have utilised advanced breast cancer cell culture models to elucidate de novo doxorubicin resistance mechanisms.MethodsThe response of breast cancer cell lines (MCF-7 and MDA-MB-231) to doxorubicin was examined in an in vitro three-dimensional (3D) cell culture model. Cells were cultured with Matrigel™ enabling cellular arrangements into a 3D architecture in conjunction with cell-to-extracellular matrix (ECM) contact.ResultsBreast cancer cells cultured in a 3D ECM-based model demonstrated altered sensitivity to doxorubicin, when compared to those grown in corresponding two-dimensional (2D) monolayer culture conditions. Investigations into the factors triggering the observed doxorubicin resistance revealed that cell-to-ECM interactions played a pivotal role. This finding correlated with the up-regulation of pro-survival proteins in 3D ECM-containing cell culture conditions following exposure to doxorubicin. Inhibition of integrin signalling in combination with doxorubicin significantly reduced breast cancer cell viability. Furthermore, breast cancer cells grown in a 3D ECM-based model demonstrated a significantly reduced proliferation rate in comparison to cells cultured in 2D conditions.ConclusionCollectively, these novel findings reveal resistance mechanisms which may contribute to reduced doxorubicin sensitivity.

PCaAnalyser: A 2D-Image Analysis Based Module for Effective Determination of Prostate Cancer Progression in 3D Culture

Three-dimensional (3D) in vitro cell based assays for Prostate Cancer (PCa) research are rapidly becoming the preferred alternative to that of conventional 2D monolayer cultures. 3D assays more precisely mimic the microenvironment found in vivo, and thus are ideally suited to evaluate compounds and their suitability for progression in the drug discovery pipeline. To achieve the desired high throughput needed for most screening programs, automated quantification of 3D cultures is required. Towards this end, this paper reports on the development of a prototype analysis module for an automated high-content-analysis (HCA) system, which allows for accurate and fast investigation of in vitro 3D cell culture models for PCa. The Java based program, which we have named PCaAnalyser, uses novel algorithms that allow accurate and rapid quantitation of protein expression in 3D cell culture. As currently configured, the PCaAnalyser can quantify a range of biological parameters including: nuclei-count, nuclei-spheroid membership prediction, various function based classification of peripheral and non-peripheral areas to measure expression of biomarkers and protein constituents known to be associated with PCa progression, as well as defining segregate cellular-objects effectively for a range of signal-to-noise ratios. In addition, PCaAnalyser architecture is highly flexible, operating as a single independent analysis, as well as in batch mode; essential for High-Throughput-Screening (HTS). Utilising the PCaAnalyser, accurate and rapid analysis in an automated high throughput manner is provided, and reproducible analysis of the distribution and intensity of well-established markers associated with PCa progression in a range of metastatic PCa cell-lines (DU145 and PC3) in a 3D model demonstrated.

A two-component regulatory system modulates twitching motility in Dichelobacter nodosus

Dichelobacter nodosus is the essential causative agent of footrot in sheep and type IV fimbriae-mediated twitching motility has been shown to be essential for virulence. We have identified a two-component signal transduction system (TwmSR) that shows similarity to chemosensory systems from other bacteria. Insertional inactivation of the gene encoding the response regulator, TwmR, led to a twitching motility defect, with the mutant having a reduced rate of twitching motility when compared to the wild-type and a mutant complemented with the wild-type twmR gene. The reduced rate of twitching motility was not a consequence of a reduced growth rate or decreased production of surface located fimbriae, but video microscopy indicated that it appeared to result from an overall loss of twitching directionality. These results suggest that a chemotactic response to environmental factors may play an important role in the D. nodosus-mediated disease process.

Abstract 4966: Influence of the microenvironment on drug sensitivity in breast cancer using a three-dimensional cell culture model.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Three-dimensional (3D) cell culture is an important research tool and an invaluable alternative to two-dimensional (2D) cell culture as it is more suitable to represent in vivo conditions. The tumor microenvironment influences cellular activity and using 3D cell culture allows these interactions to be studied in vitro. We have evaluated the efficacy of a novel 3D cell culture model for breast cancer and determined cellular responses caused by drug activity. We have recently developed and optimized a miniaturized 3D breast cancer culture model. Our model uses reconstituted basement membrane from Growth Factor Reduced Matrigel™ as a scaffold and is suitable for semi-automated early stage drug discovery. The newly developed 3D culture model has been extensively characterized for reproducibility and the activity of known anti-breast cancer therapies. Notably, anthracyclines and taxanes showed significantly altered drug sensitivity in 3D when compared to monolayer cell cultures of breast cancer cell lines. To determine the mechanisms underlying the altered sensitivity in 3D cell culture, we sought to identify the differences between untreated and drug treated spheroids, as well as the differences between 2D and 3D cell culture responses to anthracyclines and taxanes. We evaluated proliferation, penetration, cellular architecture and signaling of major cellular pathways in our 3D model. We will discuss the proliferation rate comparing 2D and 3D cell culture models and drug penetration (exemplified by doxorubicin) in 3D cell culture. The effect of anthracyclines and taxanes on the 3D cellular architecture, without the influence of extracellular matrix proteins in the microenvironment, will also be presented. In addition, we have characterized the signaling pathways in 2D and 3D cell culture commonly associated with enhanced drug resistance and other relevant proteins including those associated with apoptosis. Deconstructing the cellular processes in 3D breast cancer cell culture in response to breast cancer therapies has the potential to elucidate drug resistance mechanisms. The results obtained from this research illustrate the characteristics and mechanisms of both monolayer and 3D cell culture with respect to anthracycline and taxane drug activity. These results demonstrate the benefits of 3D cell culture in early stage novel compound evaluation for breast cancer. Citation Format: Carrie J. Lovitt, Vicky M. Avery. Influence of the microenvironment on drug sensitivity in breast cancer using a three-dimensional cell culture model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2013-4966