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

BACKGROUND: Cancer 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. METHODS: The 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. RESULTS: Breast 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. CONCLUSION: Collectively, these novel findings reveal resistance mechanisms which may contribute to reduced doxorubicin sensitivity.

Assessing Drug Efficacy in a Miniaturized Pancreatic Cancer In Vitro 3D Cell Culture Model.

Pancreatic cancer continues to have one of the poorest prognoses among all cancers. The drug discovery efforts for this disease have largely failed, with no significant improvement in survival outcomes for advanced pancreatic cancer patients over the past 20 years. Traditional in vitro cell culture techniques have been used extensively in both basic and early drug discovery; however, these systems offer poor models to assess emerging therapeutics. More predictive cell-based models, which better capture the cellular heterogeneity and complexities of solid pancreatic tumors, are urgently needed not only to improve drug discovery success but also to provide insight into the tumor biology. Pancreatic tumors are characterized by a unique micro-environment that is surrounded by a dense stroma. A complex network of interactions between extracellular matrix (ECM) components and the effects of cell-to-cell contacts may enhance survival pathways within in vivo tumors. This biological and physical complexity is lost in traditional cell monolayer models. To explore the predictive potential of a more complex cellular system, a three-dimensional (3D) micro-tumor assay was evaluated. Efficacy of six current chemotherapeutics was determined against a panel of primary and metastatic pancreatic tumor cell lines in a miniaturized ECM-based 3D cell culture system. Suitability for potential use in high-throughput screening applications was assessed, including ascertaining the effects that miniaturization and automation had on assay robustness. Cellular health was determined by utilizing an indirect population-based metabolic activity assay and a direct imaging-based cell viability assay.

Expression of the thioredoxin system in an in vivo-like cancer cell environment upon auranofin treatment.

As essential elements of the tumor microenvironment, the variable oxygenation state of the tumor tissue, the extracellular matrix (ECM) and different cell types are important determinants of carcinogenesis. These elements may also influence how tumor cells respond to therapeutic treatments. In the present study, we assessed the anti-cancer activity of auranofin and its effect on the thioredoxin (Trx) system under conditions that closely resemble the in vivo tumor microenvironment with respect to the oxygen levels and tissue architecture. We utilised an oxygen scheme involving growth of cancer cells under normoxia (20%) and hypoxia (0.1%). We also preconditioned cells with intermittent hypoxia (IH) prior to a prolonged hypoxic incubation. This oxygen scheme did not affect the cytotoxicity of auranofin; however, IH preconditioned cells were less sensitive towards the inhibition of thioredoxin reductase (TrxR) specific activity upon treatment with auranofin. IH preconditioning also upregulated Trx protein levels in auranofin treated cells. We also compared the activity of auranofin against cancer cells cultured in 2D monolayer and 3D spheroid-based culture models. Auranofin was less potent against cells grown under a more in vivo-like 3D environment. The results presented in this paper implicate the importance of the tumor oxygen environment and tissue architecture in influencing the response of cancer cells towards auranofin.

Cancer drug discovery: recent innovative approaches to tumor modeling.

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.

Development of ethynyl-2'-deoxyuridine chemical probes for cell proliferation.

A common method of evaluating cellular proliferation is to label DNA with chemical probes. 5-Ethynyl-2'-deoxyuridine (EdU) is a widely utilized chemical probe for labeling DNA, and upon incorporation, EdU treatment of cells is followed by a reaction with a small molecule fluorescent azide to allow detection. The limitations when using EdU include cytotoxicity and a reliance on nucleoside active transport mechanisms for entry into cells. Here we have developed six novel EdU pro-labels that consist of EdU modified with variable lipophilic acyl ester moieties. This pro-label:chemical probe relationship parallels the prodrug:drug relationship that is employed widely in medicinal chemistry. EdU and EdU pro-labels were evaluated for their labeling efficacy and cytotoxicity. Several EdU pro-label analogues incorporate into DNA at a similar level to EdU, suggesting that nucleoside transporters can be bypassed by the pro-labels. These EdU pro-labels also had reduced toxicity compared to EdU.

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.

Evaluation of chemotherapeutics in a three-dimensional breast cancer model.

PURPOSE: Utilization of miniaturized three-dimensional (3D) cell culture-based assays enables investigation into the anticancer activity of drug candidates and further elucidation of the anticancer profile of standard-of-care chemotherapeutic agents against tumor cells. Drug discovery assays established using 3D cell culture, which better recapitulate the tumor microenvironment, may more accurately reflect the antitumor activity of compounds. METHODS: Several standard-of-care anticancer drugs, epirubicin, paclitaxel and vinorelbine, were evaluated against a panel of breast cancer cell lines grown in a 3D cell culture microenvironment in the presence of extracellular matrix. A comparison of this antitumor activity in 3D conditions was made with that observed in traditional two-dimensional (2D) monolayer conditions. RESULTS: Examination of the above mentioned drugs against breast tumor cells cultured in 3D conditions demonstrated significantly altered potency and efficacy in comparison with cells propagated in a 2D monolayer system. The differences observed were cell line-dependent and drug-specific; the triple-negative cell line MDA-MB-231 and the endocrine receptor-positive cell line MCF-7 consistently displayed resistance to therapeutics with distinct modes of action (i.e., topoisomerase II and microtubules) in 3D cell culture in comparison with ErbB2 receptor-positive BT-474 cells. CONCLUSION: The data presented herein demonstrates the cellular viability and physical changes observed within the 3D spheroid following exposure to drug, which is not always reflected in 2D cell culture models.

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.

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.

Miniaturized three-dimensional cancer model for drug evaluation.

A more relevant in vitro cell culture model that closely mimics tumor biology and provides better predictive information on anticancer therapies has been the focus of much attention in recent years. We have developed a three-dimensional (3D) human tumor cell culture model that attempts to recreate the in vivo microenvironment and tumor biology in a miniaturized 384-well plate format. This model aims to exploit the potential of 3D cell culture as a screening tool for novel therapeutics for discovery programs. Here we have evaluated a Matrigel™ based induction of 3D tumor formation using standard labware and plate reading equipment. We have demonstrated that with an optimized protocol, reproducible proliferation, and cell viability data can be obtained across a range of cell lines and reagent batches. A panel of reference drugs was used to validate the suitability of the assays for a high throughput drug discovery program. Indicators of assay reproducibility, such as Z'-factor and coefficient of variation, as well as dose response curves confirmed the robustness of the assays. Several methods of drug activity determination were examined, including metabolic and imaging based assays. These data demonstrate this model as a robust tool for drug discovery bridging the gap between monolayer cell culture and animal models, providing insights into drug efficacy at an earlier time point, ultimately reducing costs and high attrition rates.

Copper, nickel, and zinc cyclam-amino acid and cyclam-peptide complexes may be synthesized with "click" chemistry and are noncytotoxic.

We describe the synthesis of cyclam metal complexes derivatized with amino acids or a tripeptide using a copper(I)-catalyzed Huisgen "click" reaction. The linker triazole formed during the synthesis plays an active coordinating role in the complexes. The reaction conditions do not racemize the amino acid stereocenters. However, a methylene group adjacent to the triazole is susceptible to H/D exchange under ambient conditions, an observation which has potentially important implications for structures involving stereocenters adjacent to triazoles in click-derived structures. The successful incorporation of several amino acids is described, including reactive tryptophan and cysteine side chains. All complexes are formed rapidly upon introduction of the relevant metal salt, including synthetically convenient cases where trifluoroacetate salts of cyclam derivatives are used directly in the metalation. None of the metal complexes displayed any cytotoxicity to mammalian cells, suggesting that the attachment of such complexes to amino acids and peptides does not induce toxicity, further supporting their potential suitability for labeling/imaging studies. One Cu(II)-cyclam-triazole-cysteine disulfide complex displayed moderate activity against MCF-10A breast nontumorigenic epithelial cells.