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SIMPLE SUMMARY: Three-dimensional (3D) cell culture models have been proposed as alternatives for initial drug screening to increase drug development efficiency. In addition to their ability to reproduce key aspects of the tumor architecture and microenvironment, 3D cell culture models may help decrease the use of laboratory animals in drug testing, in accordance with the 3R principles (Replacement, Reduction, Refinement). This review aims to help researchers making a transition from two-dimensional (2D) to 3D cell culture models for drug screening, by discussing the impact of 3D models on cancer research, their advantages, limitations, and compatibility with high-throughput screenings. It also outlines the relevance of available readouts provided by such models as well as the importance of incorporating key microenvironmental cues towards improving the predictive value of drug efficacy and safety. ABSTRACT: Today, innovative three-dimensional (3D) cell culture models have been proposed as viable and biomimetic alternatives for initial drug screening, allowing the improvement of the efficiency of drug development. These models are gaining popularity, given their ability to reproduce key aspects of the tumor microenvironment, concerning the 3D tumor architecture as well as the interactions of tumor cells with the extracellular matrix and surrounding non-tumor cells. The development of accurate 3D models may become beneficial to decrease the use of laboratory animals in scientific research, in accordance with the European Union's regulation on the 3R rule (Replacement, Reduction, Refinement). This review focuses on the impact of 3D cell culture models on cancer research, discussing their advantages, limitations, and compatibility with high-throughput screenings and automated systems. An insight is also given on the adequacy of the available readouts for the interpretation of the data obtained from the 3D cell culture models. Importantly, we also emphasize the need for the incorporation of additional and ...

Mushroom extracts have been studied extensively for their potential antitumor effect in tumor cell lines and in animal models [1]. Recently, some of our collaborators have described that the methanolic extract from the medicinal mushroom Cordyceps militaris (L.) Link presented tumor cell growth inhibitory activity. In particular, this extract inhibited the growth of the nonsmall cell lung cancer (NSCLC) cell line NCI-H460, presenting a GI50 of approximately 50 μg/ml [2]. The aim of this work was to further study the effect of C. militaris methanolic extract in NCIH460 cells, regarding its mechanism of action. NCI-H460 cells were treated with C. militaris methanolic extract (at 25 and 50 μg/ml) for 48 h. Viable cell number was then assessed with the Trypan blue exclusion assay. Cellular proliferation was analyzed with the BrdU incorporation assay and cell cycle profile with flow cytometry following propidium iodide (PI) labeling. Levels of apoptotic cell death were determined with flow cytometry following Annexin V-FITC /PI labeling. Treatment of cells with the C. militaris extract caused a dose-dependent decrease in viable cell number. Moreover, a clear and strong decrease in cellular proliferation was observed. In addition, alterations in cell cycle profile were found, with a strong decrease in the S and G2/M phases of the cell cycle together with an increase in G0/G1 phase. Furthermore, treatment with the extract also induced apoptosis in this cell line. In conclusion, C. militaris methanolic extract was shown to interfere with cell proliferation, cell cycle and to induce apoptosis of NCI-H460 cells. Further studies will aim at further understanding the mechanism of action of this extract.

Cordyceps militaris (L.) Link, an edible entomopathogenic fungus widely used in traditional Chinese medicine, has numerous potential medicinal properties including antitumor activity. The methanolic extract of C. militaris fruiting body was recently shown to have tumor cell growth inhibitory activity in several human tumor cell lines. Nonetheless, the mechanism of action involved is still not known. This work aimed at further studying the effect of the methanolic extract of C. militaris regarding its antitumor mechanism of action, using the non-small cell lung cancer cell line (NCI-H460) as a model. Results showed that treatment with the extract decreased cellular proliferation, induced cell cycle arrest at G0/G1 and increased apoptosis. In addition, the extract increased the levels of p53 and p21. Moreover, an increase in p-H2A.X and 53BP1 levels, together with an increase in the number of 53BP1 foci/cell (all indicative of DNA damage), were also observed after treatment with the extract. This work suggests that this extract affected NCI-H460 cellular viability through a mechanism involving DNA damage and p53 activation. This further supports the potential of this extract as a source of bioactive compounds, which may be used in anticancer strategies. ; IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE) and National Funds through the FCT, Foundation for Science and Technology, under the projects "PEst-C/SAU/LA0003/2013" and FCOMP-01-0124-FEDER-015752 (ref FCT PTDC/SAU-FAR/110848/2009). The authors also thank: FCT for the grant to R.T.L. (SFRH/BPD/68787/2010) and QREN for the grant of D.S. (NORTE-07-0124-FEDER-000023).

Mushroom extracts are recognized by their numerous potential medicinal properties. Recently, a methanolic extract from Cordyceps militaris (L.) Link (an edible entomopathogenic fungus widely used in traditional Chinese medicine) has been shown to inhibit cell growth of several human tumour cell lines [1,2]. However, its mechanism of action remained unknown. The aim of the present work was to study the antitumour mechanism of action of the methanolic extract of C. militaris, in the NCI-H460 cell line which is representative of non-small cell lung cancer. Results showed that the extract reduced viable cell number (observed with the trypan blue exclusion assay) by: i) decreasing cellular proliferation (analysed with the BrdU incorporation assay), ii) inducing cell cycle arrest at GO/GI phase (analysed by flow cytometry following propidium iodide-PI labelling) and iii) increasing apoptosis (analysed by flow cytometry following Annexin V-FITC and PI labelling). In addition, results also showed that treatment with the extract increased the cellular levels of p53 and p21. Moreover, this study also showed evidences of DNA damage caused by this extract, since an increase in the levels ofp-H2A.X and 53BP1 were observed, together with an increase in the number of 53BP1 foci/cell. In conclusion, this extract reduced NCI-H460 cellular viability, possibly through a mechanism which involves DNA damage and p53.

Cordyceps militaris (L.) Link, an edible entomopathogenic fungus widely used in traditional Chinese medicine, has numerous potential medicinal properties including antitumor activity. The methanolic extract of C. militaris fruiting body was recently shown to have tumor cell growth inhibitory activity in several human tumor cell lines. Nonetheless, the mechanism of action involved is still not known. This work aimed at further studying the effect of the methanolic extract of C. militaris regarding its antitumor mechanism of action, using the non-small cell lung cancer cell line (NCI-H460) as a model. Results showed that treatment with the extract decreased cellular proliferation, induced cell cycle arrest at G0/G1 and increased apoptosis. In addition, the extract increased the levels of p53 and p21. Moreover, an increase in p-H2A.X and 53BP1 levels, together with an increase in the number of 53BP1 foci/cell (all indicative of DNA damage), were also observed after treatment with the extract. This work suggests that this extract affected NCI-H460 cellular viability through a mechanism involving DNA damage and p53 activation. This further supports the potential of this extract as a source of bioactive compounds, which may be used in anticancer strategies.