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Three-dimensional cell culture models are increasingly adopted as preferred pre-clinical drug testing platforms, as they circumvent limitations associated with traditional monolayer cell cultures. However, many of these models are not fully characterized. This study aimed to characterize a BT-20 triple-negative breast carcinoma spheroid model and assess its susceptibility to doxorubicin in comparison to a monolayer model. Spheroids were developed using the liquid overlay method. Phenotypic attributes were analyzed by characterizing changes in size, gross morphology, protein content, metabolic activity, hypoxic status, and cell–cell junctions. The cytotoxic range of doxorubicin in monolayers was determined using the sulforhodamine B assay, and the comparative effect of toxic and sub-toxic concentrations was assessed in both spheroids and monolayers. Similar to the in vivo microenvironment, spheroids had a heterogeneous spatial cytoarchitecture, inherent hypoxia and strong adherens junctions. Doxorubicin induced dose-dependent cytotoxicity in monolayers (IC25: 130 nM, IC50: 320 nM and IC75: 1580 nM); however, these concentrations did not alter the spheroid size or acid phosphatase activity. Only concentrations ≥6 µM had any effect on spheroid integrity. In comparison to monolayers, the BT-20 spheroid model has decreased sensitivity to doxorubicin and could serve as a better model for susceptibility testing in triple-negative breast cancer. [ABSTRACT FROM AUTHOR]

Background: Tumour metastasis remains the major cause of death in cancer patients and, to date, the mechanism and signalling pathways governing this process are not completely understood. The TGF-β pathway is the most commonly mutated pathway in cancer, however its role in cancer progression is controversial as it can function as both a promoter and a suppressor of metastasis. Although previous studies have suggested a role for the molecular chaperone Hsp90 in regulating the TGF-β pathway, the level at which this occurs as well as the consequences in terms of colon cancer metastasis are unknown.Methods: The paired SW480 and SW620 colon cancer cell lines, derived from a primary tumour and its lymph node metastasis, respectively, were used as an in vitro model to study key cellular processes required for metastasis. The status of the TGF-β pathway was examined in these cells using ELISA, flow cytometry, western blot analysis and confocal microscopy. Furthermore, the effect of addition or inhibition of the TGF-β pathway and Hsp90 on adhesion, migration and anchorage-independent growth, was determined in the cell lines.Results: When comparing the canonical TGF-β1 pathway in the genetically paired cell lines our data suggests that this pathway may be constitutively active in the SW620 metastasis-derived cell line and not the SW480 primary tumour-derived line. In addition, we report that, when present in combination, TGF-β1 and Hsp90β stimulate anchorage-independent growth, reduce adhesion and stimulate migration. This effect is potentiated by inhibition of the TGF-β1 receptor and occurs via an alternate TGF-β1 pathway, mediated by αvβ6 integrin. Interestingly, in the SW620 cells, activation of this alternate TGF-β1 signalling machinery does not appear to require inhibition of the canonical TGF-β1 receptor, which would allow them to respond more effectively to the pro-metastasis stimulus of a combination of Hsp90β and TGF-β1 and this could account for the increased migratory capacity of these cells.Conclusions: In this study we report an apparent synergy between TGF-β1 and Hsp90β in stimulating migratory behaviour of colon cancer cells when signalling occurs via αvβ6 integrin as opposed to the canonical TGF-β1 pathway. [ABSTRACT FROM AUTHOR]

Background: 2-Methoxyestradiol (2ME2) is an estradiol metabolite with well documented antiproliferative properties in many cancer cell lines. However, it is rapidly metabolised in vivo which limits its clinical application. Therefore, more stable derivatives with potentially improved clinical features have been designed by our group. Here we describe an estrone-like derivative of 2ME2, namely EE-15-one, that unlike other derivatives which induce cell cycle arrest, induces a rapid loss of cell–substrate adhesion through the inactivation and disassembly of focal adhesions. Methods: To assess the effect of 2-ethyl-estra-1,3,5 (10),15-tetraen-3-ol-17-one (EE-15-one) on breast cancer cell lines, cell survival was quantified. The effect of EE-15-one on cell attachment was assessed by measuring cell adhesion and cell rounding via light microscopy. Effects on focal adhesion dynamics and actin cytoskeleton organisation were visualised by immunofluorescence while focal adhesion signalling was assessed by western blot. Cell death was quantified using a lactate dehydrogenase activity (LDH) assay. To investigate specificity towards cell–substrate over cell–cell contact inhibition, EE-15-one effects on 3D cell cultures were assessed. Results: Cell survival assays show an almost complete loss of cells within 24 h of EE-15-one exposure in contrast to published sulphamoylated 2ME2 derivatives. Cell loss is linked to rapid detachment and adhesion inhibition. Focal adhesion size and number are rapidly diminished while actin fibres became severed and disappeared within 2 h post exposure. These changes were not due to cell necrosis as LDH activity only slightly increased after 24 h. Cells grown in cell–cell adhesion dependent spheroids did not respond to EE-15-one exposure suggesting that EE-15-one specifically inhibits cell–substrate adhesions but not cell–cell adhesions and does not directly impact the actin cytoskeleton. Conclusion: We show that a novel 2ME2 derivative, EE-15-one, induces rapid loss of focal adhesion function leading to cell–substrate detachment through interference with integrin-based cell–substrate adhesions, but not cadherin dependent cell–cell adhesions. Therefore, EE-15-one is the first 2ME2 derivative that has an alternative mode of action to the antimitotic activity of 2ME2. As such EE-15-one shows potential as a lead compound for further development as an inhibitor of cell–substrate adhesion which is essential for metastatic dissemination. [ABSTRACT FROM AUTHOR]