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3D cancer cell cultures have enabled new opportunities for replacing compound testing in experimental animals. However, most solid tumors are composed of multiple cell types, including fibroblasts. In this study we developed multicellular tumor heterospheroids composed of cancer and fibroblasts cell lines. We developed heterospheroids by combining HT-29, MCF-7, PANC-1 or SW480 with 1BR.3.G fibroblasts, which we have previously reported support spheroid formation. We also tested fibroblast cell lines, MRC-5, GM00498 and HIF, but 1BR.3.G was found to best form heterospheroids with morphological similarity to in vivo tumor tissue. The architectural organization of heterospheroids was based on histological examination using immunohistochemistry. We found that HT-29 and MCF-7 cells developed spheroids with the cancer cells surrounding the fibroblasts, whereas PANC-1 cells interspersed with the fibroblasts and SW480 cells were surrounded by fibroblasts. The fibroblasts also expressed collagen-1 and FAP-α, and whole transcriptomic analysis (WTA) showed abundant ECMand EMT-related expression in heterospheroids, thus reflecting a representative tumor-like microenvironment. The WTA showed that PANC-1 heterospheroids possess a strong EMT profile with abundant Vimentin and CDH2 expression. Drug testing was evaluated by measuring cytotoxicity of 5FU and cisplatin using cell viability and apoptosis assays. We found no major impact on the cytotoxicity when fibroblasts were added to the spheroids. We conclude that the cancer cell lines together with fibroblasts shape the architectural organization of heterospheroids to form tumor-like morphology, and we propose that the various 3D tumor structures can be used for drug testing directed against the cancer cells as well as the fibroblasts. [ABSTRACT FROM AUTHOR]

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus , using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847–treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound. [ABSTRACT FROM AUTHOR]

Trastuzumab is established as standard care for patients with HER2-positive breast cancer both in the adjuvant and metastatic setting. However, 50% of the patients do not respond to the trastuzumab therapy, and therefore new predictive biomarkers are highly warranted. MicroRNAs (miRs) constitute a new group of biomarkers and their cellular expression can be determined in tumor samples by in situ hybridization (ISH) analysis. mi-21 is highly prevalent and up-regulated in breast cancer and has been linked to drug resistance in clinical and in vitro settings. To determine expression patterns of mi-21 in high-grade breast cancers, we examined mi-21 expression in 22 HER2-positive tumors and 15 HER2-negative high-grade tumors by ISH.The histological examination indicated that patient samples could be divided into three major expression patterns: mi-21 predominantly in tumor stroma, predominantly in cancer cells, or in both stromal and cancer cells. There was no obvious difference between the HER2-positive and HER2-negative tumors in terms of the mi-21 expression patterns and intensities. To explore the possibility that mi-21 expression levels and/or cellular localization could predict resistance to adjuvant trastuzumab in HER2-positive breast cancer patients, we analyzed additional 16 HER2-positive tumors from patients who were treated with trastuzumab in the adjuvant setting. Eight of the 16 patients showed clinical recurrence and were considered resistant. Examination of the mi-21 expression patterns and intensities revealed no association between the mi-21 scores in the cancer cell population (p = 0.69) or the stromal cells population (p = 0.13) and recurrent disease after adjuvant trastuzumab. Thus, our findings show that elevated mi-21 expression does not predict resistance to adjuvant trastuzumab. [ABSTRACT FROM AUTHOR]

Trastuzumab is established as standard care for patients with HER2-positive breast cancer both in the adjuvant and metastatic setting. However, approximately 50% of the patients do not respond to the trastuzumab therapy, and therefore new predictive biomarkers are highly warranted. MicroRNAs (miRs) constitute a new group of biomarkers and their cellular expression can be determined in tumor samples by in situ hybridization (ISH) analysis. miR-21 is highly prevalent and up-regulated in breast cancer and has been linked to drug resistance in clinical and in vitro settings. To determine expression patterns of miR-21 in high-grade breast cancers, we examined miR-21 expression in 22 HER2-positive tumors and 15 HER2-negative high-grade tumors by ISH. The histological examination indicated that patient samples could be divided into 3 major expression patterns: miR-21 predominantly in tumor stroma, predominantly in cancer cells or in both stromal and cancer cells. There was no obvious difference between the HER2-positive and HER2-negative tumors in terms of the miR-21 expression patterns and intensities. To explore the possibility that miR-21 expression levels and/or cellular localization could predict resistance to adjuvant trastuzumab in HER2-positive breast cancer patients, we analyzed additional 16 HER2-positive tumors from patients that were treated with trastuzumab in the adjuvant setting. Eight of the 16 patients showed clinical recurrence and were considered resistant. Examination of the miR-21 expression patterns and intensities revealed no association between the miR-21 scores in the cancer cell population (p=0.69) or the stromal cells population (p=0.13) and recurrent disease after adjuvant trastuzumab. Thus, our findings show that elevated miR-21 expression does not predict resistance to adjuvant trastuzumab. [ABSTRACT FROM AUTHOR]