Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.

• MeSH
• buněčné sféroidy patologie   MeSH
• disulfiram farmakologie   MeSH
• fluorouracil * farmakologie   terapeutické užití   MeSH
• kolorektální nádory * farmakoterapie   patologie   MeSH
• lidé MeSH
• měď farmakologie   terapeutické užití   MeSH
• nádorové buněčné linie MeSH
• nádorové mikroprostředí MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

3D cell culture methods have been an integral part of and an essential tool for mammary gland and breast cancer research for half a century. In fact, mammary gland researchers, who discovered and deciphered the instructive role of extracellular matrix (ECM) in mammary epithelial cell functional differentiation and morphogenesis, were the pioneers of the 3D cell culture techniques, including organoid cultures. The last decade has brought a tremendous increase in the 3D cell culture techniques, including modifications and innovations of the existing techniques, novel biomaterials and matrices, new technological approaches, and increase in 3D culture complexity, accompanied by several redefinitions of the terms "3D cell culture" and "organoid". In this review, we provide an overview of the 3D cell culture and organoid techniques used in mammary gland biology and breast cancer research. We discuss their advantages, shortcomings and current challenges, highlight the recent progress in reconstructing the complex mammary gland microenvironment in vitro and ex vivo, and identify the missing 3D cell cultures, urgently needed to aid our understanding of mammary gland development, function, physiology, and disease, including breast cancer.

• MeSH
• buněčná diferenciace MeSH
• buněčné kultury přístrojové vybavení   MeSH
• buněčné sféroidy patologie   MeSH
• epitelové buňky patologie   MeSH
• extracelulární matrix patologie   MeSH
• kokultivační techniky metody   MeSH
• lidé MeSH
• mléčné žlázy lidské cytologie   patologie   MeSH
• mléčné žlázy zvířat cytologie   patologie   MeSH
• modely u zvířat MeSH
• myši MeSH
• nádory prsu patologie   MeSH
• organoidy MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

BACKGROUND: Androgen receptor targeted therapies have emerged as an effective tool to manage advanced prostate cancer (PCa). Nevertheless, frequent occurrence of therapy resistance represents a major challenge in the clinical management of patients, also because the molecular mechanisms behind therapy resistance are not yet fully understood. In the present study, we therefore aimed to identify novel targets to intervene with therapy resistance using gene expression analysis of PCa co-culture spheroids where PCa cells are grown in the presence of cancer-associated fibroblasts (CAFs) and which have been previously shown to be a reliable model for antiandrogen resistance. METHODS: Gene expression changes of co-culture spheroids (LNCaP and DuCaP seeded together with CAFs) were identified by Illumina microarray profiling. Real-time PCR, Western blotting, immunohistochemistry and cell viability assays in 2D and 3D culture were performed to validate the expression of selected targets in vitro and in vivo. Cytokine profiling was conducted to analyze CAF-conditioned medium. RESULTS: Gene expression analysis of co-culture spheroids revealed that CAFs induced a significant upregulation of cholesterol and steroid biosynthesis pathways in PCa cells. Cytokine profiling revealed high amounts of pro-inflammatory, pro-migratory and pro-angiogenic factors in the CAF supernatant. In particular, two genes, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (HMGCS2) and aldo-keto reductase family 1 member C3 (AKR1C3), were significantly upregulated in PCa cells upon co-culture with CAFs. Both enzymes were also significantly increased in human PCa compared to benign tissue with AKR1C3 expression even being associated with Gleason score and metastatic status. Inhibiting HMGCS2 and AKR1C3 resulted in significant growth retardation of co-culture spheroids as well as of various castration and enzalutamide resistant cell lines in 2D and 3D culture, underscoring their putative role in PCa. Importantly, dual targeting of cholesterol and steroid biosynthesis with simvastatin, a commonly prescribed cholesterol synthesis inhibitor, and an inhibitor against AKR1C3 had the strongest growth inhibitory effect. CONCLUSIONS: From our results we conclude that CAFs induce an upregulation of cholesterol and steroid biosynthesis in PCa cells, driving them into AR targeted therapy resistance. Blocking both pathways with simvastatin and an AKR1C3 inhibitor may therefore be a promising approach to overcome resistances to AR targeted therapies in PCa. Video abstract.

• MeSH
• androgenní receptory metabolismus   MeSH
• anotace sekvence MeSH
• benzamidy farmakologie   MeSH
• biologické modely MeSH
• biosyntetické dráhy genetika   MeSH
• buněčné sféroidy metabolismus   patologie   MeSH
• buněčný cyklus genetika   MeSH
• chemorezistence účinky léků   genetika   MeSH
• cholesterol biosyntéza   MeSH
• extracelulární matrix metabolismus   MeSH
• fenotyp MeSH
• fenylthiohydantoin farmakologie   MeSH
• fibroblasty asociované s nádorem metabolismus   patologie   MeSH
• kultivační média speciální farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty rezistentní na kastraci genetika   patologie   MeSH
• nádory prostaty genetika   metabolismus   patologie   MeSH
• nitrily farmakologie   MeSH
• progrese nemoci * MeSH
• proliferace buněk genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• senioři MeSH
• simvastatin farmakologie   MeSH
• stanovení celkové genové exprese MeSH
• upregulace * MeSH
• viabilita buněk účinky léků   genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVE: Cancer stem-like cells (CSLCs) are considered a root of tumorigenicity and resistance. However, their identification remains challenging. The use of the side population (SP) assay as a credible marker of CSLCs remains controversial. The SP assay relies on the elevated activity of ABC transporters that, in turn, can be modulated by hypericin (HYP), a photosensitizer and bioactive compound of St. John's Wort (Hypericum perforatum), a popular over-the-counter antidepressant. Here we aimed to comprehensively characterize the SP phenotype of cancer cells and to determine the impact of HYP on these cells. METHODS: Flow cytometry and sorting-based assays were employed, including CD24-, CD44-, CD133-, and ALDH-positivity, clonogenicity, 3D-forming ability, ABC transporter expression and activity, and intracellular accumulation of HYP/Hoechst 33342. The tumorigenic ability of SP, nonSP, and HYP-treated cells was verified by xenotransplantation into immunodeficient mice. RESULTS: The SP phenotype was associated with elevated expression of several investigated transporters and more intensive growth in non-adherent conditions but not with higher clonogenicity, tumorigenicity or ALDH-positivity. Despite stimulated BCRP level and MRP1 activity, HYP reversibly decreased the SP proportion, presumably via competitive inhibition of BCRP. HYP-selected SP cells acquired additional traits of resistance and extensively eliminated HYP. CONCLUSIONS: Our results suggest that SP is not an unequivocal CSLC-marker. However, SP could play an important role in modulating HYP-treatment and serve as a negative predictive tool for HYP-based therapies. Moreover, the use of supplements containing HYP by cancer patients should be carefully considered, due to its proposed effect on drug efflux and complex impact on tumor cells, which have not yet been sufficiently characterized.

• MeSH
• ABC transportér z rodiny G, člen 2 metabolismus   MeSH
• aldehyddehydrogenasa metabolismus   MeSH
• analýza přežití MeSH
• buněčné klony MeSH
• buněčné sféroidy účinky léků   metabolismus   patologie   MeSH
• fenotyp MeSH
• karcinogeneze účinky léků   metabolismus   patologie   MeSH
• lidé MeSH
• myši SCID MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky účinky léků   metabolismus   patologie   MeSH
• nádorové proteiny metabolismus   MeSH
• nádory metabolismus   patologie   MeSH
• P-glykoprotein metabolismus   MeSH
• perylen analogy a deriváty   farmakologie   MeSH
• substrátová specifita účinky léků   MeSH
• vedlejší populace buněk účinky léků   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor.

• MeSH
• buněčné sféroidy patologie   MeSH
• Cytomegalovirus fyziologie   MeSH
• dospělí MeSH
• fenotyp MeSH
• glioblastom mortalita   patologie   virologie   MeSH
• Kaplanův-Meierův odhad MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorová transformace buněk metabolismus   MeSH
• nádorové kmenové buňky virologie   MeSH
• nádory mozku mortalita   patologie   virologie   MeSH
• přežití po terapii bez příznaků nemoci MeSH
• primární buněčná kultura MeSH
• prognóza MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Malignant mesothelioma (MM) is an aggressive type of tumour causing high mortality. One reason for this paradigm may be the existence of a subpopulation of tumour-initiating cells (TICs) that endow MM with drug resistance and recurrence. The objective of this study was to identify and characterise a TIC subpopulation in MM cells, using spheroid cultures, mesospheres, as a model of MM TICs. Mesospheres, typified by the stemness markers CD24, ABCG2 and OCT4, initiated tumours in immunodeficient mice more efficiently than adherent cells. CD24 knock-down cells lost the sphere-forming capacity and featured lower tumorigenicity. Upon serial transplantation, mesospheres were gradually more efficiently tumrigenic with increased level of stem cell markers. We also show that mesospheres feature mitochondrial and metabolic properties similar to those of normal and cancer stem cells. Finally, we show that mesothelioma-initiating cells are highly susceptible to mitochondrially targeted vitamin E succinate. This study documents that mesospheres can be used as a plausible model of mesothelioma-initiating cells and that they can be utilised in the search for efficient agents against MM.

• MeSH
• antigen CD24 metabolismus   MeSH
• antitumorózní látky farmakologie   MeSH
• buněčná adheze účinky léků   MeSH
• buněčné sféroidy účinky léků   patologie   MeSH
• fenotyp MeSH
• genový knockdown MeSH
• inhibiční koncentrace 50 MeSH
• invazivní růst nádoru MeSH
• lidé MeSH
• mezoteliom metabolismus   patologie   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• myši nahé MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky účinky léků   metabolismus   patologie   MeSH
• nádory plic metabolismus   patologie   MeSH
• progrese nemoci MeSH
• proliferace buněk účinky léků   MeSH
• tokoferoly farmakologie   MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH