Suchergebnisse

Primary human breast cancers invade surrounding fat and contact adipocytes, inflammatory infiltrates, and fibrous stroma. This tissue niche influences breast tumor progression. Here, we present a protocol to enable the in vitro study of the complex interactions that occur between breast cancer cells and adipose cells. We describe how to obtain different adipose cell populations, including adipose-derived stem cells, immature adipocytes, and mature adipocytes, from human breast fat tissue and detail the application for co-culture assays with breast cancer cells. For complete details on the use and execution of this protocol, please refer to Picon-Ruiz et al. (2016) and Qureshi et al. (2020). ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 845104

Nanotechnology-based approaches hold substantial potential to avoid chemoresistance and minimize side effects. In this work, we have used biocompatible iron oxide magnetic nanoparticles (MNPs) called MF66 and functionalized with the antineoplastic drug doxorubicin (DOX) against MDA-MB-231 cells. Electrostatically functionalized MNPs showed effective uptake and DOX linked to MNPs was more efficiently retained inside the cells than free DOX, leading to cell inactivation by mitotic catastrophe, senescence and apoptosis. Both effects, uptake and cytotoxicity, were demonstrated by different assays and videomicroscopy techniques. Likewise, covalently functionalized MNPs using three different linkers—disulfide (DOX-S-S-Pyr, called MF66-S-S-DOX), imine (DOX-I-Mal, called MF66-I-DOX) or both (DOX-I-S-S-Pyr, called MF66-S-S-I-DOX)—were also analysed. The highest cell death was detected using a linker sensitive to both pH and reducing environment (DOX-I-S-S-Pyr). The greatest success of this study was to detect also their activity against breast cancer stem-like cells (CSC) from MDA-MB-231 and primary breast cancer cells derived from a patient with a similar genetic profile (triple-negative breast cancer). In summary, these nanoformulations are promising tools as therapeutic agent vehicles, due to their ability to produce efficient internalization, drug delivery, and cancer cell inactivation, even in cancer stem-like cells (CSCs) from patients ; This research was funded by the European Seventh Framework Program (grant agreement number 262943); the European Union's Horizon 2020 research and innovation programme (grant agreement number 685795); Ministerio de Economía y Competitividad, Spain (grants CTQ2016-78454-C2-2-R, BIO2016-77367-C2-1-R and SAF2017-87305-R); Basque Government Elkartek KK- 2017/00008; Comunidad de Madrid (IND2017/IND-7809; S2017/BMD-3867 RENIM-CM and S2018/NMT-4321 NANOMAGCOST-CM); NIHR Manchester Biomedical Research Centre (IS-BRC-1215-20007) and Breast Cancer Now (MAN-Q2); co-financed by European Structural Cancers 2020, 12, 1397 17 of 19 and Investment Fund, Asociación Española Contra el Cáncer (Singulares 2014) and IMDEA Nanociencia. CIC biomaGUNE acknowledges Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant MDM-2017-0720). IMDEA Nanociencia acknowledges support from the 'Severo Ochoa' Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686)

In vitro cell culture is traditionally performed within two-dimensional (2D) environments, providing a quick and cheap way to study cell properties in a laboratory. However, 2D systems differ from the in vivo environment and may not mimic the physiological cell behavior realistically. For instance, 2D culture models are thought to induce cancer stem cells (CSCs) differentiation, a rare cancer cell subpopulation responsible for tumor initiation and relapse. This fact hinders the development of therapeutic strategies for tumors with a high relapse percentage, such as triple negative breast cancer (TNBC). Thus, three-dimensional (3D) scaffolds have emerged as an attractive alternative to monolayer culture, simulating the extracellular matrix structure and maintaining the differentiation state of cells. In this work, scaffolds were fabricated through electrospinning different poly("-caprolactone)-acetone solutions. Poly("-caprolactone) (PCL) meshes were seeded with triple negative breast cancer (TNBC) cells and 15% PCL scaffolds displayed significantly (p < 0.05) higher cell proliferation and elongation than the other culture systems. Moreover, cells cultured on PCL scaffolds exhibited higher mammosphere forming capacity and aldehyde dehydrogenase activity than 2D-cultured cells, indicating a breast CSCs enrichment. These results prove the powerful capability of electrospinning technology in terms of poly("-caprolactone) nanofibers fabrication. In addition, this study has demonstrated that electrospun 15% PCL scaffolds are suitable tools to culture breast cancer cells in a more physiological way and to expand the niche of breast CSCs. In conclusion, three-dimensional cell culture using PCL scaffolds could be useful to study cancer stem cell behavior and may also trigger the development of new specific targets against such malignant subpopulation ; This work was supported partially by Spanish grants from Fundación Ramón Areces, Instituto de Salud Carlos III (PI1400329) and Ministerio de Economía Y Competitividad (DPI2013-45201-P; RYC-2014-15581), and the support of the Catalonian government (2014SGR00868). The authors are grateful for the financial support from the University of Girona (MPCUdG2016/036)

In vitro cell culture is traditionally performed within two-dimensional (2D) environments, providing a quick and cheap way to study cell properties in a laboratory. However, 2D systems differ from the in vivo environment and may not mimic the physiological cell behavior realistically. For instance, 2D culture models are thought to induce cancer stem cells (CSCs) differentiation, a rare cancer cell subpopulation responsible for tumor initiation and relapse. This fact hinders the development of therapeutic strategies for tumors with a high relapse percentage, such as triple negative breast cancer (TNBC). Thus, three-dimensional (3D) scaffolds have emerged as an attractive alternative to monolayer culture, simulating the extracellular matrix structure and maintaining the differentiation state of cells. In this work, scaffolds were fabricated through electrospinning different poly("-caprolactone)-acetone solutions. Poly("-caprolactone) (PCL) meshes were seeded with triple negative breast cancer (TNBC) cells and 15% PCL scaffolds displayed significantly (p < 0.05) higher cell proliferation and elongation than the other culture systems. Moreover, cells cultured on PCL scaffolds exhibited higher mammosphere forming capacity and aldehyde dehydrogenase activity than 2D-cultured cells, indicating a breast CSCs enrichment. These results prove the powerful capability of electrospinning technology in terms of poly("-caprolactone) nanofibers fabrication. In addition, this study has demonstrated that electrospun 15% PCL scaffolds are suitable tools to culture breast cancer cells in a more physiological way and to expand the niche of breast CSCs. In conclusion, three-dimensional cell culture using PCL scaffolds could be useful to study cancer stem cell behavior and may also trigger the development of new specific targets against such malignant subpopulation ; This work was supported partially by Spanish grants from Fundación Ramón Areces, Instituto de Salud Carlos III (PI1400329) and Ministerio de Economía Y Competitividad (DPI2013-45201-P; RYC-2014-15581), and the support of the Catalonian government (2014SGR00868). The authors are grateful for the financial support from the University of Girona (MPCUdG2016/036)

Among others, expression levels of programmed cell death 1 ligand 1 (PD-L1) have been explored as biomarkers of the response to immune checkpoint inhibitors in cancer therapy. Here, we present the results of a chemical screen that interrogated how medically approved drugs influence PD-L1 expression. As expected, corticosteroids and inhibitors of Janus kinases were among the top PD-L1 downregulators. In addition, we identified that PD-L1 expression is induced by antiestrogenic compounds. Transcriptomic analyses indicate that chronic estrogen receptor alpha (ERα) inhibition triggers a broad immunosuppressive program in ER-positive breast cancer cells, which is subsequent to their growth arrest and involves the activation of multiple immune checkpoints together with the silencing of the antigen-presenting machinery. Accordingly, estrogen-deprived MCF7 cells are resistant to T-cell-mediated cell killing, in a manner that is independent of PD-L1, but which is reverted by estradiol. Our study reveals that while antiestrogen therapies efficiently limit the growth of ER-positive breast cancer cells, they concomitantly trigger a transcriptional program that favors their immune evasion. ; We would want to thank Andres J Lopez-Contreras for insightful comments on the manuscript, Hana Imrichova for help with GSEAs, and the core facility at NEO, BEA, Bioinformatics and Expression Analysis, which is supported by the board of research at the Karolinska Institute and the research committee at the Karolinska Hospital. Research was funded by grants from the Cancerfonden Foundation (CAN 2018/381) and the Swedish Research Council (VR) (538-2014-31) to OF and from the ISCIII (AES-PI16/00354; cofunded by the European Regional Development Fund) and from the Call for Coordinated Research Groups from Madrid Region, Madrid Regional Government-ERDF Funds (B2017/BMD3733) to MQ-F. This research was supported by Consejería de Sanidad, Comunidad de Madrid. ; Sí

We thank the company Nanogetic S.L. (Granada, Spain) for support with the synthesis of the SS peptides and the Centro de Instrumentacion Cientifica (CIC) of the Universidad de Granada for use of the TEM facilities. ; The main role of mitochondria, as pivotal organelles for cellular metabolism, is the production of energy (ATP) through an oxidative phosphorylation system. During this process, the electron transport chain creates a proton gradient that drives the synthesis of ATP. One of the main features of tumoral cells is their altered metabolism, providing alternative routes to enhance proliferation and survival. Hence, it is of utmost importance to understand the relationship between mitochondrial pH, tumoral metabolism, and cancer. In this manuscript, we develop a highly specific nanosensor to accurately measure the intramitochondrial pH using fluorescence lifetime imaging microscopy (FLIM). Importantly, we have applied this nanosensor to establish differences that may be hallmarks of different metabolic pathways in breast cancer cell models, leading to the characterization of different metabophenotypes. ; Spanish Ministerio de Ciencia, Innovacion y Universidades CTQ2014-56370-R CTQ2017-86568-R ; European Union (EU) ; Spanish Agencia Estatal de Investigacion ; Fundacion Ramon Areces

The epithelial-to-mesenchymal transition (EMT) and its reversion (MET) are related to tumor cell dissemination and migration, tumor circulating cell generation, cancer stem cells, chemoresistance, and metastasis formation. To identify chromatin and epigenetic factors possibly involved in the process of EMT, we compare the levels of expression of epigenetic genes in a transformed human breast epithelial cell line (HMEC-RAS) versus a stable clone of the same cell line expressing the EMT master regulator ZEB1 (HMEC-RAS-ZEB1). One of the factors strongly induced in the HMEC-RAS-ZEB1 cells was Transducin beta-like 1 (TBL1), a component of the NCoR complex, which has both corepressor and coactivator activities. We show that TBL1 interacts with ZEB1 and that both factors cooperate to repress the promoter of the epithelial gene E-cadherin (CDH1) and to autoactivate the ZEB1 promoter. Consistent with its central role, TBL1 is required for mesenchymal phenotypes of transformed breast epithelial and breast cancer cell lines of the claudin-low subtype. Importantly, a high expression of the TBL1 gene correlates with poor prognosis and increased proportion of metastasis in breast cancer patients, indicating that the level of TBL1 expression can be used as a prognostic marker. ; This work was funded by the Spanish Ministry of Economy and Competitiveness (BFU2014-53543-P and BFU2017-85420-R to J.C.R.), the Junta de Andalucía (BIO-321), and the European Union (FEDER). CABIMER is a Center partially funded by the Junta de Andalucía.

Although the anti-cancer properties of 3BP have been described previously, its selectivity for cancer cells still needs to be explained. In the work reported here we characterized the kinetic parameters of radiolabelled [14C]-3BP uptake in three breast cancer cell lines that display different levels of resistance to 3BP: ZR-75-1 < MCF-7 < SK-BR-3. At pH 6.0 the affinity of cancer cells for 3BP transport, correlates with their sensitivity, a pattern that does not occur at pH 7.4. In the three cell lines, the uptake of 3BP is dependent on the proton motive force and is decreased by MCTs inhibitors. In the SK-BR-3 cell line, a sodium-dependent transport also occurs. Butyrate promotes the localization of MCT-1 at the plasma membrane and increases the level of MCT-4 expression, leading to a higher sensitivity for 3BP. Here, we demonstrate that this phenotype is accompanied by an increase in affinity for 3BP uptake. Our results confirm the role of MCTs, especially MCT-1 in 3BP uptake and the importance of CD147 glycosylation in this process. We find that the affinity for 3BP transport is higher when the extracellular milieu is acid. This is a typical phenotype of tumor microenvironment and explains the lack of secondary effects of 3BP already described in in vivo studies. ; FEDER (Fundo Europeu deDesenvolvimento Regional), through POFC (Programa Operacional Factores de Competitividade) – COMPETE, and by Portuguese National Funds from FCT (Fundac¸˜ao para a Ciˆencia e Tecnologia) in the scope of the project PEst-OE/BIA/U14050/2014. JAS [grant number SFRH/BD/76038/2011] received a fellowship from the Portuguese government from the FCT through FSE (Fundo Social Europeu) and POPH (Programa Operacional Potencial ...

The authors acknowledge the financial support from the Ministry of Economy and Competitiveness (MINECO), Spain grants from DPI2016-77156-R, the financial support from the University of Girona (Spain) MPCUdG2016/036 and the pre-doctoral grant (IFUdG2017/62), and the support of the Catalan Government (2017SGR00385). This work was also partially supported by the grants from the Fundación Ramón Areces (Spain), the Instituto de Salud Carlos III (Spain) (PI1400329), and Fundació Oncolliga and RadikalSwim (OncoSwim)

Tumorigenesis induces actin cortex remodeling, which makes cancerous cells softer. Cell deformability is largely determined by myosin-driven cortical tension and actin fiber architecture at the cell cortex. However, it is still unclear what the weight of each contribution is, and how these contributions change during cancer development. Moreover, little attention has been paid to the effect of energy metabolism on this phenomenon and its reprogramming in cancer. Here, we perform precise two-dimensional mechanical phenotyping based on power-law rheology to unveil the contributions of myosin II, actin fiber architecture and energy metabolism to the deformability of healthy (MCF-10A), noninvasive cancerous (MCF-7), and metastatic (MDA-MB-231) human breast epithelial cells. Contrary to the perception that the actin cortex is a passive structure that provides mechanical resistance to the cell, we find that this is only true when the actin cortex is activated by metabolic processes. The results show marked differences in the nature of the active processes that build up cell stiffness, namely that healthy cells use ATP-driven actin polymerization whereas metastatic cells use myosin II activity. Noninvasive cancerous cells exhibit an anomalous behavior, as their stiffness is not as affected by the lack of nutrients and ATP, suggesting that energy metabolism reprogramming is used to sustain active processes at the actin cortex. ; This work was supported by the European Union's Horizon 2020 research and innovation program under European Research Council Grant 681275-LIQUIDMASS-ERCCoG-2015, under Grant Agreement No. 731868 – VIRUSCAN and by the Spanish Science, Innovation and Universities Ministry through project CELLTANGLE reference RTI2018-099369-B-I00; by the Comunidad de Madrid (iLUNG B2017/BMD-3884) with support from EU (FEDER, FSE) and Ramón y Cajal grant RYC-2017-21640 to P.M.K. The service from the X-SEM Laboratory is funded by MCIU (CSIC13-4E-1794) and EU (FEDER, FSE).

Cromosomes; Marcadors pronòstics ; Cromosomas; Marcadores de pronóstico ; Chromosomes; Prognostic markers ; Oxidation of H3 at lysine 4 (H3K4ox) by lysyl oxidase-like 2 (LOXL2) generates an H3 modification with an unknown physiological function. We find that LOXL2 and H3K4ox are higher in triple-negative breast cancer (TNBC) cell lines and patient-derived xenografts (PDXs) than those from other breast cancer subtypes. ChIP-seq revealed that H3K4ox is located primarily in heterochromatin, where it is involved in chromatin compaction. Knocking down LOXL2 reduces H3K4ox levels and causes chromatin decompaction, resulting in a sustained activation of the DNA damage response (DDR) and increased susceptibility to anticancer agents. This critical role that LOXL2 and oxidized H3 play in chromatin compaction and DDR suggests that functionally targeting LOXL2 could be a way to sensitize TNBC cells to conventional therapy. ; This work was supported by grants from Instituto de Salud Carlos III (ISCIII) FIS/FEDER (PI12/01250; CP08/00223; PI16/00253; and CB16/12/00449), MINECO (SAF2013-48849-C2-1-R) to SP, BFU2015-68354 to THS, Breast Cancer Research Foundation (BCRF-17-008) to JA, AGL2014-52395-C2-2-R to DA, Worldwide Cancer Research, Red Temática de Investigación Cooperativa en Cáncer (RD012/0036/005), Fundación Científica de la Asociación Española contra el Cáncer, and Fundació La Marató TV3. THS was supported by institutional funding (MINECO) through the Centres of Excellence Severo Ochoa award and the CERCA Programme of the Catalan Government, and SS-B, by a Fundació La Caixa fellowship. We thank La Caixa Foundation and Cellex Foundation for provide research facilities and equipment. GV has received funding from the MINECO (a "Juan de la Cierva Incorporation" fellowship; IJCI-2014-20723). SP was a recipient of a Miguel Servet contract (ISCIII/FIS), and AI, JPC-C, LP-G, and GS-B are supported by contracts from Worldwide Cancer Research, Fundació La Marató TV3, Fundació FERO, and a FI Fellowship from the Generalitat de ...