The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the ...implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.
Abstract
Breast cancer is the second most common cancer worldwide after lung cancer. About 70% of breast cancers express estrogen receptor α (ER+) and/or progesterone receptor (PR+), and these ...biomarkers are indicative of hormone dependence. However up to 50% acquire resistance to hormone therapy 1, 2. Estrogen independent ER+ breast cancer depends on CDK4 for tumor growth and CDK4 inhibitors have emerged as a promising approach to treat this type of tumors 3. Abemaciclib is a cell cycle inhibitor with selective activity against CDK4 and CDK6 and it is being evaluated in advanced clinical trials for its potential to reduce metastatic ER+ breast cancer growth. We have evaluated combination of abemaciclib with an anti-estrogen therapy in an in vitro breast cancer panel. Phenotypic characterization of sensitive cell lines was carried out by monitoring cell proliferation, senescence, and apoptosis markers using flow cytometry and high content imaging approaches. Using an in vitro panel with a diversity of breast cancer cell lines, a synergistic effect of abemaciclib in combination with the ER down-regulating drug fulvestrant was observed based on Bliss score. This combination treatment demonstrated effective growth inhibition in ER+ cells and exhibited synergism in MCF-7, T47D and ZR-75-1. The mechanistic analyses revealed that the combination of abemaciclib with fulvestrant promoted a decrease in cancer cell proliferation due to G1 phase arrest at doses tested. This growth inhibition was accompanied by increased hallmarks for cell senescence as observed by markers such as SA-β-galactosidase staining or morphological changes. Subsequently, an increase in biomarkers for apoptosis was also observed. These changes occurred in a time dependent manner and were significantly greater with the combination than fulvestrant single agent treatment. We conclude the combination of abemaciclib with fulvestrant better prevented proliferation of breast cancer cell lines by blocking cell proliferation and lead to induction of senescence and apoptosis as compared to fulvestrant treatment alone in ER+ cells.
Bibliography
1 American Cancer Society, Cancer Facts & Figures 2014.
2 Dixon J.M. (2014) New Journal of Science. Volume 2014, Article ID 390618.
3 Miller TW et al. (2011) Cancer Discov. Volume 1 (4): 338-51.
Citation Format: Raquel Torres, Bruna Calsina, Ana Hermoso, Carmen Baquero, Cecilia Mur, Karsten Boehnke, Joaquín Amat, Alfonso De Dios, Xueqian Gong, Sean Buchanan, Richard Paul Beckmann, Maria Jose Lallena. Characterization of the mechanism of action for abemaciclib with antiestrogen combined therapy in human breast cancer cell lines. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2836.
Abstract
Lung cancer is the most common tumor cancer worldwide and approximately 15-25% of the patients with lung adenocarcinoma have KRAS driven tumors. These malignancies involve, in the majority ...of cases, a constitutive activation of KRAS signaling pathway (1,2) and are associated with poor prognosis in patients with advanced disease (metastatic setting).
Currently there is no specific therapy to target KRAS driven tumors approved by FDA (3), then finding alternative targeted therapies is a need to cover for this disease.
Pharmacological inhibition of CDK4 was been suggested as a beneficial therapy to treat NSCLC patients carrying K-RAS oncogenes; and researchers base the potential efficacy of this approach on a synthetic lethal interaction between K-ras and CDK4 in in this type of tumors (4). Hence, CDK4/6 inhibitors appear as promising therapy to treat this type of tumors.
Abemaciclib is a cell cycle inhibitor with selective activity against CDK4 and CDK6 and is being evaluated in advanced clinical trials for its potential to reduce NSCLC cancer growth. Here we describe studies towards the in-vitro mechanism of action of abemaciclib to reduce tumor cells growth in NSCLC cell lines harboring mutations in KRAS. Overall, abemaciclib reduces NSCLC cell growth as indicated by a reduction of cell number and proliferation biomarker Ki67 upon treatment. This tumor growth inhibition is mediated by arrest of cell cycle in G1 phase as a direct consequence of Rb phosphorylation blockade. In this study we are further reporting a phenotypic characterization of sensitive cell lines monitoring cell proliferation, senescence, and apoptosis markers using flow cytometry and high content imaging approaches as well as metabolic profiling.
. Bibliography
(1)
Schubbert S, Shannon K and Bollang G (2007) Nature Rev. Cancer 7(4) 295-308.
(2)
Ihle NT et al (2012) J Natl Cancer Inst. 104(3): 228-239.
(3)
Roberts PJ et al (2010) J Clin Oncol. 28(31):4769-77
(4)
Puyol M et al (2010) Cancer Cell 1(13): 63-73.
Citation Format: Raquel Torres-Guzmán, Carmen Baquero, Carlos Marugan, Cecilia Mur, Severine I. Gharbi, Sandra Gomez, Joaquín Amat, Karsten Boehnke, Philip W. Iversen, Alfonso deDios, Xueqian Gong, Sean Buchanan, Richard P. Beckman, Maria José J. Lallena. Characterization of the mechanism of action of abemaciclib in NSCLC cell lines harboring KRAS mutation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-318. doi:10.1158/1538-7445.AM2017-LB-318
Abstract
Proper patient-tailoring strategy and the validation of novel therapeutic targets remain enormous challenges during drug discovery processes. Patient-derived three-dimensional organoid cell ...culture models possess great potential to associate compound sensitivity and disease complexity in order to provide a key missing link between compound screening and clinical trials. Abemaciclib is a reversible, ATP competitive, selective inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing advanced clinical testing.
In this study, we established and characterized three-dimensional organoid cultures from primary colorectal cancer patients and validated their use as drug sensitivity models. We aimed to explore the antitumor activity of abemaciclib in colon cancer organoid cultures by assessing markers for cell viability, proliferation, cell cycle, senescence and apoptosis. Single cell suspension of patient-derived samples were precultured for four days to allow for complete morphogenesis of three-dimensional organoid structures. Subsequently, the cultures were treated for at least two population doubling times and analyzed by luminescent cell viability, immunohistochemistry and flow cytometry assays.
Our data suggest that abemaciclib treatment decreased the cell viability of patient-derived colorectal cancer organoid cultures characterized by G1 cell cycle arrest and reduced Ki-67-positive cells. Furthermore, treated cultures showed elevated levels of reactive oxygen species and increased markers for early and late apoptosis. In summary, complex organoid models have the potential to further evaluate the antitumor activity of abemaciclib in various tumor types by enabling mechanistic studies in a patient-specific preclinical setting.
Citation Format: Karsten Boehnke, Bruna Calsina, Joaquín Amat, Ana Hermoso, Raquel Torres, Christoph Reinhard, Juan A. Velasco, Philip W. Iversen, Alfonso De Dios, Sean Buchanan, Richard P. Beckmann, Dirk Schumacher, Christian RA Regenbrecht, Marie-Laure Yaspo, Hans Lehrach, María José Lallena. Preclinical analysis and characterization of abemaciclib using three-dimensional patient-derived colorectal cancer organoid cultures. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2829.
Abstract
Dysregulation of the cell-cycle is a hallmark of cancer and genetic alterations in its regulatory machinery (or checkpoints) occur in most human tumors. The majority these defects are found ...in genes encoding for proteins regulating G1 phase progression, such as Rb, E2F1, CyclinD1, CDK4 and CDK6. Aberrant regulation of the G1 kinases CDK4 and CDK6, as well as overexpression or gene amplification of CyclinD, lead to inhibition of tumor suppressors such as Rb resulting in an accelerated cell cycle progression. Alterations in the CyclinD-CDK4/6-Rb pathway are common in breast cancer. Amplification of CCND1 gene encoding CyclinD1, occurs in 15% to 20% of breast cancers, and CyclinD1 overexpression is even more common (up to 50% of breast cancers).
Abemaciclib is a reversible, ATP competitive, kinase inhibitor selective for CDK4 and CDK6 that has been shown to prevent growth of malignant cells in-vitro and in-vivo. This antitumor activity is mediated by inhibiting the phosphorylation of Rb and subsequent blockade of tumor cell cycle progression through G1/S.
CDK4/6 inhibitors in general have shown significant potential for the treatment of metastatic breast cancer and Abemaciclib, in particular, is currently being evaluated in advanced clinical trials (Phase II as single agent and Phase III in combination with anti-hormone therapy) in hormone receptor positive metastatic breast cancer patients.
The goal of this study was to investigate the mechanism of action of Abemaciclib in ER+ luminal breast cancer. We have evaluated the response of the drug in a diversity of breast cancer cell lines. Phenotypic characterization of sensitive cell lines was carried out by monitoring proliferation, cell cycle progression and phosphorylation of Rb using High Content Imaging. Senescence markers were included in the study to monitor the final outcome of the cells upon sustained exposure to the drug.
Luminal ER+ breast cancer cells showed a marked sensitivity to treatment with Abemaciclib with IC50 values ranging from 5nM to 2uM.
Simultaneous decrease in Rb phosphorylation with sustained accumulation of the 2N subpopulation was observed. Associated to the G1S arrest phenotype, Abemaciclib treatment resulted in a decrease of cell proliferation markers (Ki67 and BrdU). Additionally, a marked hyper-methylation profile (Histone H3K9met3) and a decrease of FOXM1 expression were observed, as well as an accumulation of endogenous beta-galactosidase and p21. Taken together this profile suggests that Abemaciclib acts through promotion of senescence in breast cancer cells.
Abemaciclib prevents proliferation of breast cancer cell lines expressing D-types cyclins by promoting cell cycle arrest mediated by inhibition of Rb phosphorylation. Abemaciclib is a CDK4/6 inhibitor with potential to treat breast cancer by blocking cell proliferation leading to induction of senescence.
Citation Format: Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Ana Hermoso, Joaquin Amat, Bruna Calsina, Alfonso De Dios, Sean Buchanan, Jian Du, Richard Paul Beckmann, Xueqian Gong, Ann Mcnulty. In-vitro characterization of Abemaciclib pharmacology in ER+ breast cancer cell lines. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3101. doi:10.1158/1538-7445.AM2015-3101
Inserta : Perfil cortado por la linea 1, 2.
AGS. Secretaría de Guerra, Legajos, 05887. En carpeta con tít. : Real Orden de 10 de octubre de 99 : Para que no se establezcan las lanchas mandadas en 9 ...de agosto de 98, y que en su lugar se envien 4 obuses. Con carta de Francisco Orta y Arcos a Rafael Basco. Málaga, 5 de agosto de 1799. Inserto en el plano el calculo de las obras.
Tintas y colores a la aguada, ocre y amarillo.
Conservación Muy Buena. Explicación con clave alfabética.
Rúbrica del autor.
Manuscrito sobre papel.
•HFO-1336mzz(Z), HCFO-1233zd(E) and HCFO-1224yd(Z) are numerically studied to replace HFC-245fa.•Mapping of the minimum superheat requirement to ensure the dry compression is provided.•All ...alternatives improve the COP, being benefited from the use of an IHX.•HCFO-1233zd(E) and HCFO-1224yd(Z) present similar behaviour to HFC-245fa.•The alternatives reduce the equivalent CO2 emissions.
This paper analyses the feasibility of HCFO-1224yd(Z), HCFO-1233zd(E) and HFO-1336mzz(Z), three low global warming potential (GWP) refrigerants, as alternatives to HFC-245fa in high-temperature heat pump (HTHP) systems for low-grade waste heat recovery. HTHPs are a sustainable technology that can help to mitigate climate change through the thermal valorisation of the industrial low-grade waste heat. Before presenting and analysing the results, mapping of the minimum superheat degree requirement in the operating range, and the influence of the Internal Heat Exchanger (IHX) on each alternative are studied. The simulations were carried out at condensing temperatures from 115 to 145 °C and evaporating temperatures from 45 to 75 °C, using a single-stage cycle with and without IHX. Finally, the Total Equivalent Warming Impact (TEWI) evaluation is performed to illustrate the environmental effect of each alternative. Attending to the results, HCFO-1233zd(E) improves the COP about 27% compared to HFC-245fa, whereas HFO-1336mzz(Z) and HCFO-1224yd(Z) show an improvement of approx. 21 and 17%, respectively. Although HCFO-1233zd(E) and HCFO-1224yd(Z) present similar suction volumetric flow rate to HFC-245fa, HFO-1336mzz(Z) shows a relative increment up to 80%, and therefore, higher compressor and installation size are expected for this refrigerant. Finally, the TEWI analysis presents a significant reduction of the equivalent CO2 emissions for each low GWP alternative, between 59 and 61%. HCFO-1233zd(E) shows the highest reduction in all the simulation cases, followed by HCFO-1224yd(Z) and HFO-1336mzz(Z).
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•A reversible novel reversible HTHP-ORC system was proposed.•Heat source temperature increment benefits the energy performance for both modes.•Built-in volume ratio and IHX ...effectiveness optimization was realised.•The novel HTHP-ORC provides a COP of 2.44 and net electrical efficiency of 8.75%.•HCFO-1224yd(Z) and HCFO-1233zd(E) are appropriate alternatives for HFC-245fa.
Nowadays, a high amount of industrial thermal energy is still lost due to the lack of competitive solutions for energy revalorization. Facing this challenge, this paper presents a novel technology, based on a reversible High-Temperature Heat Pump (HTHP) and Organic Rankine Cycle (ORC). The proposed system recovers low-grade waste heat to generate electricity or useful heat in accordance with consumer demand. Compressor and expander semi-empirical models have been considered for the reversible system computational simulation, being HFC-245fa the working fluid selected. The built-in volume ratio and Internal Heat Exchanger (IHX) effectiveness have been optimized to reach the maximum energy efficiency in each operating condition. Although HFC-245fa exhibits energy performance attributes, its high Global Warming Potential (GWP) is an issue for climate change mitigation. Hence, multi-objective optimisation of the environmentally friendly working fluids Butane, Pentane, HFO-1336mzz(Z), R-514A, HCFO-1233zd(E) and HCFO-1224yd(Z) has been carried out. The results show that the system proposed, working with HFC-245fa, achieves a Coefficient of Performance (COP) of 2.44 for condensing temperature of 140 °C, operating in HTHP mode, whereas the ORC mode provides a net electrical efficiency of 8.7% at condensing temperature of 40 °C. Besides, HCFO-1233zd(E) and HCFO-1224yd(Z) are both appropriate alternatives for the HFC-245fa replacement. These working fluids provide a COP improvement of 9.7% and 5.8% and electrical net efficiency improvement of 2.1% and 0.8%, respectively, compared to HFC-245fa. This paper provides a reference study for further designs and developments of reversible HTHP-ORC systems used for industrial low-grade waste heat recovery.
La danza es una actividad física que se caracteriza por el movimiento del cuerpo mediante secuencias y ritmos, generalmente acompañada de estilos musicales variados y, por su naturaleza, se relaciona ...con beneficios psicológicos, porque se considera un medio de expresión emocional. En el presente trabajo se llevó a cabo una revisión sistemática de la literatura científica publicada en el período 2014-2023, sobre el impacto de la práctica de la danza en el bienestar. Los resultados mostraron que la práctica de la danza tiene beneficios en el bienestar en diferentes poblaciones y culturas, con efectos duraderos a corto, medio y largo plazo. La danza puede ser un recurso terapéutico útil para el tratamiento de diferentes trastornos mentales.
Objective
To assess the effectiveness and tolerability of perampanel (PER) monotherapy in routine clinical practice for the treatment of focal onset and generalized tonic‐clonic seizures (GTCS).
...Methods
This multicenter, retrospective, observational study was conducted in patients aged ≥12 years treated with PER as primary monotherapy or converted to PER monotherapy by progressive reduction of background antiepileptic drugs. Outcomes included retention, responder, and seizure‐free rate after 3, 6, and 12 months and tolerability throughout the follow‐up.
Results
A total of 98 patients (mean age = 49.6 ± 21.7 years, 51% female) with focal seizures and/or GTCS were treated with PER monotherapy for a median exposure of 14 months (range = 1‐57) with a median dose of 4 mg (range = 2‐10). The retention rates at 3, 6, and 12 months and last follow‐up were 93.8%, 89.3%, 80.9%, and 71.4%, respectively. The retention rates according to the type of monotherapy (primary vs conversion) did not differ (log‐rank P value = .57). Among the 98 patients, 61.2% patients had seizures throughout the baseline period, with a median seizure frequency of 0.6 seizures per month (range = 0.3‐26). Responder rates at 3, 6, and 12 months were 79.6%, 70.1%, and 52.8%, respectively, and seizure freedom rates at the same points were 62.7%, 56.1%, and 41.5%. Regarding the 33 patients who had GTCS in the baseline period, 87.8% were seizure‐free at 3 months, 78.1% at 6 months, and 55.1% at 12 months. Over the entire follow‐up, PER monotherapy was generally well tolerated, and only 16% of patients discontinued PER due to adverse events (AEs). Female patients were found to be at a higher risk of psychiatric AEs (female vs male odds ratio = 2.85, 95% confidence interval = 1‐8.33, P = .046).
Significance
PER demonstrated good effectiveness and a good safety profile when used as primary therapy or conversion to monotherapy at relatively low doses, in a clinical setting with patients with focal seizures and GTCS.
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