RESUMEN
This study presents an improved method for obtaining spheroids microwell arrays for histological processing and analysis, focusing on glioblastoma (U87 MG) and breast adenocarcinoma (MCF-7) tumor models. By transitioning from traditional 2D cell cultures to 3D systems, this approach overcomes the limitations of 2D cultures by more accurately replicating the tumor microenvironment. The method consists of producing homotypic and heterotypic spheroids using low-adherence agarose-coated wells, embedding these spheroids in agarose microwell arrays, and conducting immunohistochemistry (IHC) to analyze cellular and molecular profiles. Morphological analyses were performed using OrganoSeg software, and IHC staining confirmed marker expressions consistent with respective tumor types. The study details the workflow from 2D cell culture to IHC analysis, including agarose well coating, spheroid embedding, and IHC staining for markers such as EMA, p53, Ki-67, ER, PR, and HER2. Results demonstrated compact, round U87 MG spheroids and fibroblast-stabilized MCF-7 spheroids, with both types exhibiting specific marker expressions. This innovative approach significantly enhances the efficiency of producing and analyzing large volumes of spheroids, making it both quick and cost-effective. It offers a robust drug screening and cancer research platform, maintaining spheroid traceability even in bulk workflow conditions. Furthermore, this methodology supports advances in personalized medicine by providing a more physiologically relevant model than 2D cultures, which is crucial for investigating tumor behavior and therapeutic responses through IHC.
Asunto(s)
Inmunohistoquímica , Esferoides Celulares , Humanos , Esferoides Celulares/patología , Esferoides Celulares/metabolismo , Análisis Costo-Beneficio , Neoplasias de la Mama/patología , Células MCF-7 , Glioblastoma/patología , Biomarcadores de Tumor/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , FemeninoRESUMEN
Patients with chronic lymphocytic leukemia (CLL) treated with Ibrutinib often present hemorrhagic complications. Platelets dysfunction is well documented by aggregometry and flow cytometry, but the mechanisms by which Ibrutinib treatment influences the platelets status is yet to be evaluated. The aim of this study is to identify platelet membrane parameters in chronic lymphocytic leukemia (CLL) that could be altered by Ibrutinib administration. In this paper we propose a set of fluorescence measurements of the following parameters: membrane fluidity, resting membrane potential, and reactive oxygen species production of platelets suspensions obtained from CLL patients treated or not with Ibrutinib as markers for platelets status in this pathological situation. Platelets from CLL patients treated with Ibrutinib have higher membrane fluidity, lower resting membrane potential and higher level of reactive oxygen species production compared to the untreated CLL patients. These patients are also presenting higher membrane fluidity and lower resting membrane potential compared to healthy volunteers.