RESUMEN
Sarcomas are rare malignancies, the number of reports is limited, and this rarity makes further research difficult even though liposarcoma is one of major sarcomas. 2D cell culture remains an important role in establishing basic tumor biology research, but its various shortcomings and limitations are still of concern, and it is now well-accepted that the behavior of 3D-cultured cells is more reflective of in vivo cellular responses compared to 2D models. This study aimed to establish 3D cell culture of liposarcomas using two different methods: scaffold-based (Matrigel extracellular matrix [ECM] scaffold method) and scaffold-free (Ultra-low attachment [ULA] plate). Lipo246, Lipo224 and Lipo863 cell lines were cultured, and distinctive differences in structures were observed in Matrigel 3D model: Lipo224 and Lipo863 formed spheroids, whereas Lipo246 grew radially without forming spheres. In ULA plate approaches, all cell lines formed spheroids, but Lipo224 and Lipo863 spheroids showed bigger size and looser aggregation than Lipo246. Formalin fixed, paraffin embedded (FFPE) blocks were obtained from all 3D models, confirming the spheroid structures. The expression of MDM2, Ki-67 positivity and MDM2 amplification were confirmed by IHC and DNAscope™, respectively. Protein and DNA were extracted from all samples and MDM2 upregulation was confirmed by western blot and qPCR analysis. After treatment with MDM2 inhibitor SAR405838, DDLPS spheroids demonstrated different sensitivity patterns from 2D models. Taken together, we believed that 3D models would have a possibility to provide us a new predictability of efficacy and toxicity, and considered as one important process in in vitro pre-clinical phase prior to moving forward to clinical trials.
Asunto(s)
Liposarcoma , Sarcoma , Neoplasias de los Tejidos Blandos , Humanos , Liposarcoma/genética , Liposarcoma/terapia , Sarcoma/patología , Línea Celular , Esferoides Celulares/patologíaRESUMEN
EVs have emerged as an important component in tumour initiation, progression and metastasis. Although notable progresses have been made, the detection of EV cargoes remain significantly challenging for researchers to practically use; faster and more convenient methods are required to validate the EV cargoes, especially as biomarkers. Here we show, the possibility of examining embedded EVs as substrates to be used for detecting DNA amplification through ultrasensitive in situ hybridization (ISH). This methodology allows the visualization of DNA targets in a more direct manner, without time consuming optimization steps or particular expertise. Additionally, formalin-fixed paraffin-embedded (FFPE) blocks of EVs allows long-term preservation of samples, permitting future studies. We report here: (i) the successful isolation of EVs from liposarcoma tissues; (ii) the EV embedding in FFPE blocks (iii) the successful selective, specific ultrasensitive ISH examination of EVs derived from tissues, cell line, and sera; (iv) and the detection of MDM2 DNA amplification in EVs from liposarcoma tissues, cell lines and sera. Ultrasensitive ISH on EVs would enable cargo study while the application of ISH to serum EVs, could represent a possible novel methodology for diagnostic confirmation. Modification of probes may enable researchers to detect targets and specific DNA alterations directly in tumour EVs, thereby facilitating detection, diagnosis, and improved understanding of tumour biology relevant to many cancer types.
Asunto(s)
Vesículas Extracelulares , Liposarcoma , ADN/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Hibridación in Situ , Liposarcoma/diagnósticoRESUMEN
Glioblastoma (GBM) is the most frequent malignant brain tumor. It represents the most aggressive astrocytoma with an overall survival of 14 months. Despite improvements in surgery techniques, radio and chemotherapy, most patients present treatment resistance, recurrence and disease progression. Therefore, development of effective alternative therapies is essential to overcome treatment failure. The purpose of the study was to evaluate the antitumoral activity of the synthetic compound LQB118, in vitro. Monolayer and threedimensional (3D) cell culture systems of humanderived GBM cell lines were used to evaluate the effect of LQB118 on cell viability, cell death and migration. LQB118 reduced cell viability as determined by MTT and trypan blue exclusion assays and promoted apoptosis in monolayer cell lines with an intrinsic temozolomide (TMZ)resistance profile. In 3D culture models, LQB118 reduced cell viability as evaluated by APH assay and inhibited cell migration while the TMZ resistance profile was maintained. Moreover, LQB118 reduced p38 and AKT expression and phosphorylation, whereas it reduced only the phosphorylated ERK1/2 form. LQB118 reduced p38 and NRF2 expression, an axis that is associated with TMZ resistance, revealing a mechanism to overcome resistance. LQB118 also demonstrated an additional effect when combined with ionizing radiation and cisplatin. In conclusion, the present data demonstrated that LQB118 maintained its effectiveness in a 3D cell conformation, which shares more similarities with the tumor mass. LQB118 is a promising agent for GBM treatment as monotherapy and associated with radiotherapy or cisplatin. Its effect is associated with inhibition of GBMrelated survival signaling pathways.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Glioblastoma/metabolismo , Naftoquinonas/farmacología , Proteínas Quinasas/metabolismo , Pterocarpanos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Glioblastoma/tratamiento farmacológico , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Temozolomida , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the BCR-ABL oncoprotein, presents its treatment based on tyrosine kinase inhibitors (TKIs), mainly imatinib. However, despite its clinical success, almost 30% of all CML patients demand alternative therapy. In this context, the development of drugs capable of overcoming TKIs resistance is imperative. The pterocarpanquinone-LQB-118 is a novel compound with anti-tumor effect in CML cells whose mechanism of action is being elucidated. Here, we demonstrate that in two CML cell lines exhibiting different biological characteristics, LQB-118 modulates NFκB subcellular localization, apparently independently of the AKT and MAPK pathways, partially inhibits proteasome activity, and alters the expression of microRNAs -9 and -21.