DataSheet1_3D dynamic cultures of HGSOC organoids to model innovative and standard therapies.PDF

High-grade serous ovarian cancer (HGSOC) needs new technologies for improving cancer diagnosis and therapy. It is a fatal disease with few options for the patients. In this context, dynamic culture systems coupling with patient-derived cancer 3D microstructures could offer a new opportunity for exploring novel therapeutic approaches. In this study, we optimized a passive microfluidic platform with 3D cancer organoids, which allows a standardized approach among different patients, a minimum requirement of samples, multiple interrogations of biological events, and a rapid response. The passive flow was optimized to improve the growth of cancer organoids, avoiding the disruption of the extracellular matrix (ECM). Under optimized conditions of the OrganoFlow (tilting angle of 15° and an interval of rocking every 8 min), the cancer organoids grow faster than when they are in static conditions and the number of dead cells is reduced over time. To calculate the IC50 values of standard chemotherapeutic drugs (carboplatin, paclitaxel, and doxorubicin) and targeted drugs (ATRA), different approaches were utilized. Resazurin staining, ATP-based assay, and DAPI/PI colocalization assays were compared, and the IC50 values were calculated. The results showed that in the passive flow, the IC50 values are lower than in static conditions. FITC-labeled paclitaxel shows a better penetration of ECM under passive flow than in static conditions, and cancer organoids start to die after 48 h instead of 96 h, respectively. Cancer organoids are the last frontiers for ex vivo testing of drugs that replicate the response of patients in the clinic. For this study, organoids derived from ascites or tissues of patients with Ovarian Cancer have been used. In conclusion, it was possible to develop a protocol for organoid cultures in a passive microfluidic platform with a higher growth rate, faster drug response, and better penetration of drugs into ECM, maintaining the samples’ vitals and collecting the data on the same plate for up to 16 drugs.</p

ERalpha CD domain interacts with Pin1.

<p>A) Schematic representation of ERalpha domains, showing the predicted prolyl isomerase sites (Ser/Thr-Pro). B) ERalpha cDNA was split into three different domains (AB, CD, EF) and tagged with histidine. GST or GST-Pin1 pull-down assay was performed in 293FT cells and analyzed by Western blot with anti-6×His antibody. C) Site-directed mutagenesis was performed to replace Serine 294 with Alanine on the ERalphaCD domain. Plasmids with wild-type (w) or mutant (m) CD His-tagged domain were transfected in 293FT cells and pulled down with GST-Pin1 protein. Anti-His antibody was used in Western blots to detect the interaction.</p

Identification of New Fyn Kinase Inhibitors Using a FLAP-Based Approach

The abnormal activity of Fyn tyrosine kinase has been shown to be related to various human cancers. Furthermore, its involvement in signaling pathways that lead to severe pathologies, such as Alzheimer’s and Parkinson’s diseases, has also been demonstrated, thus making Fyn an attractive target for the discovery of potential novel therapeutics for brain pathologies and tumors. In this study we evaluated the reliability of various screening approaches based on the FLAP software. By the application of the best procedure, the virtual screening workflow was used to filter the Gold and Platinum database from Asinex to identify new Fyn inhibitors. Enzymatic assays revealed that among the eight top-scoring compounds five proved to efficiently inhibit Fyn activity with IC₅₀ values in the micromolar range. These results demonstrate the validity of the methodologies we followed. Furthermore, the five active compounds herein described may be considered as interesting leads for the development of new and more efficient Fyn inhibitors