3D-printed microplate inserts for long term high-resolution imaging of live brain organoids

Background: Organoids are a reliable model used in the study of human brain development and under pathological conditions. However, current methods for brain organoid culture generate tissues that range from 0.5 to 2mm of size, which need to be constantly agitated to allow proper oxygenation. The culture conditions are, therefore, not suitable for whole-brain organoid live imaging, required to study developmental processes and disease progression within physiologically relevant time frames (i.e. days, weeks, months). Results: Here we designed 3D-printed microplate inserts adaptable to standard 24 multi-well plates, which allow the growth of multiple organoids in pre-defined and fixed XYZ coordinates. This innovation facilitates highresolution imaging of whole-cerebral organoids, allowing precise assessment of organoid growth and morphology, as well as cell tracking within the organoids, over long periods. We applied this technology to track neocortex development through neuronal progenitors in brain organoids, as well as the movement of patient-derived glioblastoma stem cells within healthy brain organoids. Conclusions: This new bioengineering platform constitutes a significant advance that permits long term detailed analysis of whole-brain organoids using multimodal inverted fluorescence microscopy.Mariana Oksdath Mansilla, Camilo Salazar-Hernandez, Sally L. Perrin, Kaitlin G. Scheer, Gökhan Cildir, John Toubia, Kristyna Sedivakova, Melinda N. Tea, Sakthi Lenin, Elise Ponthier, Erica C. F. Yeo, Vinay Tergaonkar, Santosh Poonnoose, Rebecca J. Ormsby, Stuart M. Pitson, Michael P. Brown, Lisa M. Ebert, and Guillermo A. Gome

A drug screening pipeline using 2D and 3D patient-derived in vitro models for pre-clinical analysis of therapy response in glioblastoma

Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6–9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microen-vironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cul-tures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma.</p

Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy

Objectives: Targeted immunotherapies such as chimeric antigen receptor (CAR)-T cells are emerging as attractive treatment options for glioblastoma, but rely on identification of a suitable tumor antigen. We validated a new target antigen for glioblastoma, fibroblast activation protein (FAP), by undertaking a detailed expression study of human samples. Methods: Glioblastoma and normal tissues were assessed using immunostaining, supported by analyses of published transcriptomic datasets. Short-term cultures of glioma neural stem (GNS) cells were compared to cultures of healthy astrocytes and neurons using flow cytometry. Glioblastoma tissues were dissociated and analysed by high-parameter flow cytometry and single-cell transcriptomics (scRNAseq). Results: Compared to normal brain, FAP was overexpressed at the gene and protein level in a large percentage of glioblastoma tissues, with highest levels of expression associated with poorer prognosis. FAP was also overexpressed in several paediatric brain cancers. FAP was commonly expressed by cultured GNS cells but absent from normal neurons and astrocytes. Within glioblastoma tissues, the strongest expression of FAP was around blood vessels. In fact, almost every tumor vessel was highlighted by FAP expression, whereas normal tissue vessels and cultured endothelial cells (ECs) lacked expression. Single-cell analyses of dissociated tumors facilitated a detailed characterisation of the main cellular components of the glioblastoma microenvironment and revealed that vessel-localised FAP is because of expression on both ECs and pericytes. Conclusion: Fibroblast activation protein is expressed by multiple cell types within glioblastoma, highlighting it as an ideal immunotherapy antigen to target destruction of both tumor cells and their supporting vascular network.</p

Identifying the roles of amino acids, alcohols and 1,2-diamines as mediators in coupling of haloarenes to arenes

Coupling of haloarenes to arenes has been facilitated by a diverse range of organic additives in the presence of KO(t)Bu or NaO(t)Bu since the first report in 2008. Very recently, we showed that the reactivity of some of these additives (e.g., compounds 6 and 7) could be explained by the formation of organic electron donors in situ, but the role of other additives was not addressed. The simplest of these, alcohols, including 1,2-diols, 1,2-diamines, and amino acids are the most intriguing, and we now report experiments that support their roles as precursors of organic electron donors, underlining the importance of this mode of initiation in these coupling reactions