Establishment and Comprehensive Molecular Characterization of an Immortalized Glioblastoma Cell Line from a Brazilian Patient.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with few effective treatment strategies. The research on the development of new treatments is often constrained by the limitations of preclinical models, which fail to accurately replicate the disease's essential characteristics. Herein, we describe the obtention, molecular, and functional characterization of the GBM33 cell line. This cell line belongs to the GBM class according to the World Health Organization 2021 Classification of Central Nervous System Tumors, identified by methylation profiling. GBM33 expresses the astrocytic marker GFAP, as well as markers of neuronal origin commonly expressed in GBM cells, such as ßIII-tubulin and neurofilament. Functional assays demonstrated an increased growth rate when compared to the U87 commercial cell line and a similar sensitivity to temozolamide. GBM33 cells retained response to serum starvation, with reduced growth and diminished activation of the Akt signaling pathway. Unlike LN-18 and LN-229 commercial cell lines, GBM33 is able to produce primary cilia upon serum starvation. In summary, the successful establishment and comprehensive characterization of this GBM cell line provide researchers with invaluable tools for studying GBM biology, identifying novel therapeutic targets, and evaluating the efficacy of potential treatments.

Polysome-profiling in small tissue samples.

Polysome-profiling is commonly used to study translatomes and applies laborious extraction of efficiently translated mRNA (associated with >3 ribosomes) from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for efficiently translated mRNA in only one or two fractions, thereby reducing sample handling 5-10-fold. The technique generates polysome-associated RNA with a quality reflecting the starting material and, when coupled with smart-seq2 single-cell RNA sequencing, translatomes in small tissues from biobanks can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associates with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in biobanks.

Laminin-γ1 chain and stress inducible protein 1 synergistically mediate PrPC-dependent axonal growth via Ca2+ mobilization in dorsal root ganglia neurons.

Prion protein (PrP(C)) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP(C) interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP(C) co-opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross-talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrP(C)-mediated axonogenesis in peripheral neurons in response to STI1 and laminin-γ1 chain-derived peptide (Ln-γ1). STI1 and Ln-γ1 promoted robust axonogenesis in wild-type neurons, whereas no effect was observed in neurons from PrP(C) -null mice. PrP(C) binding to Ln-γ1 or STI1 led to an increase in intracellular Ca(2+) levels via distinct mechanisms: STI1 promoted extracellular Ca(2+) influx, and Ln-γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln-γ1, but depends on, C-type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrP(C)-mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrP(C). These results suggest a role for PrP(C) as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.

Aberrant expression of RSK1 characterizes high-grade gliomas with immune infiltration.

The p90 ribosomal S6 kinase (RSK) family, a downstream target of Ras/extracellular signal-regulated kinase signaling, can mediate cross-talk with the mammalian target of rapamycin complex 1 pathway. As RSK connects two oncogenic pathways in gliomas, we investigated the protein levels of the RSK isoforms RSK1-4 in nontumoral brain (NB) and grade I-IV gliomas. When compared to NB or low-grade gliomas (LGG), a group of glioblastomas (GBMs) that excluded long-survivor cases expressed higher levels of RSK1 (RSK1hi ). No difference was observed in RSK2 median-expression levels among NB and gliomas; however, high levels of RSK2 in GBM (RSK2hi ) were associated with worse survival. RSK4 expression was not detected in any brain tissues, whereas RSK3 expression was very low, with GBM demonstrating the lowest RSK3 protein levels. RSK1hi and, to a lesser extent, RSK2hi GBMs showed higher levels of phosphorylated RSK, which reveals RSK activation. Transcriptome analysis indicated that most RSK1hi GBMs belonged to the mesenchymal subtype, and RSK1 expression strongly correlated with gene expression signature of immune infiltrates, in particular of activated natural killer cells and M2 macrophages. In an independent cohort, we confirmed that RSK1hi GBMs exclude long survivors, and RSK1 expression was associated with high protein levels of the mesenchymal subtype marker lysosomal protein transmembrane 5, as well as with high expression of CD68, which indicated the presence of infiltrating immune cells. An RSK1 signature was obtained based on differentially expressed mRNAs and validated in public glioma datasets. Enrichment of RSK1 signature followed glioma progression, recapitulating RSK1 protein expression, and was associated with worse survival not only in GBM but also in LGG. In conclusion, both RSK1 and RSK2 associate with glioma malignity, but displaying isoform-specific peculiarities. The progression-dependent expression and association with immune infiltration suggest RSK1 as a potential progression marker and therapeutic target for gliomas.

Effects of tumor biobank storage on polysome stability

Background: Human biological material has become an important resource for biomedical research. Tumor Biobanks are facilities that collect, store and distribute samples of tumor and normal tissue for further use in basic and translational cancer research. mRNA-translation has been demonstrated to modulate protein levels and is considered a fundamental post-transcriptional mechanism of gene expression regulation. Thus, determining translation efficiencies of individual mRNAs in human tumors may add another layer of information that contributes to the understanding of tumorigenic pathways. To analyze the RNAs actively engaged in translation, RNAs associated with ribosomes (polysomes) are isolated, identified and compared to total RNA. However, the application of this technique in human tumors depends on the stability of the polysomal structure under Biobank storage conditions that usually consists of ultra-low temperature. Since the effect of freezing on the stability of the polysomal structure in stored tumor samples is not known, it is essential to evaluate this factor in the frozen samples, validating the use of biobank samples in studies of translational efficiency. Methods: Xenograft tumors were divided in two parts, half was subject to immediate processing, and half was frozen for posterior analysis. Both parts were subject to polysomal separation, RNA extraction and identification through RNAseq. Results: It was possible to successfully extract and identify total and polysomal RNA from both fresh and frozen tumoral tissue. The quantification of the polysome profile indicated no difference in the translational efficiency estimated in fresh versus frozen tissue. Gene expression data from the fresh versus frozen tissues were compared and the correlation between the polysome associated fresh x frozen (R = 0,89) and total fresh x frozen (0,90) mRNAs was calculated. No difference was identified between the two conditions. Conclusions: We demonstrated that tissue freezing does not affect the polysomal structure, consequently validating the viability of the use of biobank stored tissue for polysome associated RNA analysis (AU)