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.

Intraoperative radiotherapy for brain metastases: first-stage results of a single-arm, open-label, phase 2 trial.

OBJECTIVE: Focal stereotactic radiosurgery to the surgical cavity lowers local recurrence after resection of brain metastases (BM). To evaluate local control (LC) and brain disease control (BDC) after intraoperative radiotherapy (IORT) for resected BM. METHODS: Adult patients with completely resected single supratentorial BM were recruited and underwent IORT to the cavity with a prescribed dose of 18 Gy to 1 mm-depth. Primary endpoints were actuarial LC and BDC. Local failure (LF) and distant brain failure (DBF), with death as a competing risk, were estimated. Secondary endpoints were overall survival (OS) and incidence of radiation necrosis (RN). Simon's two-stage design was used and estimated an accrual of 10 patients for the first-stage analysis and a LC higher than 63% to proceed to second stage. We report the final analysis of the first stage. RESULTS: Between June 2019 to November 2020, 10 patients were accrued. Median clinical and imaging FU was 11.2 and 9.7 months, respectively. Median LC was not reached and median BDC was 5 months. The 6-month and 12-month LC was 87.5%. The 6-month and 12-month BDC was 39% and 13%, respectively. Incidence of LF at 6 and 12 months was 10% and of DBF at 6 and 12 months was 50% and 70%, respectively. Median OS was not reached. The 6-month and 12-month OS was 80%. One patient had asymptomatic RN. CONCLUSION: IORT for completely resected BM is associated with a potential high local control and low risk of RN, reaching the pre-specified criteria to proceed to second stage and warranting further studies.

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.

Prognostic impact of platinum sensitivity in ovarian carcinoma patients with brain metastasis.

BACKGROUND: Brain metastasis (BM) is a rare event in ovarian cancer patients. The current prognostic scores that have been used for other tumors do not account for specific characteristics of ovarian cancer, such as platinum sensitivity. METHODS: This retrospective cohort study examined patients with ovarian carcinoma and BM who were treated at a single institution from January 2007 to December 2017. Clinical data on the diagnosis of BM and follow-up were collected. Cox regression was used to evaluate prognostic factors for overall survival (OS). RESULTS: Of 560 patients, 26 presented with BM. Eight patients were treated with surgery, 15 with whole-brain radiotherapy (RT), and 5 with stereotactic RT, and 4 patients received systemic treatment at the diagnosis of BM. The median OS was 10.8 months. The following factors were associated with OS: platinum-sensitive recurrence (HR 0.34, 95% CI 0.12-0.99; p = 0.049), higher number of previous treatment lines (HR 1.57, 95% CI 1.12-2.19; p = 0.008), ECOG performance status (HR 2.52, 95% CI 1.24-5.09; p = 0.010), and longer interval from initial diagnosis to BM (p = 0.025). Notably, the number of brain metastasis, the largest tumor size, and progression outside of the CNS were not related to survival. Platinum sensitivity was not associated with any of the classic prognostic factors in brain metastasis patients such as number or size of brain metastasis or disease progression outside the CNS strengthening the hypothesis of the importance of platinum sensitivity to the prognosis of ovarian cancer patients with BM. CONCLUSIONS: The factors related to the biological behavior of the ovarian cancer such as platinum sensitivity at the time of BM diagnosis, fewer number of previous treatment lines and interval from initial diagnosis were associated with survival in ovarian cancer patients with BM, while factors that are usually related to survival in BM in other cancers were not associated with survival in this cohort of ovarian cancer patients. The small number of patients did not allow us to exclude the prognostic role of these former factors that were not associated with survival in the present cohort.

Polysome Profiling of a Human Glioblastoma Reveals Intratumoral Heterogeneity.

Glioblastoma (GBM) is one of the most aggressive cancers, with median survival of less than 2 years. Despite of considerable advance in molecular classification of GBMs, no improvements in therapy have been described. The scenario is further complicated by tumor heterogeneity and the relationship among genetic, transcriptional and functional findings. Classically, gene expression has been evaluated by steady-state mRNA, however, this does not take translational control into consideration, which contributes considerably to the composition of the proteome. In this study, we evaluated the transcriptomic and translatomic signature of a GBM obtained from a single patient focusing in tumor heterogeneity. In a sampling of eight fragments, we investigated the translation rates, mTORC1 and ERK1/2 pathways and identified both total and polysome associated mRNAs. An increased translation rate was observed in fragments with high-grade histological features. High-grade histology was also associated with the expression of genes related to extracellular matrix (ECM) and angiogenesis, in both transcriptomes and translatomes. However, genes associated with epithelial to mesenchymal transition and stress response, were observed only in translatomes from high-grade fragments. Overall, our results demonstrate that isolation of translated mRNA can be used to identify biomarkers and reveal previously unrecognized determinants of heterogeneity in GBMs.

Nuclear unphosphorylated STAT3 correlates with a worse prognosis in human glioblastoma.

Glioblastoma (GBM) is currently the most aggressive form of brain tumor identified, and STAT3 is known to play an important role in gliomagenesis. Moreover, while several studies have used pharmacological approaches to modulate STAT3 activity, the results have been contradictory. In this study, expressions of STAT3, pSTAT3 (Y705), and pSTAT3 (S727) were evaluated using immunohistochemistry assays of tissue microarrays containing non-neoplastic tissue (NN, n=12), grade II astrocytomas (n=33), grade III astrocytomas (n=12), and GBM (n=85) specimens. In GBM specimens, STAT3 was overexpressed and exhibited greater nuclear localization compared with lower grade astrocytomas and NN. Conversely, nuclear localization of pSTAT3 (Y705) and pSTAT3 (S727) exhibited a similar phenotype in both GBMs and NNs. MET was also detected as a non-canonical pathway marker for STAT3. For tumors with higher levels of STAT3 nuclear localization, and not pSTAT3 (Y705) and pSTAT3 (S727), these specimens exhibited increased levels of MET expression. Thus, a non-canonical pathway may mediate a proportion of the STAT3 that translocates to the nucleus. Moreover, tumors which exhibited greater nuclear localization of STAT3 corresponded with patients that presented with lower rates of recurrence-free survival and overall survival. In contrast, the phosphorylated forms of STAT3 did not correlate with patient survival. These findings suggest that phosphorylation-independent mechanisms may mediate the nuclear translocation and activation of STAT3. Further studies are needed to identify the mechanisms involved, especially those that provide targets to achieve efficient inhibition and control of GBM progression.

Local control after radiosurgery for brain metastases: predictive factors and implications for clinical decision.

BACKGROUND: To evaluate the local control of brain metastases (BM) in patients treated with stereotactic radiosurgery (SRS), correlate the outcome with treatment parameters and lesion characteristics, and define its implications for clinical decisions. METHODS: Between 2007 and 2012, 305 BM in 141 consecutive patients were treated with SRS. After exclusions, 216 BM in 100 patients were analyzed. Doses were grouped as follows: ≤15 Gy, 16-20 Gy, and ≥21 Gy. Sizes were classified as ≤10 mm and >10 mm. Local control (LC) and overall survival (OS) were estimated using the Kaplan-Meier method. Log-rank statistics were used to identify the prognostic factors affecting LC and OS. For multivariate analyses, a Cox proportional model was applied including all potentially significant variables reached on univariate analyses. RESULTS: Median age was 54 years (18-80). Median radiological follow-up of the lesions was 7 months (1-66). Median LC and the LC at 1 year were 22.3 months and 69.7%, respectively. On univariate analysis, tumor size, SRS dose, and previous whole brain irradiation (WBRT) were significant factors for LC. Patients with lesions >10 and ≤10 mm had an LC at 1 year of 58.6% and 79.1%, respectively (p = 0.008). In lesions receiving ≤15 Gy, 16-20 Gy, and ≥21 Gy, the 1-year LC rates were 39.6%, 71.7%, and 92.3%, respectively (p < 0.001). When WBRT was done previously, LC at 1 year was 57.9% compared with 78.4% for those who did not undergo WBRT (p = 0.004). On multivariate analysis, dose remained the single most powerful prognostic factor for LC. Median OS for all patients was 17 months, with no difference among the groups. CONCLUSIONS: Dose is the most important predictive factor for LC of BM. Doses below 16 Gy correlated with poor LC. The SRS dose as salvage treatment after previous WBRT should not be reduced unless there is a pressing reason to do so.

Regulation of stress-inducible phosphoprotein 1 nuclear retention by protein inhibitor of activated STAT PIAS1.

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450-480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.