Extracellular vesicles (EVs) are membrane-delimited vesicular bodies carrying different molecules, classified according to their size, density, cargo, and origin. Research on this topic has been ...actively growing through the years, as EVs are associated with critical pathological processes such as neurodegenerative diseases and cancer. Despite that, studies exploring the physiological functions of EVs are sparse, with particular emphasis on their role in organismal development, initial cell differentiation, and morphogenesis. In this review, we explore the topic of EVs from a developmental perspective, discussing their role in the earliest cell-fate decisions and neural tissue morphogenesis. We focus on the function of EVs through development to highlight possible conserved or novel processes that can impact disease progression. Specifically, we take advantage of what was learned about their role in development so far to discuss EVs impact on glioblastoma, a particular brain tumor of stem-cell origin and poor prognosis, and how their function can be hijacked to improve current therapies.
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Glioblastoma (GBM) is the most common and aggressive stage IV brain cancer with a poor prognosis and survival rate. The blood–brain barrier (BBB) in GBM prevents the entry and exit of biomarkers, ...limiting its treatment options. Hence, GBM diagnosis is pivotal for timely clinical management. Currently, there exists no clinically validated biomarker for GBM diagnosis. T cells exhibit the potential to escape a leaky BBB in GBM patients. These T cells infiltrating the GBM interact with the heterogeneous population of tumor cells, display a symbiotic interaction resulting in intertwined molecular crosstalk, and display a GBM-associated signature while entering the peripheral circulation. Therefore, we hypothesize that studying these distinct molecular changes is critical to enable T cells to be a diagnostic marker for accurate detection of GBM from patient blood. We demonstrated this by utilizing the phenotypic and immunological landscape changes in T cells associated with glioblastoma tumors. GBM exhibits a high level of heterogeneity with diverse subtypes of cells within the tumor, enabling immune infiltration and different degrees of interactions with the tumor. To accurately detect these subtle molecular differences in T cells, we designed an immunosensor with a high detection sensitivity and repeatability. Hence in this study, we investigated the characteristic behavior of T cells to establish two preclinically validated biomarkers: GBM-associated T cells (GBMAT) and GBM stem cell-associated T cells (GSCAT). A comprehensive investigation was conducted by mimicking the tumor microenvironment in vitro by coculturing T cells with cancer cells and cancer stem cells to study the distinct variation in GBMAT and GSCAT. Preclinical investigation of T cells from GBM patient blood shows similar characteristics to our established biomarkers (GBMAT, GSCAT). Further evaluating the relative attributes of T cells in patient blood and tissue biopsy confirms the infiltrating ability of T cells across the BBB. A pilot validation using a SERS-based machine learning algorithm was accomplished by training the model with GBMAT and GSCAT as diagnostic markers. Using GBMAT as a biomarker, we achieved a sensitivity and specificity of 93.3% and 97.4%, respectively, whereas applying GSCAT yielded a sensitivity and specificity of 100% and 98.7%, respectively. We also validated this diagnostic methodology by using conventional biological assays to study the change in expression levels of T cell surface markers (CD4 and CD8) and cytokine levels in T cells (IL6, IL10, TNFα, INFγ) from GBM patients. This study introduces T cells as GBM-specific immune biomarkers to diagnose GBM using patient liquid biopsy. This preclinical validation study presents a better translatability into clinical reality that will enable rapid and noninvasive glioblastoma detection from patient blood.
Glioblastoma poses a formidable challenge among primary brain tumors: its tumorigenic stem cells, capable of self-renewal, proliferation, and differentiation, contribute substantially to tumor ...initiation and therapy resistance. These glioblastoma stem cells (GSCs), resembling conventional stem and progenitor cells, adopt pathways critical for tissue development and repair, promoting uninterrupted tumor expansion. Long non-coding RNAs (lncRNAs), a substantial component of the human transcriptome, have garnered considerable interest for their pivotal roles in normal physiological processes and cancer pathogenesis. They display cell- or tissue-specific expression patterns, and extensive investigations have highlighted their impact on regulating GSC properties and cellular differentiation, thus offering promising avenues for therapeutic interventions. Consequently, lncRNAs, with their ability to exert regulatory control over tumor initiation and progression, have emerged as promising targets for innovative glioblastoma therapies. This review explores notable examples of GSC-associated lncRNAs and elucidates their functional roles in driving glioblastoma progression. Additionally, we delved deeper into utilizing a 3D in vitro model for investigating GSC biology and elucidated four primary methodologies for targeting lncRNAs as potential therapeutics in managing glioblastoma.
Purpose
Iron acquisition is key to maintaining cell survival and function. Cancer cells in general are considered to have an insatiable iron need. Iron delivery via the transferrin/transferrin ...receptor pathway has been the canonical iron uptake mechanism. Recently, however, our laboratory and others have explored the ability of ferritin, particularly the H-subunit, to deliver iron to a variety of cell types. Here, we investigate whether Glioblastoma (GBM) initiating cells (GICs), a small population of stem-like cells, are known for their iron addiction and invasive nature acquire exogenous ferritin, as a source of iron. We further assess the functional impact of ferritin uptake on the invasion capacity of the GICs.
Methods
To establish that H-ferritin can bind to human GBM, tissue-binding assays were performed on samples collected at the time of surgery. To interrogate the functional consequences of H-ferritin uptake, we utilized two patient-derived GIC lines. We further describe H-ferritin’s impact on GIC invasion capacity using a 3D invasion assay.
Results
H-ferritin bound to human GBM tissue at the amount of binding was influenced by sex. GIC lines showed uptake of H-ferritin protein via transferrin receptor. FTH1 uptake correlated with a significant decrease in the invasion capacity of the cells. H-ferritin uptake was associated with a significant decrease in the invasion-related protein Rap1A.
Conclusion
These findings indicate that extracellular H-ferritin participates in iron acquisition to GBMs and patient-derived GICs. The functional significance of the increased iron delivery by H-ferritin is a decreased invasion capacity of GICs potentially via reduction of Rap1A protein levels.
The current standard therapy for GBM includes maximal surgical resection, followed by concurrent radiotherapy and oral chemotherapy; however, the median post-diagnosis survival period is only 15–18 ...months. Nanoparticles (NPs) and their composite drug delivery systems (DDSs)—particularly those with liposomes and polymeric nanoparticles (PNPs)—have become an increasingly popular route for therapeutic delivery in glioblastoma multiforme (GBM). These systems also have the added benefit of enabling combinational therapies, which is particularly necessary for more resilient cancers like GBM. To ensure the most effective means of targeting the tumor microenvironment (TME), GBM stem cells (GSCs) can be used, both as therapeutic targets and as delivery vectors—both avenues showing promising results. However, DDSs directed towards the brain must overcome many barriers associated with cranial administration—including the blood-brain-barrier (BBB) and its derivatives, intracranial fluid pressure, induced regression from NP activating protocols involving heat or electromagnetic radiation, and extended retention of synthetic DDSs within cranial tissue—before they can finally circulate in the clinical domain. In this article, we review the progress made in incorporating GSCs within DDSs design, as well as the challenges that need to be addressed to facilitate a widespread regimen of successfully treating GBM.
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•Targeting tumor microenvironment via glioblastoma stem cells helps reduce resistance and relapse.•Biomarkers linked with CD133 are most effective in the directed targeting of glioblastoma stem cells.•Drug delivery systems designed with nanoparticles allow for less invasive targeting systems.•High-efficacy nanoparticle vectors have been designed to target biomarkers in human glioblastoma stem cells.
In the glioblastoma (GBM) microenvironment, tumor-associated macrophages (TAMs) are prominent components and facilitate tumor growth. The exact molecular mechanisms underlying TAMs' function in ...promoting glioma stem cells (GSCs) maintenance and tumor growth remain largely unknown. We found a candidate molecule, transforming growth factor beta-induced (TGFBI), that was specifically expressed by TAMs and extremely low in GBM and GSC cells, and meanwhile closely related to glioma WHO grades and patient prognosis. The exact mechanism of TGFBI linking TAM functions to GSC-driven tumor growth was explored.
Western blot, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), immunohistochemistry staining (IHC) and public datasets were used to evaluate TGFBI origin and level in GBM. The response of GSCs to recombinant human TGFBI was assessed
and orthotopic xenografts were established to investigate the function and mechanism
.
M2-like TAMs infiltration was elevated in high-grade gliomas. TGFBI was preferentially secreted by M2-like TAMs and associated with a poor prognosis for patients with GBM. TGFBI promoted the maintenance of GSCs and GBM malignant growth through integrin αvβ5-Src-Stat3 signaling
and
. Of clinical relevance, TGFBI was enriched in the serum and CSF of GBM patients and significantly decreased after tumor resection.
TAM-derived TGFBI promotes GSC-driven tumor growth through integrin αvβ5-Src-Stat3 signaling. High serum or CSF TGFBI may serve as a potential diagnostic and prognostic bio-index for GBMs.
Combination therapy is increasingly becoming the cornerstone of current day antitumor therapy. Glioblastoma multiforme is an aggressive brain tumor with a dismal median survival post diagnosis and a ...high rate of disease recurrence. The poor prognosis can be attributed to unique treatment limitations, which include the infiltrative nature of tumor cells, failure of anti-glioma drugs to cross the blood–brain barrier, tumor heterogeneity and the highly metastatic and angiogenic nature of the tumor making cells resistant to chemotherapy. Combination therapy approach is being developed against glioblastoma with new innovative combination drug regimens being tested in preclinical and clinical trials. In this review, we discuss the pathophysiology of glioblastoma, diagnostic markers, therapeutic targeting strategies, current treatment limitations, novel combination therapies in the context of current treatment options and the ongoing clinical trials for glioblastoma therapy.
•TMZ sensitivity of GSCs is correlated to their MGMT methylation status.•TMZ sensitivity of GSCs ex vivo is predictive of improved patient survival.•Individualized GSC cultures can be utilized for ...functional precision medicine.
Serum-free culturing of patient-derived glioblastoma biopsies enrich for glioblastoma stem cells (GSCs) and is recognized as a disease-relevant model system in glioblastoma (GBM). We hypothesized that the temozolomide (TMZ) drug sensitivity of patient-derived GSC cultures correlates to clinical sensitivity patterns and has clinical predictive value in a cohort of GBM patients. To this aim, we established 51 individual GSC cultures from surgical biopsies from both treatment-naïve primary and pretreated recurrent GBM patients. The cultures were evaluated for sensitivity to TMZ over a dosing range achievable in normal clinical practice. Drug efficacy was quantified by the drug sensitivity score. MGMT-methylation status was investigated by pyrosequencing. Correlative, contingency, and survival analyses were performed for associations between experimental and clinical data. We found a heterogeneous response to temozolomide in the GSC culture cohort. There were significant differences in the sensitivity to TMZ between the newly diagnosed and the TMZ-treated recurrent disease (p <0.01). There was a moderate correlation between MGMT-status and sensitivity to TMZ (r=0.459, p=0.0009). The relationship between MGMT status and TMZ efficacy was statistically significant on multivariate analyses (p=0.0051). We found a predictive value of TMZ sensitivity in individual GSC cultures to patient survival (p=0.0089). We conclude that GSC-enriched cultures hold clinical and translational relevance by their ability to reflect the clinical heterogeneity in TMZ-sensitivity, substantiate the association between TMZ-sensitivity and MGMT-promotor methylation status and appear to have a stronger predictive value than MGMT-promotor methylation on clinical responses to TMZ.
Glioblastoma is the most aggressive primary brain tumour and has a very poor prognosis. Inhibition of c-Src activity in glioblastoma stem cells (GSCs, responsible for glioblastoma lethality) and ...primary glioblastoma cells by the peptide TAT-Cx43266–283 reduces tumorigenicity, and boosts survival in preclinical models. Because c-Src can modulate cell metabolism and several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells, here we explored the inhibition of advantageous GSC metabolic plasticity by the c-Src inhibitor TAT-Cx43266-283.
Metabolic impairment induced by the c-Src inhibitor TAT-Cx43266-283 in vitro was assessed by fluorometry, western blotting, immunofluorescence, qPCR, enzyme activity assays, electron microscopy, Seahorse analysis, time-lapse imaging, siRNA, and MTT assays. Protein expression in tumours from a xenograft orthotopic glioblastoma mouse model was evaluated by immunofluorescence.
TAT-Cx43266–283 decreased glucose uptake in human GSCs and reduced oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on brain cell metabolism, including rat neurons, human and rat astrocytes, and human neural stem cells. TAT-Cx43266-283 impaired metabolic plasticity, reducing GSC growth and survival under different nutrient environments. Finally, GSCs intracranially implanted with TAT-Cx43266–283 showed decreased levels of important metabolic targets for cancer therapy, such as hexokinase-2 and GLUT-3.
The reduced ability of TAT-Cx43266-283–treated GSCs to survive in metabolically challenging settings, such as those with restricted nutrient availability or the ever-changing in vivo environment, allows us to conclude that the advantageous metabolic plasticity of GSCs can be therapeutically exploited through the specific and cell-selective inhibition of c-Src by TAT-Cx43266-283.
Spanish Ministerio de Economía y Competitividad (FEDER BFU2015-70040-R and FEDER RTI2018-099873-B-I00), Fundación Ramón Areces. Fellowships from the Junta de Castilla y León, European Social Fund, Ministerio de Ciencia and Asociación Española Contra el Cáncer (AECC).
Purpose
Constructed from a theoretical framework, the coordinated undermining of survival paths in glioblastoma (GBM) is a combination of nine drugs approved for non-oncological indications (CUSP9; ...aprepitant, auranofin, captopril, celecoxib, disulfiram, itraconazole, minocycline, quetiapine, and sertraline) combined with temozolomide (TMZ). The availability of these drugs outside of specialized treatment centers has led patients to embark on combination treatments without systematic follow-up. However, no experimental data on efficacy using the CUSP9 strategy in GBM have been reported.
Methods
Using patient-derived glioblastoma stem cell (GSC) cultures from 15 GBM patients, we described stem cell properties of individual cultures, determined the dose–response relationships of the drugs in the CUSP9, and assessed the efficacy the CUSP9 combination with TMZ in concentrations clinically achievable. The efficacy was evaluated by cell viability, cytotoxicity, and sphere-forming assays in both primary and recurrent GSC cultures.
Results
We found that CUSP9 with TMZ induced a combination effect compared to the drugs individually (
p
< 0.0001). Evaluated by cell viability and cytotoxicity, 50% of the GSC cultures displayed a high sensitivity to the drug combination. In clinical plasma concentrations, the effect of the CUSP9 with TMZ was superior to TMZ monotherapy (
p
< 0.001). The Wnt-signaling pathway has been shown important in GSC, and CUSP9 significantly reduces Wnt-activity.
Conclusions
Adding experimental data to the theoretical rationale of CUSP9, our results demonstrate that the CUSP9 treatment strategy can induce a combination effect in both treatment-naïve and pretreated GSC cultures; however, predicting response in individual cultures will require further profiling of GSCs.
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