Comprehensive analysis of the impact of procedure time and the 'golden hour' in subpopulations of stroke thrombectomy patients.

OBJECTIVE: To evaluate the effect of procedure time on thrombectomy outcomes in different subpopulations of patients undergoing endovascular thrombectomy (EVT), given the recently expanded indications for EVT. METHODS: This multicenter study included patients undergoing EVT for acute ischemic stroke at 35 centers globally. Procedure time was defined as time from groin puncture to successful recanalization (Thrombolysis in Cerebral Infarction score ≥2b) or abortion of procedure. Patients were stratified based on stroke location, use of IV tissue plasminogen activator (tPA), Alberta Stroke Program Early CT score, age group, and onset-to-groin time. Primary outcome was the 90-day modified Rankin Scale (mRS) score, with scores 0-2 designating good outcome. Secondary outcome was postprocedural symptomatic intracranial hemorrhage (sICH). Multivariate analyses were performed using generalized linear models to study the impact of procedure time on outcomes in each subpopulation. RESULTS: Among 8961 patients included in the study, a longer procedure time was associated with higher odds of poor outcome (mRS score 3-6), with 10% increase in odds for each 10 min increment. When procedure time exceeded the 'golden hour', poor outcome was twice as likely. The golden hour effect was consistent in patients with anterior and posterior circulation strokes, proximal or distal occlusions, in patients with large core infarcts, with or without IV tPA treatment, and across age groups. Procedures exceeding 1 hour were associated with a 40% higher sICH rate. Posterior circulation strokes, delayed presentation, and old age were the variables most sensitive to procedure time. CONCLUSIONS: In this work we demonstrate the universality of the golden hour effect, in which procedures lasting more than 1 hour are associated with worse clinical outcomes and higher rates of sICH across different subpopulations of patients undergoing EVT.

The effect of occlusion location and technique in mechanical thrombectomy for minor stroke.

INTRODUCTION: Endovascular mechanical thrombectomy (MT) is an established treatment for large vessel occlusion strokes with a National Institutes of Health Stroke Scale (NIHSS) score of 6 or higher. Data pertaining to minor strokes, medium, or distal vessel occlusions, and most effective MT technique is limited and controversial. METHODS: A multicenter retrospective study of all patients treated with MT presenting with NIHSS score of 5 or less at 29 comprehensive stroke centers. The cohort was dichotomized based on location of occlusion (proximal vs. distal) and divided based on MT technique (direct aspiration first-pass technique [ADAPT], stent retriever [SR], and primary combined [PC]). Outcomes at discharge and 90 days were compared between proximal and distal occlusion groups, and across MT techniques. RESULTS: The cohort included 759 patients, 34% presented with distal occlusion. Distal occlusions were more likely to present with atrial fibrillation (p = 0.008) and receive IV tPA (p = 0.001). Clinical outcomes at discharge and 90 days were comparable between proximal and distal groups. Compared to SR, patients managed with ADAPT were more likely to have a modified Rankin Scale of 0-2 at discharge and at 90 days (p = 0.024 and p = 0.013). Primary combined compared to ADAPT, prior stroke, multiple passes, older age, and longer procedure time were independently associated with worse clinical outcome, while successful recanalization was positively associated with good clinical outcomes. CONCLUSIONS: Proximal and distal occlusions with low NIHSS have comparable outcomes and safety profiles. While all MT techniques have a similar safety profile, ADAPT was associated with better clinical outcomes at discharge and 90 days.

Risk factors and predictors of intracranial hemorrhage after mechanical thrombectomy in acute ischemic stroke: insights from the Stroke Thrombectomy and Aneurysm Registry (STAR).

BACKGROUND: Reducing intracranial hemorrhage (ICH) can improve patient outcome in acute ischemic stroke (AIS) intervention. We sought to identify ICH risk factors after AIS thrombectomy. METHODS: This is a retrospective review of the Stroke Thrombectomy and Aneurysm Registry (STAR) database. All patients who underwent AIS thrombectomy with available ICH data were included. Multivariable regression models were developed to identify predictors of ICH after thrombectomy. Subgroup analyses were performed stratified by symptom status and European Cooperative Acute Stroke Study (ECASS) grade. RESULTS: The study cohort comprised 6860 patients. Any ICH and symptomatic ICH (sICH) occurred in 25% and 7% of patients, respectively. Hemorrhagic infarction 1 (HI1) occurred in 36%, HI2 in 24%, parenchymal hemorrhage 1 (PH1) in 22%, and PH2 in 17% of patients classified by ECASS grade. Intraprocedural complications independently predicted any ICH (OR 3.8083, P<0.0001), PH1 (OR 1.9053, P=0.0195), and PH2 (OR 2.7347, P=0.0004). Race also independently predicted any ICH (black: OR 0.5180, P=0.0017; Hispanic: OR 0.4615, P=0.0148), sICH (non-white: OR 0.4349, P=0.0107), PH1 (non-white: OR 3.1668, P<0.0001), and PH2 (non-white: OR 1.8689, P=0.0176), with white as the reference. Primary mechanical thrombectomy technique also independently predicted ICH. ADAPT (A Direct Aspiration First Pass Technique) was a negative predictor of sICH (OR 0.2501, P<0.0001), with stent retriever as the reference. CONCLUSIONS: This study identified ICH risk factors after AIS thrombectomy using real-world data. There was a propensity towards a reduced sICH risk with direct aspiration. Procedural complications and ethnicity were predictors congruent between categories of any ICH, sICH, PH1, and PH2. Further investigation of technique and ethnicity effects on ICH and outcomes after AIS thrombectomy is warranted.

Mechanical thrombectomy for large vessel occlusion strokes beyond 24 hours.

BACKGROUND: Recent clinical trials have shown that mechanical thrombectomy is superior to medical management for large vessel occlusion for up to 24 hours from onset. Our objective is to examine the safety and efficacy of thrombectomy beyond the standard of care window. METHODS: A retrospective review was undertaken of the multicenter Stroke Thrombectomy and Aneurysm Registry (STAR). We identified patients who underwent mechanical thrombectomy for large vessel occlusion beyond 24 hours. We selected a matched control group from patients who underwent thrombectomy in the 6-24-hour window. We used functional independence at 3 months as our primary outcome measure. RESULTS: We identified 121 patients who underwent thrombectomy beyond 24 hours and 1824 in the 6-24-hour window. We selected a 2:1 matched group of patients with thrombectomy 6-24 hours as a comparison group. Patients undergoing thrombectomy beyond 24 hours were less likely to be independent at 90 days (18 (18.8%) vs 73 (34.9%), P=0.005). They had higher odds of mortality at 90 days in the adjusted analysis (OR 2.34, P=0.023). Symptomatic intracerebral hemorrhage and other complications were similar in the two groups. In a multivariate analysis only lower number of attempts was associated with good outcomes (OR 0.27, P=0.022). CONCLUSIONS: Mechanical thrombectomy beyond 24 hours appears to be safe and tolerable with no more hemorrhages or complications compared with standard of care thrombectomy. Outcomes and mortality in this time window are worse compared with an earlier time window, but the rates of good outcomes may justify this therapy in selected patients.

Multicenter investigation of technical and clinical outcomes after thrombectomy for distal vessel occlusion by frontline technique.

BACKGROUND: Endovascular thrombectomy (EVT) is the standard-of-care for proximal large vessel occlusion (LVO) stroke. Data on technical and clinical outcomes in distal vessel occlusions (DVOs) remain limited. METHODS: This was a retrospective study of patients undergoing EVT for stroke at 32 international centers. Patients were divided into LVOs (internal carotid artery/M1/vertebrobasilar), medium vessel occlusions (M2/A1/P1) and isolated DVOs (M3/M4/A2/A3/P2/P3) and categorized by thrombectomy technique. Primary outcome was a good functional outcome (modified Rankin Scale ≤2) at 90 days. Secondary outcomes included recanalization, procedure-time, thrombectomy attempts, hemorrhage, and mortality. Multivariate logistic regressions were used to evaluate the impact of technical variables. Propensity score matching was used to compare outcome in patients with DVO treated with aspiration versus stent retriever RESULTS: We included 7477 patients including 213 DVOs. Distal location did not independently predict good functional outcome at 90 days compared with proximal (p=0.467). In distal occlusions, successful recanalization was an independent predictor of good outcome (adjusted odds ratio (aOR) 5.11, p<0.05) irrespective of technique. Younger age, bridging therapy, and lower admission National Institutes of Health Stroke Scale (NIHSS) were also predictors of good outcome. Procedure time ≤1 hour or ≤3 thrombectomy attempts were independent predictors of good outcomes in DVOs irrespective of technique (aOR 4.5 and 2.3, respectively, p<0.05). There were no differences in outcomes in a DVO matched cohort of aspiration versus stent retriever. Rates of hemorrhage and good outcome showed an exponential relationship to procedural metrics, and were more dependent on time in the aspiration group and attempts in the stent retriever group. CONCLUSIONS: Outcomes following EVT for DVO are comparable to LVO with similar results between techniques. Techniques may exhibit different futility metrics; stent retriever thrombectomy was influenced by attempts whereas aspiration was more dependent on procedure time.

Multicenter investigation of technical and clinical outcomes after thrombectomy for Proximal Medium Vessel Occlusion (pMeVO) by frontline technique.

BACKGROUND: Endovascular thrombectomy(EVT) is the standard of care for large vessel occlusion(LVO) stroke. Data on technical and clinical outcome in proximal medium vessel occlusions(pMeVOs) comparing frontline techniques remain limited. METHODS: We report an international multicenter retrospective study of patients undergoing EVT for stroke at 32 centers between 2015-2021. Patients were divided into LVOs(ICA/M1/Vertebrobasilar) or pMeVOs(M2/A1/P1) and categorized by thrombectomy technique. Primary outcome was 90-day good functional outcome(mRS ≤ 2). Multivariate logistic regressions were used to evaluate the impact of technical variables on clinical outcomes. Propensity score matching was used to compare outcome in patients with pMeVO treated with aspiration versus stent-retriever. RESULTS: In the cohort of 5977 LVO and 1287 pMeVO patients, pMeVO did not independently predict good-outcome(p = 0.55). In pMeVO patients, successful recanalization irrespective of frontline technique(aOR = 3.2,p < 0.05), procedure time ≤ 1-h(aOR = 2.2,p < 0.05), and thrombectomy attempts ≤ 4(aOR = 2.8,p < 0.05) were independent predictors of good-outcomes.In a propensity-matched cohort of aspiration versus stent-retriever pMeVO patients, there was no difference in good-outcomes. The rates of hemorrhage were higher(9%vs.4%,p < 0.01) and procedure time longer(51-min vs. 33-min,p < 0.01) with stent-retriever, while the number of attempts was higher with aspiration(2.5vs.2,p < 0.01). Rates of hemorrhage and good-outcome showed an exponential relationship to procedural metrics, and were more dependent on time in the aspiration group compared to attempts in the stent-retriever group. CONCLUSIONS: Clinical outcomes following EVT for pMeVO are comparable to those in LVOs. The golden hour or 3-pass rules in LVO thrombectomy still apply to pMeVO thrombectomy. Different techniques may exhibit different futility metrics; SR thrombectomy was more influenced by attempts whereas aspiration was more dependent on procedure time.

Drug versus non-drug behaviors: A dual-reward model of sex differences and neurobiological mechanisms in rats.

Substance Use Disorders (SUDs) are an impactful problem characterized by chronic relapse and engagement in drug-related behaviors at the expense of non-drug behaviors. Brain regions implicated in drug and non-drug-related behaviors often overlap, complicating investigations of neurobiological mechanisms underlying SUDs. Here we presented a within-subject model for studying self-administration, reinforcer competition, extinction, and cued reinstatement of cocaine- and food-seeking in rats. Due to differences in cocaine- and food-reinforced behavior, we transformed data to proportions of baseline, revealing increased resistance to extinction and disproportionately greater cued reinstatement of cocaine seeking relative to food seeking. Consistent with previous reports, females showed greater preference for cocaine reinforcement than males, though these findings failed to reach statistical significance. To demonstrate the model's utility for investigating neurobiological mechanisms, we included proof-of-concept calcium imaging data demonstrating the utility of the behavioral model for detecting cellular activity patterns associated with cocaine- and food-seeking behaviors. Future studies utilizing this model should improve understanding of the development and expression of pathological behaviors characteristic of SUDs in humans, sex differences in these behaviors, and their neurobiological correlates. Thus, the model has utility for improving understanding of SUDs, leading to novel treatments to reduce the pathological behaviors associated with SUDs.

Accumbens D2-MSN hyperactivity drives antipsychotic-induced behavioral supersensitivity.

Antipsychotic-induced dopamine supersensitivity, or behavioral supersensitivity, is a problematic consequence of long-term antipsychotic treatment characterized by the emergence of motor abnormalities, refractory symptoms, and rebound psychosis. The underlying mechanisms are unclear and no approaches exist to prevent or reverse these unwanted effects of antipsychotic treatment. Here we demonstrate that behavioral supersensitivity stems from long-lasting pre, post and perisynaptic plasticity, including insertion of Ca2+-permeable AMPA receptors and loss of D2 receptor-dependent inhibitory postsynaptic currents (IPSCs) in D2 receptor-expressing medium spiny neurons (D2-MSNs) in the nucleus accumbens core (NAcore). The resulting hyperexcitability, prominent in a subpopulation of D2-MSNs (21%), caused locomotor sensitization to cocaine and was associated with behavioral endophenotypes of antipsychotic treatment resistance and substance use disorder, including disrupted extinction learning and augmented cue-induced cocaine-seeking behavior. Chemogenetic restoration of IPSCs in D2-MSNs in the NAcore was sufficient to prevent antipsychotic-induced supersensitivity, pointing to an entirely novel therapeutic direction for overcoming this condition.

Complement Drives Synaptic Degeneration and Progressive Cognitive Decline in the Chronic Phase after Traumatic Brain Injury.

Cognitive deficits following traumatic brain injury (TBI) remain a major cause of disability and early-onset dementia, and there is increasing evidence that chronic neuroinflammation occurring after TBI plays an important role in this process. However, little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation after TBI. Here, we identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response that is associated with cognitive decline. Three months after an initial insult, there is ongoing complement activation in the injured brain of male C57BL/6 mice, which drives a robust chronic neuroinflammatory response extending to both hemispheres. This chronic neuroinflammatory response promotes synaptic degeneration and predicts progressive cognitive decline. Synaptic degeneration was driven by microglial phagocytosis of complement-opsonized synapses in both the ipsilateral and contralateral brain, and complement inhibition interrupted the degenerative neuroinflammatory response and reversed cognitive decline, even when therapy was delayed until 2 months after TBI. These findings provide new insight into our understanding of TBI pathology and its management; and whereas previous therapeutic investigations have focused almost exclusively on acute treatments, we show that all phases of TBI, including at chronic time points after TBI, may be amenable to therapeutic interventions, and specifically to complement inhibition.SIGNIFICANCE STATEMENT There is increasing evidence of a chronic neuroinflammatory response after traumatic brain injury (TBI), but little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation. We identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response, and further that this response is associated with cognitive decline. Complement inhibition interrupted this response and reversed cognitive decline, even when therapy was delayed until 2 months after injury. The data further support the concept that TBI should be considered a chronic rather than an acute disease condition, and have implications for the management of TBI in the chronic phase of injury, specifically with regard to the therapeutic application of complement inhibition.

Stem Cells: In Sickness and in Health.

Stem cells are undifferentiated cells with the ability to proliferate and convert to different types of differentiated cells that make up the various tissues and organs in the body. They exist both in embryos as pluripotent stem cells that can differentiate into the three germ layers and as multipotent or unipotent stem cells in adult tissues to aid in repair and homeostasis. Perturbations in these cells' normal functions can give rise to a wide variety of diseases. In this review, we discuss the origin of different stem cell types, their properties and characteristics, their role in tissue homeostasis, current research, and their potential applications in various life-threatening diseases. We focus on neural stem cells, their role in neurogenesis and how they can be exploited to treat diseases of the brain including neurodegenerative diseases and cancer. Next, we explore current research in Induced Pluripotent Stem Cells (iPSC) techniques and their clinical applications in regenerative and personalized medicine. Lastly, we tackle a special type of stem cells called Cancer Stem Cells (CSCs) and how they can be responsible for therapy resistance and tumor recurrence and explore ways to target them.

Tideglusib attenuates growth of neuroblastoma cancer stem/progenitor cells in vitro and in vivo by specifically targeting GSK-3ß.

BACKGROUND: Neuroblastoma (NB) is the most frequently diagnosed extracranial solid tumor among the pediatric population. It is an embryonic tumor with high relapse rates pertaining to the presence of dormant slowly dividing cancer stem cells (CSC) within the tumor bulk that are responsible for therapy resistance. Therefore, there is a dire need to develop new therapeutic approaches that specifically target NB CSCs. Glycogen synthase kinase (GSK)-3ß is a serine/threonine kinase that represents a common signaling node at the intersection of many pathways implicated in NB CSCs. GSK-3ß sustains the survival and maintenance of CSCs and renders them insensitive to chemotherapeutic agents and radiation. METHODS: In our study, we aimed at evaluating the potential anti-tumor effect of Tideglusib (TDG), an irreversible GSK-3ß inhibitor drug, on three human NB cell lines, SK-N-SH, SH-SY5Y, and IMR-32. RESULTS: Our results showed that TDG significantly reduced cell proliferation, viability, and migration of the NB cells, in a dose- and time-dependent manner, and also significantly hindered the neurospheres formation eradicating the self-renewal ability of highly resistant CSCs. Besides, TDG potently reduced CD133 cancer stem cell marker expression in both SH-SY5Y cells and G1 spheres. Lastly, TDG inhibited NB tumor growth and progression in vivo. CONCLUSION: Collectively, we concluded that TDG could serve as an effective treatment capable of targeting the NB CSCs and hence overcoming therapy resistance. Yet, future studies are warranted to further investigate its potential role in NB and decipher the subcellular and molecular mechanisms underlying this role.

The potential use of tideglusib as an adjuvant radio-therapeutic treatment for glioblastoma multiforme cancer stem-like cells.

BACKGROUND: Glioblastoma multiforme (GBM), a stage IV astrocytoma, is the most common brain malignancy among adults. Conventional treatments of surgical resection followed by radio and/or chemotherapy fail to completely eradicate the tumor. Resistance to the currently available therapies is mainly attributed to a subpopulation of cancer stem cells (CSCs) present within the tumor bulk that self-renew leading to tumor relapse with time. Therefore, identification of characteristic markers specific to these cells is crucial for the development of targeted therapies. Glycogen synthase kinase 3 (GSK-3), a serine-threonine kinase, is deregulated in a wide range of diseases, including cancer. In GBM, GSK-3ß is overexpressed and its suppression in vitro has been shown to induce apoptosis of cancer cells. METHODS: In our study, we assessed the effect of GSK-3ß inhibition with Tideglusib (TDG), an irreversible non-ATP competitive inhibitor, using two human GBM cell lines, U-251 MG and U-118 MG. In addition, we combined TDG with radiotherapy to assess whether this inhibition enhances the effect of standard treatment. RESULTS: Our results showed that TDG significantly reduced cell proliferation, cell viability, and migration of both GBM cell lines in a dose- and time-dependent manner in vitro. Treatment with TDG alone and in combination with radiation significantly decreased the colony formation of U-251 MG cells and the sphere formation of both cell lines, by targeting and reducing their glioblastoma cancer stem-like cells (GSCs) population. Finally, cells treated with TDG showed an increased level of unrepaired radio-induced DNA damage and, thus, became sensitized toward radiation. CONCLUSIONS: In conclusion, TDG has proven its effectiveness in targeting the cancerous properties of GBM in vitro and may, hence, serve as a potential adjuvant radio-therapeutic agent to better target this deadly tumor.

Repeatability of Zone Averages Compared to Single-Point Measurements of Maximal Curvature in Keratoconus.

PURPOSE: To evaluate the repeatability of curvature zone averages centered on the point of maximum curvature (Kmax) compared to that of the single-point Kmax. DESIGN: Comparative reliability analysis. METHODS: Setting: American University of Beirut Medical Center, Beirut, Lebanon. STUDY POPULATION: Sixty-five eyes of 65 adult keratoconus patients. Patients with other ocular disease, history of ocular surgery or trauma, and contact lens wear within 2 weeks of image acquisition were excluded. OBSERVATION PROCEDURES: Eyes were evaluated with 3 consecutive scans using the Galilei dual Scheimpflug-Placido system. MAIN OUTCOME MEASURES: Repeatability of axial and instantaneous Kmax single points, and zone averages with radii of 0.1-2.0 mm, centered on them. Repeatability was assessed by within-subject standard deviations, repeatability limits (r), and intraclass correlation coefficients. RESULTS: Axial curvature zone averaging yielded clinically acceptable repeatability only in eyes with Kmax ≤50 diopters (D), for radii of 1.5 mm and 2.0 mm (r = 0.87 D and r = 0.76, respectively, vs r = 0.91 for the single-point axial Kmax). Compared to instantaneous Kmax, clinically acceptable repeatability was achieved with instantaneous zone averages of at least 1.5 mm radius in eyes with Kmax ≤50 D (r = 0.99 D and r = 0.70 D, respectively) and 2.0 mm radius in eyes with Kmax >50 D (r = 2.28 D and r = 0.87 D, respectively). For all eyes, the repeatability limit of the location of Kmax was 0.82 mm and 0.80 mm for axial and instantaneous curvature, respectively. CONCLUSIONS: Instantaneous curvature zone averages centered on Kmax yielded a greater improvement in repeatability than axial zone averages and reached clinical adequacy with radii of at least 1.5 mm, for eyes with Kmax ≤50 D, and with a 2.0 mm radius for eyes with Kmax >50 D.

Non-Opioid Treatments for Opioid Use Disorder: Rationales and Data to Date.

Opioid use disorder (OUD) represents a major public health problem that affects millions of people in the USA and worldwide. The relapsing and recurring aspect of OUD, driven by lasting neurobiological adaptations at different reward centres in the brain, represents a major obstacle towards successful long-term remission from opioid use. Currently, three drugs that modulate the function of the opioidergic receptors, methadone, buprenorphine and naltrexone have been approved by the US Food and Drug Administration (FDA) to treat OUD. In this review, we discuss the limitations and challenges associated with the current maintenance and medication-assisted withdrawal strategies commonly used to treat OUD. We further explore the involvement of glutamatergic, endocannabinoid and orexin signaling systems in the development, maintenance and expression of addiction-like behaviours in animal models of opioid addiction, and as potential and novel targets to expand therapeutic options to treat OUD. Despite a growing preclinical literature highlighting the role of these potential targets in animal models of opioid addiction, clinical and translational studies for novel treatments of OUD remain limited and inconclusive. Further preclinical and clinical investigations are needed to expand the arsenal of primary treatment options and adjuncts to maximise efficacy and prevent relapse.

Opposing Regulation of Cocaine Seeking by Glutamate and GABA Neurons in the Ventral Pallidum.

Projections from the nucleus accumbens to the ventral pallidum (VP) regulate relapse in animal models of addiction. The VP contains GABAergic (VPGABA) and glutamatergic (VPGlu) neurons, and a subpopulation of GABAergic neurons co-express enkephalin (VPPenk). Rabies tracing reveals that VPGlu and VPPenk neurons receive preferential innervation from upstream D1- relative to D2-expressing accumbens neurons. Chemogenetic stimulation of VPGlu neurons inhibits, whereas stimulation of VPGABA and VPPenk neurons potentiates cocaine seeking in mice withdrawn from intravenous cocaine self-administration. Calcium imaging reveals cell type-specific activity patterns when animals learn to suppress drug seeking during extinction training versus engaging in cue-induced cocaine seeking. During cued seeking, VPGABA neurons increase their overall activity, and VPPenk neurons are selectively activated around nose pokes for cocaine. In contrast, VPGlu neurons increase their spike rate following extinction training. These data show that VP subpopulations differentially encode and regulate cocaine seeking, with VPPenk and VPGABA neurons facilitating and VPGlu neurons inhibiting cocaine seeking.

Cancer Stem Cells in Neuroblastoma: Expanding the Therapeutic Frontier.

Neuroblastoma (NB) is the most common extracranial solid tumor often diagnosed in childhood. Despite intense efforts to develop a successful treatment, current available therapies are still challenged by high rates of resistance, recurrence and progression, most notably in advanced cases and highly malignant tumors. Emerging evidence proposes that this might be due to a subpopulation of cancer stem cells (CSCs) or tumor-initiating cells (TICs) found in the bulk of the tumor. Therefore, the development of more targeted therapy is highly dependent on the identification of the molecular signatures and genetic aberrations characteristic to this subpopulation of cells. This review aims at providing an overview of the key molecular players involved in NB CSCs and focuses on the experimental evidence from NB cell lines, patient-derived xenografts and primary tumors. It also provides some novel approaches of targeting multiple drivers governing the stemness of CSCs to achieve better anti-tumor effects than the currently used therapeutic agents.

The Akt/mTOR pathway in cancer stem/progenitor cells is a potential therapeutic target for glioblastoma and neuroblastoma.

Nervous system tumors represent some of the highly aggressive cancers in both children and adults, particularly neuroblastoma and glioblastoma. Many studies focused on the pathogenic role of the Akt pathway and the mechanistic target of Rapamycin (mTOR) complex in mediating the progression of various types of cancer, which designates the Akt/mTOR signaling pathway as a master regulator for cancer. Current studies are also elucidating the mechanisms of cancer stem cells (CSCs) in replenishing tumors and explicating the strong correlation between the Akt/mTOR pathway and CSC biology. This instigates the development of novel treatments that target CSCs via inhibiting this pathway to prevent recurrence in various cancer subtypes. In accordance, neuroblastoma and glioblastoma tumors are believed to originate from stem/progenitor cells or dedifferentiated mature neural/glial cells transformed into CSCs, which warrants targeting this subpopulation of CSCs in these tumors. In our study, Triciribine and Rapamycin were used to assess the role of inhibiting two different points of the Akt/mTOR pathway in vitro on U251 (glioblastoma) and SH-SY5Y (neuroblastoma) human cell lines and their CSCs. We showed that both drugs minimally decrease the survival of U251 and SH-SY5Y cells in a 2D model, while this effect was much more pronounced in a 3D culture model. Triciribine and Rapamycin decreased migratory abilities of both cell lines and decreased their sphere-forming units (SFU) by extinguishing their CSC populations. Together, we concluded that Rapamycin and Triciribine proved to be effective in the in vitro treatment of glioblastoma and neuroblastoma, by targeting their CSC population.

Sphere-Formation Assay: Three-Dimensional in vitro Culturing of Prostate Cancer Stem/Progenitor Sphere-Forming Cells.

Cancer Stem Cells (CSCs) are a sub-population of cells, identified in most tumors, responsible for the initiation, recurrence, metastatic potential, and resistance of different malignancies. In prostate cancer (PCa), CSCs were identified and thought to be responsible for the generation of the lethal subtype, commonly known as Castration-Resistant Prostate Cancer (CRPC). In vitro models to investigate the properties of CSCs in PCa are highly required. Sphere-formation assay is an in vitro method commonly used to identify CSCs and study their properties. Here, we report the detailed methodology on how to generate and propagate spheres from PCa cell lines and from murine prostate tissue. This model is based on the ability of stem cells to grow in non-adherent serum-free gel matrix. We also describe how to use these spheres in histological and immuno-fluorescent staining assays to assess the differentiation potential of the CSCs. Our results show the sphere-formation Assay (SFA) as a reliable in vitro assay to assess the presence and self-renewal ability of CSCs in different PCa models. This platform presents a useful tool to evaluate the effect of conventional or novel agents on the initiation and self-renewing properties of different tumors. The effects can be directly evaluated through assessment of the sphere-forming efficiency (SFE) over five generations or other downstream assays such as immuno-histochemical analysis of the generated spheres.