CD146 increases stemness and aggressiveness in glioblastoma and activates YAP signaling.

Glioblastoma (GBM), a highly malignant and lethal brain tumor, is characterized by diffuse invasion into the brain and chemo-radiotherapy resistance resulting in poor prognosis. In this study, we examined the involvement of the cell adhesion molecule CD146/MCAM in regulating GBM aggressiveness. Analyses of GBM transcript expression databases revealed correlations of elevated CD146 levels with higher glioma grades, IDH-wildtype and unmethylated MGMT phenotypes, poor response to chemo-radiotherapy and worse overall survival. In a panel of GBM stem cells (GSCs) variable expression levels of CD146 were detected, which strongly increased upon adherent growth. CD146 was linked with mesenchymal transition since expression increased in TGF-ß-treated U-87MG cells. Ectopic overexpression of CD146/GFP in GG16 cells enhanced the mesenchymal phenotype and resulted in increased cell invasion. Conversely, GSC23-CD146 knockouts had decreased mesenchymal marker expression and reduced cell invasion in transwell and GBM-cortical assembloid assays. Moreover, using GSC23 xenografted zebrafish, we found that CD146 depletion resulted in more compact delineated tumor formation and reduced tumor cell dissemination. Stem cell marker expression and neurosphere formation assays showed that CD146 increased the stem cell potential of GSCs. Furthermore, CD146 mediated radioresistance by stimulating cell survival signaling through suppression of p53 expression and activation of NF-κB. Interestingly, CD146 was also identified as an inducer of the oncogenic Yes-associated protein (YAP). In conclusion, CD146 carries out various pro-tumorigenic roles in GBM involving its cell surface receptor function, which include the stimulation of mesenchymal and invasive properties, stemness, and radiotherapy resistance, thus providing an interesting target for therapy.

The Unfolded Protein Response Sensor PERK Mediates Stiffness-Dependent Adaptation in Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults. In addition to genetic causes, the tumor microenvironment (TME), including stiffening of the extracellular matrix (ECM), is a main driver of GBM progression. Mechano-transduction and the unfolded protein response (UPR) are essential for tumor-cell adaptation to harsh TME conditions. Here, we studied the effect of a variable stiff ECM on the morphology and malignant properties of GBM stem cells (GSCs) and, moreover, examined the possible involvement of the UPR sensor PERK herein. For this, stiffness-tunable human blood plasma (HBP)/alginate hydrogels were generated to mimic ECM stiffening. GSCs showed stiffness-dependent adaptation characterized by elongated morphology, increased proliferation, and motility which was accompanied by F-Actin cytoskeletal remodeling. Interestingly, in PERK-deficient GSCs, stiffness adaptation was severely impaired, which was evidenced by low F-Actin levels, the absence of F-Actin remodeling, and decreased cell proliferation and migration. This impairment could be linked with Filamin-A (FLN-A) expression, a known interactor of PERK, which was strongly reduced in PERK-deficient GSCs. In conclusion, we identified a novel PERK/FLNA/F-Actin mechano-adaptive mechanism and found a new function for PERK in the cellular adaptation to ECM stiffening.

Evaluation of Ac-Lys0(IRDye800CW)Tyr3-octreotate as a novel tracer for SSTR2-targeted molecular fluorescence guided surgery in meningioma.

PURPOSE: Meningioma recurrence rates can be reduced by optimizing surgical resection with the use of intraoperative molecular fluorescence guided surgery (MFGS). We evaluated the potential of the fluorescent tracer 800CW-TATE for MFGS using in vitro and in vivo models. It targets somatostatin receptor subtype 2 (SSTR2), which is overexpressed in all meningiomas. METHODS: Binding affinity of 800CW-TATE was evaluated using [177Lu] Lu-DOTA-Tyr3-octreotate displacement assays. Tumor uptake was determined by injecting 800CW-TATE in (SSTR2-positive) NCI-H69 or (SSTR2-negative) CH-157MN xenograft bearing mice and FMT2500 imaging. SSTR2-specific binding was measured by comparing tumor uptake in NCI-H69 and CH-157MN xenografts, blocking experiments and non-targeted IRDye800CW-carboxylate binding. Tracer distribution was analyzed ex vivo, and the tumor-to-background ratio (TBR) was calculated. SSTR2 expression was determined by immunohistochemistry (IHC). Lastly, 800CW-TATE was incubated on frozen and fresh meningioma specimens and analyzed by microscopy. RESULTS: 800CW-TATE binding affinity assays showed an IC50 value of 72 nM. NCI-H69 xenografted mice showed a TBR of 21.1. 800CW-TATE detection was reduced after co-administration of non-fluorescent DOTA-Tyr3-octreotate or administration of IRDye800CW. CH-157MN had no tumor specific tracer staining due to absence of SSTR2 expression, thereby serving as a negative control. The tracer bound specifically to SSTR2-positive meningioma tissues representing all WHO grades. CONCLUSION: 800CW-TATE demonstrated sufficient binding affinity, specific SSTR2-mediated tumor uptake, a favorable biodistribution, and high TBR. These features make this tracer very promising for use in MFGS and could potentially aid in safer and a more complete meningioma resection, especially in high-grade meningiomas or those at complex anatomical localizations.

Understanding Lung Carcinogenesis from a Morphostatic Perspective: Prevention and Therapeutic Potential of Phytochemicals for Targeting Cancer Stem Cells.

Lung cancer is still one of the deadliest cancers, with over two million incidences annually. Prevention is regarded as the most efficient way to reduce both the incidence and death figures. Nevertheless, treatment should still be improved, particularly in addressing therapeutic resistance due to cancer stem cells-the assumed drivers of tumor initiation and progression. Phytochemicals in plant-based diets are thought to contribute substantially to lung cancer prevention and may be efficacious for targeting lung cancer stem cells. In this review, we collect recent literature on lung homeostasis, carcinogenesis, and phytochemicals studied in lung cancers. We provide a comprehensive overview of how normal lung tissue operates and relate it with lung carcinogenesis to redefine better targets for lung cancer stem cells. Nine well-studied phytochemical compounds, namely curcumin, resveratrol, quercetin, epigallocatechin-3-gallate, luteolin, sulforaphane, berberine, genistein, and capsaicin, are discussed in terms of their chemopreventive and anticancer mechanisms in lung cancer and potential use in the clinic. How the use of phytochemicals can be improved by structural manipulations, targeted delivery, concentration adjustments, and combinatorial treatments is also highlighted. We propose that lung carcinomas should be treated differently based on their respective cellular origins. Targeting quiescence-inducing, inflammation-dampening, or reactive oxygen species-balancing pathways appears particularly interesting.

Novel insights into vascularization patterns and angiogenic factors in glioblastoma subclasses.

Glioblastoma (GBM) is a highly vascularized and aggressive type of primary brain tumor in adults with dismal survival. Molecular subtypes of GBM have been identified that are related to clinical outcome and response to therapy. Although the mesenchymal type has been ascribed higher angiogenic activity, extensive characterization of the vascular component in GBM subtypes has not been performed. Therefore, we aimed to investigate the differential vascular status and angiogenic signaling levels in molecular subtypes. GBM tissue samples representing proneural IDH1 mutant, classical-like and mesenchymal-like subtypes were analyzed by morphometry for the number of vessels, vessel size and vessel maturity. Also the expression levels of factors from multiple angiogenic signaling pathways were determined. We found that necrotic and hypoxic areas were relatively larger in mesenchymal-like tumors and these tumors also had larger vessels. However, the number of vessels, basement membrane deposition and pericyte coverage did not vary between the subtypes. Regarding signaling patterns the majority of factors were expressed at similar levels in the subtypes, and only ANGPT2, MMP2, TIMP1, VEGFA and MMP9/TIMP2 were higher expressed in GBMs of the classical-like subtype. In conclusion, although morphological differences were observed between the subtypes, the angiogenic signaling status of GBM subtypes seemed to be rather similar. These results challenge the concept of mesenchymal GBMs being more angiogenic than other subclasses.

BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity.

Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MTT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasion and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma.

Kinome profiling of non-canonical TRAIL signaling reveals RIP1-Src-STAT3-dependent invasion in resistant non-small cell lung cancer cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) triggers apoptosis selectively in tumor cells through interaction with TRAIL-R1/DR4 or TRAIL-R2/DR5 and this process is considered a promising avenue for cancer treatment. TRAIL resistance, however, is frequently encountered and hampers anti-cancer activity. Here we show that whereas H460 non-small cell lung cancer (NSCLC) cells display canonical TRAIL-dependent apoptosis, A549 and SW1573 NSCLC cells are TRAIL resistant and display pro-tumorigenic activity, in particular invasion, following TRAIL treatment. We exploit this situation to contrast TRAIL effects on the kinome of apoptosis-sensitive cells to that of NSCLC cells in which non-canonical effects predominate, employing peptide arrays displaying 1024 different kinase pseudosubstrates more or less comprehensively covering the human kinome. We observed that failure of a therapeutic response to TRAIL coincides with the activation of a non-canonical TRAIL-induced signaling pathway involving, amongst others, Src, STAT3, FAK, ERK and Akt. The use of selective TRAIL variants against TRAIL-R1 or TRAIL-R2 subsequently showed that this non-canonical migration and invasion is mediated through TRAIL-R2. Short-hairpin-mediated silencing of RIP1 kinase prevented TRAIL-induced Src and STAT3 phosphorylation and reduced TRAIL-induced migration and invasion of A549 cells. Inhibition of Src or STAT3 by shRNA or chemical inhibitors including dasatinib and 5,15-diphenylporphyrin blocked TRAIL-induced invasion. FAK, AKT and ERK were activated in a RIP1-independent way and inhibition of AKT sensitized A549 cells to TRAIL-induced apoptosis. We thus identified RIP1-dependent and -independent non-canonical TRAIL kinase cascades in which Src and AKT are instrumental and could be exploited as co-targets in TRAIL therapy for NSCLC.

Loss of CD44 and SOX2 expression is correlated with a poor prognosis in esophageal adenocarcinoma patients.

BACKGROUND: It has been suggested that markers associated with cancer stem cells (CSC) may play a role in esophageal cancer. Our aim was to investigate the expression pattern of proposed CSC markers ALDH1, Axin2, BMI1, CD44, and SOX2 in esophageal adenocarcinoma (EAC) and to relate their expression to survival. METHODS: In this study we included 94 EAC patients and examined the expression of the above-mentioned markers by using immunohistochemistry on tissue microarrays. Expression was scored as positive or negative or categorized as low or high in terms of an immunoreactivity score (IRS). Expression rates were related to clinicopathologic characteristics and overall and disease-free survival (DFS). RESULTS: In a multivariate analysis, negative expression of CD44 and of SOX2 were both significant prognostic factors for DFS [hazard ratio (HR), 1.73; 95 % confidence interval (CI), 1.00-2.96; P = 0.046 and HR, 2.06; 95 % CI 1.14-3.70 P = 0.016). When CD44 and SOX2 expression were analyzed together, negative SOX2 expression was an independent prognostic factor for DFS (HR, 1.91; 95 % CI 1.05-3.46; P = 0.034). Low IRS scores for ALDH1 or Axin2 were associated with a reduced median survival (12.8 vs. 28.7 and 12.1 vs. 25.5 months, respectively). However, these markers and BMI1 were not prognostic factors for survival. CONCLUSIONS: Loss of CD44 expression and loss of SOX2 expression are prognostic factors of poor survival in EAC patients. This suggests a role of these proteins in EAC that requires further investigation.

Targeting FLIP and Mcl-1 using a combination of aspirin and sorafenib sensitizes colon cancer cells to TRAIL.

The multikinase inhibitor sorafenib is highly effective against certain types of cancer in the clinic and prevents colon cancer cell proliferation in vitro. Non-steroidal anti-inflammatory drugs, such as acetylsalicylic acid (aspirin), have shown activity against colon cancer cells. The aims of this study were to determine whether the combination of aspirin with sorafenib has enhanced anti-proliferative effects and increases recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL)-induced apoptosis in the human SW948, Lovo, Colo205, Colo320, Caco-2 and HCT116 colon cancer cell lines. In four cell lines, aspirin strongly stimulated the anti-proliferative effects of sorafenib (∼four-fold enhancement) by inducing cell cycle arrest. Furthermore, combining low doses of aspirin (≤ 5 mm) and sorafenib (≤ 2.5 µm) greatly sensitized TRAIL-sensitive and TRAIL-resistant colon cancer cells to rhTRAIL, much more potently than either drug combined with rhTRAIL. The increase in rhTRAIL sensitivity was due to inhibition of FLIP and Mcl-1 protein expression following aspirin and sorafenib co-treatment, as confirmed by knock-down studies. Next, the clinical relevance of targeting FLIP and Mcl-1 in colon cancer was examined. Using immunohistochemistry, we found that Mcl-1 expression was significantly increased in colon adenoma and carcinoma patient material compared to healthy colonic epithelium, similar to the enhanced FLIP expression we recently observed in colon cancer. These results underscore the potential of combining low doses of aspirin with sorafenib to inhibit proliferation and target the anti-apoptotic proteins FLIP and Mcl-1 in colon cancer cells.

Mechanisms underlying reversed TRAIL sensitivity in acquired bortezomib-resistant non-small cell lung cancer cells.

Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

Bevacizumab-IRDye800CW for tumor detection in fluorescence-guided meningioma surgery (LUMINA trial): a single-center phase I study.

OBJECTIVE: Meningiomas are one of the most frequently occurring brain tumors and can be curatively treated with gross-total resection. A subtotal resection increases the chances of recurrence. The intraoperative identification of invisible tumor remnants by using a fluorescent tracer targeting an upregulated biomarker could help to optimize meningioma resection. This is called molecular fluorescence-guided surgery (MFGS). Vascular endothelial growth factor α (VEGFα) has been identified as a suitable meningioma biomarker and can be targeted with bevacizumab-IRDye800CW. METHODS: The aim of this prospective phase I trial was to determine the safety and feasibility of bevacizumab-IRDye800CW for MFGS for intracranial meningiomas by administering 4.5, 10, or 25 mg of the tracer 2-4 days prior to surgery. Fluorescence was verified during the operation with the standard neurosurgical microscope, and tissue specimens were postoperatively analyzed with fluorescence imaging systems (Pearl and Odyssey CLx) and spectroscopy to determine the optimal dose. Uptake was compared in several tissue types and correlated with VEGFα expression. RESULTS: No adverse events related to the use of bevacizumab-IRDye800CW occurred. After two interim analyses, 10 mg was the optimal dose based on ex vivo tumor-to-background ratio. Although the standard intraoperative imaging revealed no fluorescence, postoperative analyses with tailored imaging systems showed high fluorescence uptake in tumor compared with unaffected dura mater and brain. Additionally, tumor invasion of the dura mater (dural tail) and invasion of bone could be distinguished using fluorescence imaging. Fluorescence intensity showed a good correlation with VEGFα expression. CONCLUSIONS: Bevacizumab-IRDye800CW can be safely used in patients with meningioma; 10 mg bevacizumab-IRDye800CW provided an adequate tumor-to-background ratio. Adjustments of the currently available neurosurgical microscopes are needed to achieve visualization of targeted IRDye800CW intraoperatively. A phase II/III trial is needed to methodically investigate the benefit of MFGS with bevacizumab-IRDye800CW for meningioma surgery in a larger cohort of patients.

MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce both caspase-dependent apoptosis and kinase activation in tumor cells. Here, we examined the consequences and mechanisms of TRAIL-induced MAPKs p38 and JNK in non-small cell lung cancer (NSCLC) cells. In apoptosis sensitive H460 cells, these kinases were phosphorylated, but not in resistant A549 cells. Time course experiments in H460 cells showed that induction of p38 phosphorylation preceded that of JNK. To explore the function of these kinases in apoptosis activation by TRAIL, chemical inhibitors or siRNAs were employed to impair JNK or p38 functioning. JNK activation counteracted TRAIL-induced apoptosis whereas activation of p38 stimulated apoptosis. Notably, the serine/threonine kinase RIP1 was cleaved following TRAIL treatment, concomitant with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA-dependent knockdown or inhibition by necrostatin-1 showed that p38 can be phosphorylated in both RIP1-dependent and -independent manner, whereas JNK phosphorylation occurred independent of RIP1. On the other hand JNK appeared to suppress RIP1 cleavage via an unknown mechanism. In addition, only the activation of JNK by TRAIL was caspase-8-dependent. Finally, we identified Mcl-1, a known substrate for p38 and JNK, as a downstream modulator of JNK or p38 activity. Collectively, our data suggest in a subset of NSCLC cells a model in which TRAIL-induced activation of p38 and JNK have counteracting effects on Mcl-1 expression leading to pro- or anti-apoptotic effects, respectively. Strategies aiming to stimulate p38 and inhibit JNK may have benefit for TRAIL-based therapies in NSCLC.

Bioinspired Processing: Complex Coacervates as Versatile Inks for 3D Bioprinting.

3D bioprinting is a powerful fabrication technique in biomedical engineering, which is currently limited by the number of available materials that meet all physicochemical and cytocompatibility requirements for biomaterial inks. Inspired by the key role of coacervation in the extrusion and spinning of many natural materials, hyaluronic acid-chitosan complex coacervates are proposed here as tunable biomaterial inks. Complex coacervates are obtained through an associative liquid-liquid phase separation driven by electrostatic attraction between oppositely charged macromolecules. They offer bioactive properties and facile modulation of their mechanical properties through mild physicochemical changes in the environment, making them attractive for 3D bioprinting. Fine-tuning the salt concentration, pH, and molecular weight of the constituent polymers results in biomaterial inks that are printable in air and water. The biomaterial ink, initially a viscoelastic fluid, transitions into a viscoelastic solid upon printing due to dehydration (for printing in air) or due to a change in pH and ionic composition (for printing in solution). Consequently, scaffolds printed using the complex coacervate inks are stable without the need for post-printing processing. Fabricated cell culture scaffolds are cytocompatible and show long-term topological stability. These results pave the way to a new class of easy-to-handle tunable biomaterials for biofabrication.

Feasibility of bevacizumab-IRDye800CW as a tracer for fluorescence-guided meningioma surgery.

OBJECTIVE: Meningiomas are frequently occurring, often benign intracranial tumors. Molecular fluorescence can be used to intraoperatively identify residual meningioma tissue and optimize safe resection; however, currently no clinically approved agent is available for this specific tumor type. In meningiomas, vascular endothelial growth factor α (VEGFα) is upregulated, and this biomarker could be targeted with bevacizumab-IRDye800CW, a fluorescent agent that is already clinically applied for the resection of other tumors and neoplasms. Here, the authors investigated the feasibility of using bevacizumab-IRDye800CW to target VEGFα in a CH-157MN xenografted mouse model. METHODS: Five mice with CH-157MN xenografts with volumes of 500 mm3 were administered intravenous bevacizumab-IRDye800CW. Mice were imaged in vivo at 24 hours, 48 hours, and 72 hours after injection with the FMT2500 fluorescence imaging system. Biodistribution was determined ex vivo using the Pearl fluorescent imager at 72 hours after injection. To mimic a clinical scenario, 2 animals underwent postmortem xenograft resection using both white-light and fluorescence guidance. Lastly, fresh and frozen human meningioma specimens were incubated ex vivo with bevacizumab-IRDye800CW, stained with anti-VEGFα, and microscopically examined. RESULTS: In vivo, tumors fluoresced at all time points after tracer administration and background fluorescence decreased with time. Ex vivo analyses of tracer biodistribution showed the highest fluorescence in resected tumor tissue. Brain, skull, and muscle tissue showed very low fluorescence. Microscopically, fluorescence was observed in the cytoplasm and was correlated with VEGFα expression patterns. During postmortem surgery, both the tumor bulk and a small tumor remnant were detected. Bevacizumab-IRDye800CW bound specifically to all tested human meningioma samples, as indicated by a high fluorescent signal in the tumor bulk compared with the surrounding healthy dura mater. CONCLUSIONS: Bevacizumab-IRDye800CW showed meningioma specificity, as illustrated by high VEGFα-mediated uptake in the meningioma xenograft mouse model. Small tumor lesions were detected using fluorescence guidance. Thus, the next step will be to assess the feasibility of using already available clinical grade bevacizumab-IRDye800CW to optimize meningioma resection in a human trial.

Physics of Brain Cancer: Multiscale Alterations of Glioblastoma Cells under Extracellular Matrix Stiffening.

The biology and physics underlying glioblastoma is not yet completely understood, resulting in the limited efficacy of current clinical therapy. Recent studies have indicated the importance of mechanical stress on the development and malignancy of cancer. Various types of mechanical stress activate adaptive tumor cell responses that include alterations in the extracellular matrix (ECM) which have an impact on tumor malignancy. In this review, we describe and discuss the current knowledge of the effects of ECM alterations and mechanical stress on GBM aggressiveness. Gradual changes in the brain ECM have been connected to the biological and physical alterations of GBM cells. For example, increased expression of several ECM components such as glycosaminoglycans (GAGs), hyaluronic acid (HA), proteoglycans and fibrous proteins result in stiffening of the brain ECM, which alters inter- and intracellular signaling activity. Several mechanosensing signaling pathways have been identified that orchestrate adaptive responses, such as Hippo/YAP, CD44, and actin skeleton signaling, which remodel the cytoskeleton and affect cellular properties such as cell-cell/ECM interactions, growth, and migration/invasion of GBM cells. In vitro, hydrogels are used as a model to mimic the stiffening of the brain ECM and reconstruct its mechanics, which we also discuss. Overall, we provide an overview of the tumor microenvironmental landscape of GBM with a focus on ECM stiffening and its associated adaptive cellular signaling pathways and their possible therapeutic exploitation.

TGF-ß promotes microtube formation in glioblastoma through thrombospondin 1.

BACKGROUND: Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular, glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation. METHODS: Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate MT formation under transforming growth factor-beta (TGF-ß) stimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-ß. RESULTS: Analysis of TCGA data showed that the TGF-ß pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-ß1 stimulation of GBM cell lines promotes enhanced MT formation and communication via calcium signaling. Inhibition of the TGF-ß pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-ß, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-ß stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. CONCLUSION: TGF-ß and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT-driven invasion/resistance network.

Playing the DISC: turning on TRAIL death receptor-mediated apoptosis in cancer.

Formation of the pro-apoptotic death-inducing signaling complex (DISC) can be initiated in cancer cells via binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to its two pro-apoptotic receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Primary components of the DISC are trimerized TRAIL-R1/-R2, FADD, caspase 8 and caspase 10. The anti-apoptotic protein FLIP can also be recruited to the DISC to replace caspase 8 and form an inactive complex. Caspase 8/10 processing at the DISC triggers the caspase cascade, which eventually leads to apoptotic cell death. Besides TRAIL, TRAIL-R1- or TRAIL-R2-selective variants of TRAIL and agonistic antibodies have been designed. These ligands are of interest as anti-cancer agents since they selectively kill tumor cells. To increase tumor sensitivity to TRAIL death receptor-mediated apoptosis and to overcome drug resistance, TRAIL receptor ligands have already been combined with various therapies in preclinical models. In this review, we discuss factors influencing the initial steps of the TRAIL apoptosis signaling pathway, focusing on mechanisms modulating DISC assembly and caspase activation at the DISC. These insights will direct rational design of drug combinations with TRAIL receptor ligands to maximize DISC signaling.

Three-dimensional culture models to study glioblastoma - current trends and future perspectives.

Glioblastoma (GBM) is the most prevalent form of primary malignant brain tumor in adults and remains almost invariably lethal owing to its aggressive and invasive nature. There have only been marginal improvements in its bleak survival rate of 12-15 months over the last four decades. The lack of preclinical models that efficiently recapitulate tumor biology and the tumor microenvironment is also in part responsible for the slow phase of translational GBM research. Emerging three-dimensional (3D) organoids and cell culture systems offer new and innovative possibilities for GBM modelling. These 3D models find their application to engineer the disease, screen drugs, establishing live biobank, and explore personalized therapy. Furthermore, these models can also be genetically modified by using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, which would allow one to study the specific role of key genes associated with gliomagenesis. Establishment of a coculture system with GBM cells to understand its invasive behavior is yet another major application of this model. Despite these merits, the organoid models also have certain limitations, including the absence of immune responses and vascular systems. In recent years, major progress has been made in the development and refinement of 3D models of GBM. In this review, we intend to highlight these recent advances and the potential future implications of this rapidly evolving field, which should facilitate a better understanding of GBM biology.

The unfolded protein response as regulator of cancer stemness and differentiation: Mechanisms and implications for cancer therapy.

The unfolded protein response (UPR) is an adaptive mechanism that regulates protein and cellular homeostasis. Three endoplasmic reticulum (ER) membrane localized stress sensors, IRE1, PERK and ATF6, coordinate the UPR in order to maintain ER proteostasis and cell survival, or induce cell death when homeostasis cannot be restored. However, recent studies have identified alternative functions for the UPR in developmental biology processes and cell fate decisions under both normal and cancerous conditions. In cancer, increasing evidence points towards the involvement of the three UPR sensors in oncogenic reprogramming and the regulation of tumor cells endowed with stem cell properties, named cancer stem cells (CSCs), that are considered to be the most malignant cells in tumors. Here we review the reported roles and underlying molecular mechanisms of the three UPR sensors in regulating stemness and differentiation, particularly in solid tumor cells, processes that have a major impact on tumor aggressiveness. Mainly PERK and IRE1 branches of the UPR were found to regulate CSCs and tumor development and examples are provided for breast cancer, colon cancer and aggressive brain tumors, glioblastoma. Although the underlying mechanisms and interactions between the different UPR branches in regulating stemness in cancer need to be further elucidated, we propose that PERK and IRE1 targeted therapy could inhibit self-renewal of CSCs or induce differentiation that is predicted to have therapeutic benefit. For this, more specific UPR modulators need to be developed with favorable pharmacological properties that together with patient stratification will allow optimal evaluation in clinical studies.