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Background: Diffuse midline gliomas (DMG) are pediatric tumors with negligible 2-year survival after diagnosis characterized by their ability to infiltrate the central nervous system. In the hope of controlling the local growth and slowing the disease, all patients receive radiotherapy. However, distant progression occurs frequently in DMG patients. Current clues as to what causes tumor infiltration circle mainly around the tumor microenvironment, but there are currently no known determinants to predict the degree of invasiveness., Methods: In this study, we use patient-derived glioma stem cells (GSCs) to create patient-specific 3D avatars to model interindividual invasion and elucidate the cellular supporting mechanisms., Results: We show that GSC models in 3D mirror the invasive behavior of the parental tumors, thus proving the ability of DMG to infiltrate as an autonomous characteristic of tumor cells. Furthermore, we distinguished 2 modes of migration, mesenchymal and ameboid-like, and associated the ameboid-like modality with GSCs derived from the most invasive tumors. Using transcriptomics of both organoids and primary tumors, we further characterized the invasive ameboid-like tumors as oligodendrocyte progenitor-like, with highly contractile cytoskeleton and reduced adhesion ability driven by crucial over-expression of bone morphogenetic pathway 7 (BMP7). Finally, we deciphered MEK, ERK, and Rho/ROCK kinases activated downstream of the BMP7 stimulation as actionable targets controlling tumor cell motility., Conclusions: Our findings identify 2 new therapeutic avenues. First, patient-derived GSCs represent a predictive tool for patient stratification in order to adapt irradiation strategies. Second, autocrine and short-range BMP7-related signaling becomes a druggable target to prevent DMG spread and metastasis., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background: Ultrasound-guided fine needle aspiration cytology (FNAC US) has been proven to be an accurate and efficient tool in thyroid nodule evaluation. Thyroid nodule aspiration can be acquired with either of two techniques: the short axis, in which only the tip of the needle is observed, and the long axis, in which the entire length of the needle is observed. Our retrospective study aimed to compare the adequacy of the two techniques., Methods: FNAC US was performed in 538 thyroid nodules between January 2019 and December 2021. Data on the technique and the diagnostic accuracy were collected., Results: A total of 273 nodules were aspirated using the long axis technique, and 265 nodules were aspirated using the short axis technique. The diagnostic adequacies of the long axis technique were significantly higher than those of the short axis technique (92 % versus 86 %, respectively, p < 0.025)., Conclusion: In our study, the long axis technique provided more accurate cytological evaluation than the short axis technique., Level of Evidence: level 3., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Patients with advanced salivary gland malignancies (SGCs) have few therapy options. Although results from newly published trials suggest that checkpoint inhibition may be useful in a subgroup of patients, there are no clear criteria for PD-L1 score in SGCs. Chemotherapy benefits were observed to be limited, with a dismal prognosis in unresectable and high-grade SGC. Immunotherapies have demonstrated extraordinary efficacy in a variety of cancers, including non-small cell lung cancer and malignant melanoma. Anti-PD-1 antibody pembrolizumab has been shown to have potent anti-tumor action in a number of clinical trials., Case Presentation: We report a unique case of advanced high grade mucoepidermoid carcinoma of the parotid salivary gland after Pembrolizumab treatment as a first line therapy. The tumor was downstaged as a result of the pembrolizumab treatment, allowing for a successful surgical excision with no facial nerve sacrifice and no major neoadjuvant treatment adverse effects, and the final specimen pathology was tumor-free. In these types of malignancies, a similar technique resulted in a complete response (CR) radiologically and pathologically has never been discussed before., Conclusions: In pretreated patients with high-grade salivary gland mucoepidermoid carcinoma, pembrolizumab showed good anticancer activity and provided a clinically, radiologically, and pathological response with a viable treatment choice. More research is needed to bring Pembrolizumab to the front-line of treatment. The time and duration of medication should be compared to the time required for surgery in these investigations., (© 2022. The Author(s).)

Migrating cells navigate in complex environments through sensing and interpreting biochemical and/or mechanical cues. Here, we report that recently identified tubular clathrin/AP-2 lattices (TCALs), a subset of clathrin-coated structures (CCSs) that pinch collagen fibers, mechanically control directed migration along fibers decorated with ligands of CCS cargoes in three-dimensional (3D) environments. We observed that epidermal growth factor or low-density lipoprotein bound to collagen fibers leads to increased local nucleation and accumulation of TCALs. By using engineered, mixed collagen networks, we demonstrate that this mechanism selectively increases local forces applied on ligand-decorated fibers. We show that these effects depend on the ligand’s receptors but do not rely on their ability to trigger signaling events. We propose that the preferential accumulation of TCALs along ligand-decorated fibers steers migration in 3D environments. We conclude that ligand-regulated, local TCAL accumulation results in asymmetric force distribution that orients cell migration in 3D environments.

Cell migration is oriented by cues from the environment. Such cues are read and interpreted by the cell and translated into a reorganization of the migration machinery to steer migration. Receptors at the cell surface are central to detect these cues. These receptors can be internalized and this plays an important role in the decision-making process leading to choosing a migration direction. Independently of endocytosis, recent findings suggest that regulation of these receptors and translation of the information they carry into a phenotype is facilitated by their clustering at discrete locations of the plasma membrane. Clathrin-coated structures are archetypal clustering assemblies and thus provide the cell with a finely tunable mechanism for controlling receptor availability. In addition, clathrin-coated structures can be regulated by many factors playing a role in cell migration and thus take part in feedback loop mechanisms that are instrumental in defining a migration direction., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Under conditions of starvation, normal and tumor epithelial cells can rewire their metabolism toward the consumption of extracellular proteins, including extracellular matrix-derived components as nutrient sources. The mechanism of pericellular matrix degradation by starved cells has been largely overlooked. Here it is shown that matrix degradation by breast and pancreatic tumor cells and patient-derived xenograft explants increases by one order of magnitude upon amino acid and growth factor deprivation. In addition, it is found that collagenolysis requires the invadopodia components, TKS5, and the transmembrane metalloproteinase, MT1-MMP, which are key to the tumor invasion program. Increased collagenolysis is controlled by mTOR repression upon nutrient depletion or pharmacological inhibition by rapamycin. The results reveal that starvation hampers clathrin-mediated endocytosis, resulting in MT1-MMP accumulation in arrested clathrin-coated pits. The study uncovers a new mechanism whereby mTOR repression in starved cells leads to the repurposing of abundant plasma membrane clathrin-coated pits into robust ECM-degradative assemblies., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Cells experience mechanical stresses in different physiological and pathological settings. Clathrin-coated structures (CCSs) are sensitive to such perturbations in a way that often results in a mechanical impairment of endocytic budding. Compressive stress is a mechanical perturbation that leads to increased membrane tension and promotes proliferative signals. Here, we report that compression leads to frustration of CCSs and that CCSs are required to potentiate receptor-mediated signaling in these conditions. We show that cell compression stalled CCS dynamics and slowed down the dynamic exchange of CCS components. As previously reported, compression-induced paracrine activation of the epidermal growth factor receptor (EGFR) was the primary cause of ERK (ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively) activation in these conditions. We observed that EGFR was efficiently recruited at CCSs upon compression and that CCSs were required for full ERK activation. In addition, we demonstrated that compression-induced frustrated CCSs could also increase ligand-dependent signaling of other receptors. We thus propose that CCS frustration resulting from mechanical perturbations can potentiate signaling through different receptors, with potential important consequences for the adaptation of the cell to its environment.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

It is generally assumed that cells interrogate the mechanical properties of their environment by pushing and pulling on the extracellular matrix (ECM). For instance, acto-myosin-dependent contraction forces exerted at focal adhesions (FAs) allow the cell to actively probe substrate elasticity. Here, we report that a subset of long-lived and flat clathrin-coated structures (CCSs), also termed plaques, are contractility-independent mechanosensitive signaling platforms. We observed that plaques assemble in response to increasing substrate rigidity and that this is independent of FAs, actin and myosin-II activity. We show that plaque assembly depends on αvβ5 integrin, and is a consequence of frustrated endocytosis whereby αvβ5 tightly engaged with the stiff substrate locally stalls CCS dynamics. We also report that plaques serve as platforms for receptor-dependent signaling and are required for increased Erk activation and cell proliferation on stiff environments. We conclude that CCSs are mechanotransduction structures that sense substrate rigidity independently of cell contractility.

Cancer-associated fibroblasts (CAFs) are the most abundant cells of the tumor stroma. Their capacity to contract the matrix and induce invasion of cancer cells has been well documented. However, it is not clear whether CAFs remodel the matrix by other means, such as degradation, matrix deposition, or stiffening. We now show that CAFs assemble fibronectin (FN) and trigger invasion mainly via integrin-αvβ3. In the absence of FN, contractility of the matrix by CAFs is preserved, but their ability to induce invasion is abrogated. When degradation is impaired, CAFs retain the capacity to induce invasion in an FN-dependent manner. The level of expression of integrins αv and β3 and the amount of assembled FN are directly proportional to the invasion induced by fibroblast populations. Our results highlight FN assembly and integrin-αvβ3 expression as new hallmarks of CAFs that promote tumor invasion., (© 2017 Attieh et al.)

The interaction between circulating tumor cells (CTC) and endothelial cells during extravasation is a critical process during metastatic colonization, but its mechanisms remain poorly characterized. Here we report that the luminal side of liver blood vessels contains fibronectin deposits that are enriched in mice bearing primary tumors and are also present in vessels from human livers affected with metastases. Cancer cells attached to endothelial fibronectin deposits via talin1, a major component of focal adhesions. Talin1 depletion impaired cancer cell adhesion to the endothelium and transendothelial migration, resulting in reduced liver metastasis formation in vivo Talin1 expression levels in patient CTC's correlated with prognosis and therapy response. Together, our findings uncover a new mechanism for liver metastasis formation involving an active contribution of hepatic vascular fibronectin and talin1 in cancer cells. Cancer Res; 77(13); 3431-41. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Migrating cells often use focal adhesions in order to move. Focal adhesions are less prominent in cells migrating in three-dimensional (3D) as compared with 2D environments. We looked for alternative adhesion structures supporting cell migration. We analyzed the dynamics of clathrin-coated pits in cells migrating in a 3D environment of collagen fibers. Both topological cues and local engagement of integrins triggered the accumulation of clathrin-coated structures on fibers. Clathrin/adaptor protein 2 (AP-2) lattices pinched collagen fibers by adopting a tube-like morphology and regulated adhesion to fibers in an endocytosis-independent manner. During migration, tubular clathrin/AP-2 lattices stabilized cellular protrusions by providing anchoring points to collagen fibers. Thus, tubular clathrin/AP-2 lattices promote cell adhesion that, in coordination with focal adhesions, supports cell migration in 3D., (Copyright © 2017, American Association for the Advancement of Science.)

Acetylation of the lysine 40 of α-tubulin (K40) is a post-translational modification occurring in the lumen of microtubules (MTs) and is controlled by the α-tubulin acetyl-transferase αTAT1. How αTAT1 accesses the lumen and acetylates α-tubulin there has been an open question. Here, we report that acetylation starts at open ends of MTs and progressively spreads longitudinally from there. We observed acetylation marks at the open ends of in vivo MTs re-growing after a Nocodazole block, and acetylated segments growing in length with time. Bias for MTs extremities was even more pronounced when using non-dynamic MTs extracted from HeLa cells. In contrast, K40 acetylation was mostly uniform along the length of MTs reconstituted from purified tubulin in vitro. Quantitative modelling of luminal diffusion of αTAT1 suggested that the uniform acetylation pattern observed in vitro is consistent with defects in the MT lattice providing lateral access to the lumen. Indeed, we observed that in vitro MTs are permeable to macromolecules along their shaft while cellular MTs are not. Our results demonstrate αTAT1 enters the lumen from open extremities and spreads K40 acetylation marks longitudinally along cellular MTs. This mode of tip-directed microtubule acetylation may allow for selective acetylation of subsets of microtubules.

Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions., Competing Interests: The authors declare no competing financial interests.

Migrating cells nucleate focal adhesions (FAs) at the cell front and disassemble them at the rear to allow cell translocation. FAs are made of a multiprotein complex, the adhesome, which connects integrins to stress fibers made of mixed-polarity actin filaments [1-5]. Myosin II-driven contraction of stress fibers generates tensile forces that promote adhesion growth [6-9]. However, tension must be tightly controlled, because if released, FAs disassemble [3, 10-12]. Conversely, excess tension can cause abrupt cell detachment resulting in the loss of a major part of the adhesion [9, 12]. Thus, both adhesion growth and disassembly depend on tensile forces generated by stress fiber contraction, but how this contractility is regulated remains unclear. Here, we show that the actin-bundling protein fascin crosslinks the actin filaments into parallel bundles at the stress fibers' termini. Fascin prevents myosin II entry at this region and inhibits its activity in vitro. In fascin-depleted cells, polymerization of actin filaments at the stress fiber termini is slower, the actin cytoskeleton is reorganized into thicker stress fibers with a higher number of myosin II molecules, FAs are larger and less dynamic, and consequently, traction forces that cells exert on their substrate are larger. We also show that fascin dissociation from stress fibers is required to allow their severing by cofilin, leading to efficient disassembly of FAs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

During metastasis, cancer cells breach the basement membrane and migrate through the stroma mostly composed of a network of collagen I fibers. Cell migration on 2D is initiated by protrusion of the cell membrane followed by formation of adhesions that link the actin cytoskeleton to the extracellular matrix (ECM). Cells then move forwards by exerting traction forces on the adhesions at its front and by disassembling adhesions at the rear. In 2D, only the ventral surface of a migrating cell is in contact with the ECM, where cell-matrix adhesions are assembled. In 3D matrices, even though the whole surface of a migrating cell is available for interacting with the ECM, it is unclear whether discrete adhesion structures actually exist. Using high-resolution confocal microscopy we imaged the endogenous adhesome proteins in three different cancer cell types embedded in non-pepsinized collagen type I, polymerized at a slow rate, to allow the formation of a network that resembles the organization of EMC observed in vivo. Vinculin aggregates were detected in the cellular protrusions, frequently colocalizing with collagen fibers, implying they correspond to adhesion structures in 3D. As the distance from the substrate bottom increases, adhesion aggregates become smaller and almost undetectable in some cell lines. Using intravital imaging we show here, for the first time, the existence of adhesome proteins aggregates in vivo. These aggregates share similarities with the ones found in 3D collagen matrices. It still remains to be determined if adhesions assembled in 3D and in vivo share functional similarities to the well-described adhesions in 2D. This will provide a major step forward in understanding cell migration in more physiological environments., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The dramatic cell shape changes during cytokinesis require the interplay between microtubules and the actomyosin contractile ring, and addition of membrane to the plasma membrane. Numerous membrane-trafficking components localize to the central spindle during cytokinesis, but it is still unclear how this machinery is targeted there and how membrane trafficking is coordinated with cleavage furrow ingression. Here we use an arf6 null mutant to show that the endosomal GTPase ARF6 is required for cytokinesis in Drosophila spermatocytes. ARF6 is enriched on recycling endosomes at the central spindle, but it is required neither for central spindle nor actomyosin contractile ring assembly, nor for targeting of recycling endosomes to the central spindle. However, in arf6 mutants the cleavage furrow regresses because of a failure in rapid membrane addition to the plasma membrane. We propose that ARF6 promotes rapid recycling of endosomal membrane stores during cytokinesis, which is critical for rapid cleavage furrow ingression.

ARHGAP21 is a Rho family GTPase-activating protein (RhoGAP) that controls the Arp2/3 complex and F-actin dynamics at the Golgi complex by regulating the activity of the small GTPase Cdc42. ARHGAP21 is recruited to the Golgi by binding to another small GTPase, ARF1. Here, we present the crystal structure of the activated GTP-bound form of ARF1 in a complex with the Arf-binding domain (ArfBD) of ARHGAP21 at 2.1 A resolution. We show that ArfBD comprises a PH domain adjoining a C-terminal alpha helix, and that ARF1 interacts with both of these structural motifs through its switch regions and triggers structural rearrangement of the PH domain. We used site-directed mutagenesis to confirm that both the PH domain and the helical motif are essential for the binding of ArfBD to ARF1 and for its recruitment to the Golgi. Our data demonstrate that two well-known small GTPase-binding motifs, the PH domain and the alpha helical motif, can combine to create a novel mode of binding to Arfs.

Fibroblast growth factor (FGF)-1 and -2 have potent biological activities implicated in malignant tumor development. Their autocrine and nonautocrine activity in tumor progression of carcinoma was investigated in the NBT-II cell system. Cells were manipulated to either produce and be autocrine for FGF-1 or -2 or to only produce but not respond to these factors. The autocrine cells are highly invasive and tumorigenic and the determination of specific targets of FGF/fibroblast growth factor receptor (FGFR) signaling was assessed. In vitro studies showed that nonautocrine cells behave like epithelial parental cells, whereas autocrine cells have a mesenchymal phenotype correlated with the overexpression of urokinase plasminogen activator receptor (uPAR), the internalization of E-cadherin, and the redistribution of beta-catenin from the cell surface to the cytoplasm and nucleus. uPAR was defined as an early target, whereas E-cadherin and the leukocyte common antigen-related protein-tyrosine phosphatase (LAR-PTP) were later targets of FGF signaling, with FGFR1 activation more efficient than FGFR2 at modulating these targets. Behavior of autocrine cells was consistent with a decrease of tumor-suppressive activities of both E-cadherin and LAR-PTP. These molecular analyses show that the potential of these two growth factors in tumor progression is highly dependent on specific FGFR signaling and highlights its importance as a target for antitumor therapy.