Microfluidic Lab-on-a-Chip Based on UHF-Dielectrophoresis for Stemness Phenotype Characterization and Discrimination among Glioblastoma Cells.

Glioblastoma (GBM) is one of the most aggressive solid tumors, particularly due to the presence of cancer stem cells (CSCs). Nowadays, the characterization of this cell type with an efficient, fast and low-cost method remains an issue. Hence, we have developed a microfluidic lab-on-a-chip based on dielectrophoresis (DEP) single cell electro-manipulation to measure the two crossover frequencies: fx01 in the low-frequency range (below 500 kHz) and fx02 in the ultra-high-frequency range (UHF, above 50 MHz). First, in vitro conditions were investigated. An U87-MG cell line was cultured in different conditions in order to induce an undifferentiated phenotype. Then, ex vivo GBM cells from patients' primary cell culture were passed through the developed microfluidic system and characterized in order to reflect clinical conditions. This article demonstrates that the usual exploitation of low-frequency range DEP does not allow the discrimination of the undifferentiated GBM cells from the differentiated one. However, the presented study highlights the use of UHF-DEP as a relevant discriminant parameter. The proposed microfluidic lab-on-a-chip is able to follow the kinetics of U87-MG phenotype transformation in a CSC enrichment medium and the cancer stem cells phenotype acquirement.

Characterization of Glioblastoma Cancer Stem Cells Sorted by Sedimentation Field-Flow Fractionation Using an Ultrahigh-Frequency Range Dielectrophoresis Biosensor.

Cancer stem cells (CSCs) appear to be an essential target for cancer therapies, in particular, in brain tumors such as Glioblastoma. Nevertheless, their isolation is made difficult by their low content in culture or tumors (<5% of the tumor mass) and is essentially based on the use of fluorescent or magnetic labeling techniques, increasing the risk of differentiation induction. The use of label-free separation methods such as sedimentation field-flow fractionation (SdFFF) is promising, but it becomes necessary to consider a coupling with a detection and characterization method for future identification and purification of CSCs from patient-derived tumors. In this study, we demonstrate for the first time the capability of using an ultrahigh-frequency range dielectrophoresis fluidic biosensor as a detector. This implies an important methodological adaptation of SdFFF cell sorting by the use of a new compatible carrier liquid DEP buffer (DEP-B). After SdFFF sorting, subpopulations derived from U87-MG and LN18 cell lines undergo biological characterization, demonstrating that using DEP-B as a carrier liquid, we sorted by SdFFF subpopulations with specific differentiation characteristics: F1 = differentiated cells/F2 = CSCs. These subpopulations presented high-frequency crossover (HFC) values similar to those measured for standard differentiated (around 110 MHz) and CSC (around 80 MHz) populations. This coupling appeared as a promising solution for the development of an online integration of these two complementary label-free separation/detection technologies.

Autophagy inhibition reinforces stemness together with exit from dormancy of polydisperse glioblastoma stem cells.

Therapeutic resistance and infiltrative capacities justify the aggressiveness of glioblastoma. This is due to cellular heterogeneity, especially the presence of stemness-related cells, i.e. Cancer Stem Cells (CSC). Previous studies focused on autophagy and its role in CSCs maintenance; these studies gave conflicting results as they reported either sustaining or disruptive effects. In the present work, we silenced two autophagy related genes -either Beclin1 or ATG5- by shRNA and we explored the ensuing consequences on CSCs markers' expression and functionalities. Our results showed that the down regulation of autophagy led to enhancement in expression of CSCs markers, while proliferation and clonogenicity were boosted. Temozolomide (TMZ) treatment failed to induce apoptotic death in shBeclin1-transfected cells, contrary to control. We optimized the cellular subset analysis with the use of Sedimentation Field Flow Fractionation, a biological event monitoring- and cell sorting-dedicated technique. Fractograms of both shBeclin1 and shATG5 cells exhibited a shift of elution peak as compared with control cells, showing cellular dispersion and intrinsic sub-fraction modifications. The classical stemness fraction (i.e. F3) highlighted data obtained with the overall cellular population, exhibiting enhancement of stemness markers and escape from dormancy. Our results contributed to illustrate CSCs polydispersity and to show how these cells develop capacity to bypass autophagy inhibition, thanks to their acute adaptability and plasticity.

Overexpression of sortilin is associated with 5-FU resistance and poor prognosis in colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide. Even if 5-fluorouracil (5-FU) is used as the first-line chemotherapeutic drug, responsiveness is only 20-30%. Acquired resistance to 5-FU contributes to both poor patient prognosis and relapse, emphasizing the need to identify biomarkers. Sortilin, a vacuolar protein sorting 10 protein (Vps10p), implicated in protein trafficking, is over expressed in CRC cell lines cultured 72 hours in presence of 5-FU. This overexpression was also observed in 5-FU-resistant cells derived from these cell lines as well as in CRC primary cultures (or patients derived cell lines). A significantly higher expression of sortilin was observed in vivo, in 5-FU-treated tumours engrafted in Nude mice, as compared with non-treated tumour. A study of transcriptional regulation allowed identifying a decrease in ATF3 expression, as an explanation of sortilin overexpression following 5-FU treatment. In silico analysis revealed SORT1 expression correlation with poor prognosis. Moreover, sortilin expression was found to be positively correlated with CRC tumour grades. Collectively, our findings identify sortilin as a potential biomarker of 5-FU resistance associated with poor clinical outcomes and aggressiveness in CRC. As a new prognostic factor, sortilin expression could be used to fight against CRC.

BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells.

Evading apoptosis and sustained survival signaling pathways are two central hallmarks of B-cell chronic lymphocytic leukemia (B-CLL) cells. In this regard, nurse-like cells (NLC), the monocyte-derived type 2 macrophages, deliver stimulatory signals via B-cell activating factor (BAFF), a proliferation-inducing ligand (APRIL), and the C-X-C Motif Chemokine Ligand 12 (CXCL12). Previously, we demonstrated that brain-derived neurotrophic factor (BDNF) protects B-CLL cells from spontaneous apoptosis by activating the oncogenic complex NTSR2-TrkB (neurotensin receptor 2-tropomyosin-related kinase receptor B), only overexpressed in B-CLL cells, inducing anti-apoptotic protein Bcl-2 (B-cell lymphoma 2) expression and Src kinase survival signaling pathways. Herein, we demonstrate that BDNF belongs to the NLC secretome and promotes B-CLL survival. This was demonstrated in primary B-CLL co-cultured with their autologous NLC, compared to B-CLL cells cultured alone. Inhibition of BDNF in co-cultures, enhances B-CLL apoptosis, whereas its exogenous recombinant activates pro-survival pathways in B-CLL cultured alone (i.e. Src activation and Bcl-2 expression), at a higher level than those obtained by the exogenous recombinant cytokines BAFF, APRIL and CXCL12, the known pro-survival cytokines secreted by NLC. Together, these results showed that BDNF release from NLC trigger B-CLL survival. Blocking BDNF would support research strategies against pro-survival cytokines to limit sustained B-CLL cell survival.

Human Medulloblastoma Cell Lines: Investigating on Cancer Stem Cell-Like Phenotype.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite the progress of new treatments, the risk of recurrence, morbidity, and death remains significant and the long-term adverse effects in survivors are substantial. The fraction of cancer stem-like cells (CSCs) because of their self-renewal ability and multi-lineage differentiation potential is critical for tumor initiation, growth, and resistance to therapies. For the development of new CSC-targeted therapies, further in-depth studies are needed using enriched and stable MB-CSCs populations. This work, aimed at identifying the amount of CSCs in three available human cell lines (DAOY, D341, and D283), describes different approaches based on the expression of stemness markers. First, we explored potential differences in gene and protein expression patterns of specific stem cell markers. Then, in order to identify and discriminate undifferentiated from differentiated cells, MB cells were characterized using a physical characterization method based on a high-frequency dielectrophoresis approach. Finally, we compared their tumorigenic potential in vivo, through engrafting in nude mice. Concordantly, our findings identified the D283 human cell line as an ideal model of CSCs, providing important evidence on the use of a commercial human MB cell line for the development of new strategic CSC-targeting therapies.

A New Label-Free Approach to Glioblastoma Cancer Stem Cell Sorting and Detection.

Cancer stem cells (CSCs) play critical roles in cancer, making them important targets for new diagnostic and therapeutic approaches. Since CSCs are heterogeneous and not abundant in tumors, and few specific markers for these cells currently exist, new methods to isolate and characterize them are required. To address this issue, we developed a new label-free methodology to isolate, enrich, and identify CSCs from an heterogeneous tumor cell subpopulation using a cell sorting method (sedimentation field flow fractionation, SdFFF) and a biosensor as a detector. Enrichment was optimized using an original protocol and U87-MG glioblastoma cells cultured in a normal (N) or defined (D) medium (± fetal bovine serum, FBS) under normoxic (N, pO2 = 20%) or hypoxic (H, pO2 < 2%) conditions to obtain four cell populations: NN, NH, DN, and DH. After elution of CSCs via SdFFF using the hyperlayer mode (inertial elution mode for micrometer-sized species), we isolated eight subpopulations with distinct CSC contents based on phenotypical and functional properties, ranging from NN F1 with a lower CSC content to DH F3 with a higher CSC content. Reflecting biological differences, the intrinsic intracellular dielectric permittivity increased from NN to DH conditions. The largest difference in electromagnetic signature was observed between NN F1 and DH F3, in which the CSC content was lowest and highest, respectively. The results demonstrate that microwave dielectric spectroscopy can be used to reliably and efficiently distinguish stem cell characteristics. This new instrumental and methodological approach is an important innovation that allows both enrichment and detection of CSCs, opening the door to novel diagnostic and therapeutic approaches.

CD226 opposes TIGIT to disrupt Tregs in melanoma.

CD4+ Tregs impede T cell responses to tumors. They express multiple inhibitory receptors that support their suppressive functions, including T cell Ig and ITIM domain (TIGIT). In melanoma patients, we show that Tregs exhibit increased TIGIT expression and decreased expression of its competing costimulatory receptor CD226 as compared with CD4+ effector T cells, resulting in an increased TIGIT/CD226 ratio. Tregs failed to upregulate CD226 upon T cell activation. TIGIT+ Tregs are highly suppressive, stable, and enriched in tumors. TIGIT and CD226 oppose each other to augment or disrupt, respectively, Treg suppression and stability. A high TIGIT/CD226 ratio in Tregs correlates with increased Treg frequencies in tumors and poor clinical outcome upon immune checkpoint blockade. Altogether, our findings show that a high TIGIT/CD226 ratio in Tregs regulates their suppressive function and stability in melanoma. They provide the rationale for novel immunotherapies to activate CD226 in Tregs together with TIGIT blockade to counteract Treg suppression in cancer patients.

Regulatory Roles of Sortilin and SorLA in Immune-Related Processes.

Sortilin, also known as Neurotensin Receptor-3, and the sorting-related receptor with type-A repeats (SorLA) are both members of the Vps10p domain receptor family. Initially identified in CNS cells, they are expressed in various other cell types where they exert multiple functions. Although mostly studied for its involvement in Alzheimer's disease, SorLA has recently been shown to be implicated in immune response by regulating IL-6-mediated signaling, as well as driving monocyte migration. Sortilin has been shown to act as a receptor, as a co-receptor and as an intra- and extracellular trafficking regulator. In the last two decades, deregulation of sortilin has been demonstrated to be involved in many human pathophysiologies, including neurodegenerative disorders (Alzheimer and Parkinson diseases), type 2 diabetes and obesity, cancer, and cardiovascular pathologies such as atherosclerosis. Several studies highlighted different functions of sortilin in the immune system, notably in microglia, pro-inflammatory cytokine regulation, phagosome fusion and pathogen clearance. In this review, we will analyze the multiple roles of sortilin and SorLA in the human immune system and how their deregulation may be involved in disease development.

p75 neurotrophin receptor and pro-BDNF promote cell survival and migration in clear cell renal cell carcinoma.

p75NTR, a member of TNF receptor family, is the low affinity receptor common to several mature neurotrophins and the high affinity receptor for pro-neurotrophins. Brain-Derived Neurotrophic Factor (BDNF), a member of neurotrophin family has been described to play an important role in development and progression of several cancers, through its binding to a high affinity tyrosine kinase receptor B (TrkB) and/or p75NTR. However, the functions of these two receptors in renal cell carcinoma (RCC) have never been investigated. An overexpression of p75NTR, pro-BDNF, and to a lesser extent for TrkB and sortilin, was detected by immunohistochemistry in a cohort of 83 clear cell RCC tumors. p75NTR, mainly expressed in tumor tissues, was significantly associated with higher Fuhrman grade in multivariate analysis. In two derived-RCC lines, 786-O and ACHN cells, we demonstrated that pro-BDNF induced cell survival and migration, through p75NTR as provided by p75NTR RNA silencing or blocking anti-p75NTR antibody. This mechanism is independent of TrkB activation as demonstrated by k252a, a tyrosine kinase inhibitor for Trk neurotrophin receptors. Taken together, these data highlight for the first time an important role for p75NTR in renal cancer and indicate a putative novel target therapy in RCC.

Anti-apoptotic role and clinical relevance of neurotrophins in diffuse large B-cell lymphomas.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a fatal malignancy that needs to identify new targets for additional therapeutic options. This study aimed to clarify the clinical and biological significance of endogenous neurotrophin (nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF)) in DLBCL biopsy samples and cell lines. METHODS: We analysed expression of NGF, BDNF, and their receptors (Trk, p75(NTR)) in 51 biopsies and cell lines by immunohistochemistry, immunofluorescence, and western blotting. To investigate the biological role of BDNF/TrkB/p75(NTR) axis, effects of neurotrophin signalling inhibition were determined on tumour cell survival and vascular endothelial growth factor (VEGF) secretion. The pharmacological pan-Trk inhibitor K252a was used for in vitro and in vivo studies. RESULTS: A BDNF/TrkB axis was expressed in all biopsies, which was independent of the germinal centre B-cell (GCB)/non-GCB profile. p75(NTR), TrkB, and BDNF tumour scores were significantly correlated and high NGF expression was significantly associated with MUM1/IRF4, and the non-GCB subtype. Diffuse large B-cell lymphoma cell lines co-expressed neurotrophins and their receptors. The full-length TrkB receptor was found in all cell lines, which was also phosphorylated at Tyr-817. p75(NTR) was associated to Trk and not to its cell death co-receptor sortilin. In vitro, inhibition of neurotrophin signalling induced cell apoptosis. K252a caused cell apoptosis, decreased VEGF secretion, and potentiated rituximab effect, notably in less rituximab-sensitive cells. In vivo, K252a significantly reduced tumour growth and potentiated the effects of rituximab in a GCB-DLBCL xenograft model. CONCLUSIONS: This work argues for a pro-survival role of endogenous neurotrophins in DLBCLs and inhibition of Trk signalling might be a potential treatment strategy for rituximab resistant subgroups.

Neurotrophins are expressed in giant cell arteritis lesions and may contribute to vascular remodeling.

INTRODUCTION: Giant cell arteritis (GCA) is characterized by intimal hyperplasia leading to ischaemic manifestations that involve large vessels. Neurotrophins (NTs) and their receptors (NTRs) are protein factors for growth, differentiation and survival of neurons. They are also involved in the migration of vascular smooth muscle cells (VSMCs). Our aim was to investigate whether NTs and NTRs are involved in vascular remodelling of GCA. METHODS: We included consecutive patients who underwent a temporal artery biopsy for suspected GCA. We developed an enzymatic digestion method to obtain VSMCs from smooth muscle cells in GCA patients and controls. Neurotrophin protein and gene expression and functional assays were studied from these VSMCs. Neurotrophin expression was also analysed by immunohistochemistry in GCA patients and controls. RESULTS: Whereas temporal arteries of both GCA patients (n = 22) and controls (n = 21) expressed nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB) and sortilin, immunostaining was more intense in GCA patients, especially in the media and intima, while neurotrophin-3 (NT-3) and P75 receptor (P75NTR) were only detected in TA from GCA patients. Expression of TrkB, a BDNF receptor, was higher in GCA patients with ischaemic complications. Serum NGF was significantly higher in GCA patients (n = 28) vs. controls (n = 48), whereas no significant difference was found for BDNF and NT-3. NGF and BDNF enhanced GCA-derived temporal artery VSMC proliferation and BDNF facilitated migration of temporal artery VSMCs in patients with GCA compared to controls. CONCLUSIONS: Our results suggest that NTs and NTRs are involved in vascular remodelling of GCA. In GCA-derived temporal artery VSMC, NGF promoted proliferation and BDNF enhanced migration by binding to TrkB and p75NTR receptors. Further experiments are needed on a larger number of VSMC samples to confirm these results.

The implications of sortilin/vps10p domain receptors in neurological and human diseases.

The neurotensin receptor-3 also known as sortilin is part of the new receptor family of vacuolar protein sorting 10 protein domain. Growing evidence show that the vacuolar protein sorting 10 protein domain family is implicated as a genetic risk factor for neurodegenerative diseases such as Alzheimer's disease, frontotemporal lobar degeneration, and Parkinson's disease, in addition to links associated with type 2 diabetes mellitus, lysosomal disorders, cardiovascular disease and atherosclerosis. In fact, sortilin expression is elevated in many human cell lines controlling the trafficking and release of neurotrophins. Hence, not surprisingly the imbalance of neurotrophin signaling is implicated in several human diseases. The fine regulation of the growth factor, brain derived nerve factor by sortilin mediates both neuronal and tumor cell survival, whereas in Alzheimer's disease sortilin mediated beta secretase-1 trafficking increases the cleavage of the beta-amyloid precursor protein. Perturbation of the autocrine/paracrine loop of neurotrophins in combination with the cell surface interaction of sortilin with neurotensin receptor 1 or 2 or tyrosine kinase receptor A or B are dramatically upregulated in both neurodegenerative diseases and cancer. In cardiovascular diseases, the circulatory low-density lipoprotein is closely correlated with sortilin expression in hepatocytes. Herein, this review discusses the multifaceted role played by sortilin and its interacting partners in human disease which could be interesting novel target(s) in drug discovery. Nevertheless, completely challenging the function of sortilin could prove unfavorable given the important universal role of sortilin plays in the body. Hence, metabolism disorders could be relieved with specific targeted therapeutic challenge of sortilin function.

Differential expression of neurotensin and specific receptors, NTSR1 and NTSR2, in normal and malignant human B lymphocytes.

Neurotensin, a neuropeptide growth factor, and its two specific neurotensin receptors, NTSR1 and NTSR2, were shown to be expressed by human B cell lines. Another NTSR, sortilin, which is common to neurotensin and neurotrophins, was also detected as we have previously described. Neurotensin was functional in B cell lines; it induced their proliferation and inhibited apoptosis induced by serum deprivation or Fas activation. Quantitative study of gene expression in two malignant B cell diseases showed that NTSR2 was overexpressed, NTSR1 decreased, and neurotensin was unexpressed in B cell leukemia patient's cells, as compared with healthy B cells. However, these expressions did not significantly change in large diffuse B cell lymphoma lymph nodes compared with benign ones. This study points out that neurotensin and its two specific receptors are expressed in human B lymphocytes. Such expressions were not described, and their relationship in B cell diseases, especially in chronic B cell leukemia, needs to be considered further in regard to these findings.