Cancer stem cells (CSCs) remain challenging to isolate and characterize because of their plastic phenotype. To overcome this issue, we used a microfluidic lab-on-a-chip analysis approach based on ultra-high frequency dielectophoresis (UHF-DEP) to measure the dielectrophoretic signature of colorectal cancer cells. We demonstrated that CSCs exhibit a distinct and lower frequency signature than differentiated cancer cells. Extracellular vesicles (EVs) released by tumor cells are implicated in tumor progression and metastasis. As CSC-derived EVs carry a more aggressive cargo, we hypothesized that treating differentiated colorectal cancer cells with these vesicles might affect their phenotype which would be detected by our lab on a chip. Indeed, the dielectrophoretic signature of cells treated with those EVs was altered in comparison to untreated cells, even in cases where no detectable biological changes were observed. Compared to conventional approaches using biomarkers to characterize CSCs, this UHF-DEP lab on a chip is a label-free method providing rapid and relevant results. Such a method could be useful in the clinic for the early detection of CSCs in the tumor mass, as well as for monitoring CSC-derived EVs in the bloodstream in order to study responses to therapy and prevent relapses.

In this article, we introduce a method to exploit ultra-high dielectrophoresis (UHF-DEP) with a microfluidic radio frequency device able to sort biological cells by deflecting the trajectory of the targeted population in a continuous flow. This study highlights the capability of a high frequency lab-on-chip implemented UHF-DEP cytometer to isolate cancer stem cells (CSCs). Actually, CSCs sorting by an efficient, fast and low-cost method remains an issue. Above 20 MHz, the intracellular dielectric properties of cells determine the dielectrophoretic behavior of cells and is then very sensitive to the cell cytoplasm content. The proposed cell sorting principle exploits the combination of the fluidic drag and the repulsive dielectrophoretic forces in order to isolate CSCs hidden within differentiated cancer cells. We previously demonstrated the UHF-DEP behavior of cells, as a new type of electromagnetic biomarkers to discriminate cancer stem cells among a tumor population from colorectal cancer cell line.

Cancer stem cells remain a challenge to isolate and characterize because of their plastic phenotype. Using a microfluidic lab-on-a-chip based on ultra-high frequency dielectophoresis, we measured the electromagnetic signature of colorectal cancer cells and demonstrated that cancer stem cells show a distinct and lower electromagnetic signature than differentiated cells. The release of extracellular vesicles from tumor cells can drive tumor progression and metastasis development. As extracellular vesicles from cancer stem cells carry more aggressive content, we treated colorectal cancer cells with these vesicles to test whether the lab-on-a-chip can detect a change in phenotype. The electromagnetic signature of treated cells is modified in comparison to untreated cells and sometimes even when no biological change is observed. The lab-on-a-chip provides rapid and relevant result without prior labeling compared to conventional biological approaches. It could be useful in the clinic for early detection of cancer stem cells in the tumor mass and for monitoring the aggressive potential of extracellular vesicles in the bloodstream in order to adapt therapeutic management and prevent relapse.

This paper demonstrates the great potential of Ultra-High Frequency dielectrophoresis (UHF-DEP) to monitor the kinetics of glioblastoma (GBM) cells stemness phenotype transformation. Above 20 MHz, the method is able to probe the intracellular content and to be sensitive to its conductivity and permittivity values. U87-MG GBM cell line was cultured in different conditions in order to induce a differentiation gradient phenotype among the cell population. Using the presented characterization technic, it is shown than undifferentiated cells can be discriminated.

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.

Background Glioblastoma (GBM) is the most aggressive and frequent primary brain tumor during adulthood. One of the major treatments is the association of surgery and a combination of chemo and radiotherapies. Despite its immediate efficiency, it fails to prevent the cancer recurrence in the irradiated area due to radioresistance mechanisms. MicroRNAs (miRNAs or miR) are small non-coding, single strand RNA molecules encoding to various specific genes and able to regulate their expression and induce the tumor cell survival leading to radioresistance. Small extracellular vesicles (EVs), or exosomes released by tumor cells in tumor microenvironment and blood circulation are able to transport and diffuse miRNAs and affect the microenvironment by spreading the miRNAs, which drive radioresistance. Aims To identify the variations of miRNAs expression induced by irradiation in human glioblastoma U87-MG cells and their secreted exosomes collected in supernatants. To analyze the miRNAs variations in EVs-derived from the plasma of patients during radiotherapy, in order to identify a miRNA signature induced by radiotherapy in a liquid biopsy. Materiel and methods U87-MG cells were cultured on plates and exposed to irradiation. miRNAs analyzes were performed in cells and in EVs isolated from cell supernatants to determine miRNAs expressions both in cells and in secreted exosomes before and after irradiation. Plasma-derived EVs were collected from 4 glioblastoma patients before and after surgery and radiotherapy treatments. Conclusion The analysis of miRNAs expression profiles in both GBM cells and their derived EVs revealed that miR profile changes after irradiation. However, the number of similar miR between cells or EVs, following cell irradiation, was restricted to 3 miRs alone suggesting that the irradiation-induced changes in the miR profile in the cells and their EVs are not closely linked. In this context, the miR profile in EVs from patients plasma was investigated to establish a potential link with the miRNAs profile observed in EVs from irradiated cells and to assess its relationship with the response to radiotherapy. Three miRs (different from those identified in cells) were common between EVs derived from cells and patients derived-exosomes. These miRs detected in circulating EVs could provide a specific and reliable signature in response to ionizing radiation, which could be useful for monitoring the effectiveness of radiotherapy. Further experiments on a larger patients population with clinical data could also help to define whether this signature might have a prognostic value on the response to radiotherapy.

This paper presents a microfluidic radiofrequency device operating at a few hundred MHz, which is able to sort biological cells. It uses a non-invasive and label-free technic based on intracellular dielectric specificities of biological cells. The sorting principle relies on a dynamic dielectrophoresis (DEP) deviation resulting from the interaction between a high frequency electric signal and the cell cytoplasm content. Driven in a microfluidic channel by a continuous flow, cells are individually deflected from their primary trajectories after having entered a non-uniform electric field generated by a microelectrode system. Designed with different slopes, these electrodes allow a selective guiding of cells to different outlets depending on the dielectrophoresis deviation efficiency. To allow a successful cell sorting, the intensity of deviation forces acting on cells is modulated according to the particle speed, the dielectrophoresis signal frequency and the electrode slope angles related to the Clausius-Mossotti factor of each cell. As proof of concept, experiments with cells from glioblastoma line were carried out, using different DEP signal frequencies to highlight system ability to sort cells from heterogeneous basal population into less disparate sub-populations.

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.

This paper introduces results about characterizations of different tumor cell lines, using intracellular sensing based on Ultra High Frequency dielectrophoresis (UHF-DEP). Above 50 MHz, presented technic allows to probe internal cell content to characterize its cytoplasm permittivity and conductivity properties. The measured frequency-dependent cell behavior results on a distinctive DEP signature according to the cell type and its biological specificity. This paper illustrates the high potential of UHF lab-on-chip sensor to discriminate various tumor cell types derived from different tissues.

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.

Glioblastoma (GBM) is one of the most frequent and the most aggressive tumor of the central nervous system. About 240,000 brain tumor new cases were diagnosed over the worldwide; the majority are GBMs with an incidence of 3–4 per 100 000 persons per year [2]. Medulloblastoma (MB) is the most common malignant pediatric brain tumor [3]. Despite the progress of new treatments, the risk of recurrence, morbidity, and death remains significant. Hence, the dark prognosis of these diseases, especially for GBM, is primarily due to the recurrence of tumor, which can be resistant to conventional treatments closely linked to the existence of very immature and undifferentiated cells, as cancer stem-like cells (CSC) [3]. Conventional markers for normal stem cells as CD133, CD44, OCT-4, SOX2, pSTAT3 and NANOG are currently used and combined to have clues of CSC occurrence. However, CSCs subpopulation are very rare in tumors and their isolation often requires enriching them in specific culture medium followed with functional test to established with certainty the diagnosis. Such strategy is really time consuming and makes the results longer. Consequently, alternatives cell analysis methods are required to get around this issue in order to efficiently discriminate and identify undifferentiated cancer cell populations from the whole heterogeneity that occurs in the tumor biopsies. Accepted presentation during the BIOEM 2020 conference: Oxford June 2020 High frequency dielectrophoresis might be one of them. Hence, this study discusses about the significant correlation between dielectrophoresis signatures in the UHF frequency domain we have established related to own CSC biological specificities [4-5]. Especially, we will discuss about how relevant the UHF signatures vs the conventionally measured ones in the kHz range are. Such new physical marker can be used to isolate CSC from others cells and applied to characterize various patient tumors. Original lab on chip concepts to exploit such physical properties will be also introduced.

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.

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.

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.

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.

This paper introduces the first results of dielectric spectroscopy characterization of glioblastoma cells, measuring their crossover frequencies in the ultra-high-frequency range (above 50 MHz) by dielectrophoresis (DEP) techniques. Experiments were performed on two glioblastoma lines U87-MG and LN18 that were cultured following different conditions, in order to achieve different phenotypic profiles. We demonstrate here that the presented DEP electrokinetic method can be used to discriminate the undifferentiated from the differentiated cells. In this study, microfluidic lab-on-chip systems implemented on bipolar-complementary oxide semiconductor technology are used allowing single cell handling and analysis. Based on the characterizations of their own intracellular features, both the selected glioblastoma (GBM) cell lines cultured in distinct culture conditions have shown clear differences of DEP crossover frequency signatures compared to the differentiated cells cultured in a normal medium. These results support the concept and validate the efficiency for cell characterization in glioblastoma pathology.

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.

Ce papier demontre le fort potentiel et les capacites de la dielectrophorese Ultra-Haute Frequence (DEP-UHF) pour trier des populations cellulaires. Le concept propose combine des forces hydrodynamiques et dielectrophoretiques majoritairement repulsives au sein d'un cytometre de type laboratoire-sur-puce. L'objectif principal est de separer differents types de cellules en suspension dans un flux continu sur la base de leurs differences de proprietes dielectriques. Pour cela nous montrons qu'il est important d'appliquer aux cellules un champ electrique adequat dans une gamme de frequences particulieres afin de produire differentes intensites de forces et de deviations de trajectoire associees. L'efficacite du dispositif sera illustree au travers l'exemple d'un test de tri de cellules mesenchymateuses.