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.

KLRC3, a Natural Killer receptor gene, is a key factor involved in glioblastoma tumourigenesis and aggressiveness.

Glioblastoma is the most lethal brain tumour with a poor prognosis. Cancer stem cells (CSC) were proposed to be the most aggressive cells allowing brain tumour recurrence and aggressiveness. Current challenge is to determine CSC signature to characterize these cells and to develop new therapeutics. In a previous work, we achieved a screening of glycosylation-related genes to characterize specific genes involved in CSC maintenance. Three genes named CHI3L1, KLRC3 and PRUNE2 were found overexpressed in glioblastoma undifferentiated cells (related to CSC) compared to the differentiated ones. The comparison of their roles suggest that KLRC3 gene coding for NKG2E, a protein initially identified in NK cells, is more important than both two other genes in glioblastomas aggressiveness. Indeed, KLRC3 silencing decreased self-renewal capacity, invasion, proliferation, radioresistance and tumourigenicity of U87-MG glioblastoma cell line. For the first time we report that KLRC3 gene expression is linked to glioblastoma aggressiveness and could be a new potential therapeutic target to attenuate glioblastoma.

New ex-ovo colorectal-cancer models from different SdFFF-sorted tumor-initiating cells.

Despite effective treatments, relapse of colorectal cancer (CRC) is frequent, in part caused by the existence of tumor-initiating cells (TICs). Different subtypes of TICs, quiescent and activated, coexist in tumors, defining the tumor aggressiveness and therapeutic response. These subtypes have been sorted by hyperlayer sedimentation field-flow fractionation (SdFFF) from WiDr and HCT116 cell lines. On the basis of a new strategy, including TIC SdFFF sorting, 3D Matrigel amplification, and grafting of corresponding TIC colonies on the chick chorioallantoic membrane (CAM), specific tumor matrices could be obtained. If tumors had similar architectural structure with vascularization by the host system, they had different proliferative indices in agreement with their initial quiescent or activated state. Protein analysis also revealed that tumors obtained from a population enriched for "activated" TICs lost "stemness" properties and became invasive. In contrast, tumors obtained from a population enriched for "quiescent" TICs kept their stemness properties and seemed to be less proliferative and invasive. Then, it was possible to produce different kinds of tumor which could be used as selective supports to study carcinogenesis and therapy sensitivity.

Cancer stem cell sorting from colorectal cancer cell lines by sedimentation field flow fractionation.

Recently, cancer stem cells (CSCs) have been identified in many types of cancers, such as colorectal cancer (CRC). CSCs seem to be involved in initiation, growth, and tumor metastasis, as well as in radio- and chemotherapy failures. CSCs appears as new biological targets for cancer therapy, requiring the development of noninvasive cell sorting methods. In this study, we used sedimentation field flow fractionation (SdFFF) to prepare enriched populations of CSCs from eight cell lines corresponding to different CRC grades. On the basis of phenotypic and functional characterizations, "hyperlayer" elution resulted in a fraction overexpressing CSC markers (CD44, CD166, EpCAM) for all cell lines. CSCs were eluted in the last fraction for seven out of eight cell lines, but in the first for HCT116. These results suggest, according to the literature, that two different pools of CSCs exist, quiescent and activated, which can both be sorted by SdFFF. Moreover, according to CSC properties, enriched fractions are able to form colonies.

SCO-spondin oligopeptide inhibits angiogenesis in glioblastoma.

Angiogenesis plays a critical role in glioblastoma growth and progression. We therefore aimed at evaluating the anti-angiogenic properties of an oligopeptide originating from SCO-spondin (NX) on a model of human glioblastoma. To this end, we studied the impact of NX treatment on human brain endothelial cells (HBMECs) alone or co-cultured with glioblastoma cells (U87-MG) on apoptosis, proliferation, migration and release of angiogenic factors. We further investigated the anti-angiogenic potential of NX on human glioblastoma cells grown on chorio-allantoic membrane (CAM) or in glioblastoma xenografts. The results of our experiments showed that NX treatment impaired the microvascular network and induced a decrease in cell proliferation, vascularization and tumor growth in the CAM model as well as in xenotransplants. Interestingly, our in vitro experiments showed that NX impairs HBMECs migration but also regulates the release of angiogenic factors from U87-MG. These results are confirmed by the profiling of NX-treated U87-MG grown on CAM that highlighted modifications of several genes involved in angiogenesis. In conclusion, NX inhibits tumorigenesis by impairing the ability of glioblastoma cells to induce angiogenesis and by inhibiting endothelial cell migration. This molecule might therefore be an interesting candidate for future cancer therapies.

Sedimentation field flow fractionation monitoring of in vitro enrichment in cancer stem cells by specific serum-free culture medium.

The development of methods to enrich cell populations for cancer stem cells (CSC) is urgently needed to help understand tumor progression, therapeutic escape and to evaluate new drugs, in particular for colorectal cancer (CRC). In this work, we describe the in vitro use of OncoMiD for colon, a CRC-specific primary cell culture medium, to enrich CRC cell lines in CSC. Sedimentation field flow fractionation (SdFFF) was used to monitor the evolution of subpopulations composition. In these models, medium induced a loss of adherence properties associated with a balance between proliferation and apoptosis rates and, more important, an increased expression of relevant CSC markers, leading to specific SdFFF elution profile changes.

Hallmarks in colorectal cancer: angiogenesis and cancer stem-like cells.

Carcinogenesis is a multistep process that requires the accumulation of various genetic and epigenetic aberrations to drive the progressive malignant transformation of normal human cells. Two major hallmarks of carcinogenesis that have been described are angiogenesis and the stem cell characteristic of limitless replicative potential. These properties have been targeted over the past decade in the development of therapeutic treatments for colorectal cancer (CRC), one of the most commonly diagnosed and lethal cancers worldwide. The treatment of solid tumor cancers such as CRC has been challenging due to the heterogeneity of the tumor itself and the chemoresistance of the malignant cells. Furthermore, the same microenvironment that maintains the pool of intestinal stem cells that contribute to the continuous renewal of the intestinal epithelia also provides the necessary conditions for proliferative growth of cancer stem-like cells. These cancer stem-like cells are responsible for the resistance to therapy and cancer recurrence, though they represent less than 2.5% of the tumor mass. The stromal environment surrounding the tumor cells, referred to as the tumor niche, also supports angiogenesis, which supplies the oxygen and nutrients needed for tumor development. Anti-angiogenic therapy, such as with bevacizumab, a monoclonal antibody against vascular-endothelial growth factor, significantly prolongs the survival of metastatic CRC patients. However, such treatments are not completely curative, and a large proportion of patient tumors retain chemoresistance or show recurrence. This article reviews the current knowledge regarding the molecular phenotype of CRC cancer cells, as well as discusses the mechanisms contributing to their maintenance. Future personalized therapeutic approaches that are based on the interaction of the carcinogenic hallmarks, namely angiogenic and proliferative attributes, could improve survival and decrease adverse effects induced by unnecessary chemotherapy.

Improved sedimentation field-flow fractionation separation channel for concentrated cellular elution.

SdFFF is now commonly used for cell sorting. Nevertheless, as with many other separation methods, SdFFF Hyperlayer elution leads (1) to sample dilution resulting in cell loss which could restrict further use; and (2) to a high output flow rate impacting detector sensitivity and selectivity. In order to limit these problems, we proposed modifications of the SdFFF separation channel consisting both in downscaling and the insertion of an outlet stream splitter. This last system corresponded to a strip which divides the flow rate output into two parts, one containing concentrated cells in a reduced volume and flow rate, the other containing the excess mobile phase useless for further cell manipulation, detection and characterization. For the first time we have shown that splitter implementation and downscaling respected channel flowing and resulted in Hyperlayer elution of around 95% of cells in less than 50% of input flow rate. Improved cell sorting was demonstrated by enrichment (∼10 times) of cancer stem cells from WiDr cells with two times less quantity of injected cells.

Fine-tuning roles of endogenous brain-derived neurotrophic factor, TrkB and sortilin in colorectal cancer cell survival.

BACKGROUND: Neurotrophin receptors were initially identified in neural cells. They were recently detected in some cancers in association with invasiveness, but the function of these tyrosine kinase receptors was not previously investigated in colorectal cancer (CRC) cells. METHODS AND FINDINGS: We report herein that human CRC cell lines synthesize the neural growth factor Brain-derived neurotrophic factor (BDNF) under stress conditions (serum starvation). In parallel, CRC cells expressed high- (TrkB) and low-affinity (p75(NTR)) receptors at the plasma membrane, whereas TrkA and TrkC, two other high affinity receptors for NGF and NT-3, respectively, were undetectable. We demonstrate that BDNF induced cell proliferation and had an anti-apoptotic effect mediated through TrkB, as assessed by K252a, a Trk pharmacologic inhibitor. It suppressed both cell proliferation and survival of CRC cells that do not express TrkA nor TrkC. In parallel to the increase of BDNF secretion, sortilin, a protein acting as a neurotrophin transporter as well as a co-receptor for p75(NTR), was increased in the cytoplasm of primary and metastatic CRC cells, which suggests that sortilin could regulate neurotrophin transport in these cells. However, pro-BDNF, also detected in CRC cells, was co-expressed with p75(NTR) at the cell membrane and co-localized with sortilin. In contrast to BDNF, exogenous pro-BDNF induced CRC apoptosis, which suggests that a counterbalance mechanism is involved in the control of CRC cell survival, through sortilin as the co-receptor for p75(NTR), the high affinity receptor for pro-neurotrophins. Likewise, we show that BDNF and TrkB transcripts (and not p75(NTR)) are overexpressed in the patients' tumors by comparison with their adjacent normal tissues, notably in advanced stages of CRC. CONCLUSION: Taken together, these results highlight that BDNF and TrkB are essential for CRC cell growth and survival in vitro and in tumors. This autocrine loop could be of major importance to define new targeted therapies.