Barbara Bessette

EGFR Soluble Isoforms and Their Transcripts Are Expressed in Meningiomas

The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%.Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types.

TrkB-containing exosomes promote the transfer of glioblastoma aggressiveness to YKL-40-inactivated glioblastoma cells

The neurotrophin receptors are known to promote growth and proliferation of glioblastoma cells. Their functions in spreading glioblastoma cell aggressiveness to the microenvironment through exosome release from glioblastoma cells are unknown. Considering previous reports demonstrating that YKL-40 expression is associated with undifferentiated glioblastoma cancer stem cells, we used YKL-40-silenced cells to modulate the U87-MG differentiated state and their biological aggressiveness. Herein, we demonstrated a relationship between neurotrophin-receptors and YKL-40 expression in undifferentiated cells. Differential functions of cells and derived-exosomes were evidenced according to neurotrophin receptor content and differentiated cell state by comparison with control pLKO cells. YKL-40 silencing of glioblastoma cells impairs proliferation, neurosphere formation, and their ability to induce endothelial cell (HBMEC) migration. The modulation of differentiated cell state in YKL-40-silenced cells induces a decrease of TrkB, sortilin and p75NTR cellular expressions, associated with a low-aggressiveness phenotype. Interestingly, TrkB expressed in exosomes derived from control cells was undetectable in exosomes from YKL-40 -silenced cells. The transfer of TrkB-containing exosomes in YKL-40-silenced cells contributed to restore cell proliferation and promote endothelial cell activation. Interestingly, in U87 MG xenografted mice, TrkB-depleted exosomes from YKL-40-silenced cells inhibited tumor growth in vivo. These data highlight that TrkB-containing exosomes play a key role in the control of glioblastoma progression and aggressiveness. Furthermore, TrkB expression was detected in exosomes isolated from plasma of glioblastoma patients, suggesting that this receptor may be considered as a new biomarker for glioblastoma diagnosis.

IL22/IL-22R pathway induces cell survival in human glioblastoma cells.

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.

Oncogramme, a new individualized tumor response testing method: application to colon cancer

Colon cancer is the second leading cause of cancer-related death in industrialized countries. Many anti-cancer researches are consequently performed and individualized tumor response testing (ITRT) methods are now used to individualize patient chemotherapeutic administrations. Then, a new ITRT method, Oncogramme, was developed for colon cancer. Colon tumor fragments from different patients were dissociated and seeded in a defined culture medium. Cell preparation process as well as culture medium allowed high cell viability and a good primary culture success rate. After treatment of isolated tumoral cells by chemotherapeutics alone or in combination, cytotoxicity was determined by cell death assay allowing the Oncogramme establishment, which was validated by statistical analysis. Indeed, significant results were obtained such as different profile for each patient’s cells with various drugs, and variability between patient’s cells in the response to each drug. Procedure described here to obtain the Oncogramme is a new, fast and technically reliable ITRT method applied to colon cancer. For an individualized cancer treatment use, this test should be further validated by a phase I clinical trial.

Autophagy and TrkC/NT-3 signaling joined forces boost the hypoxic glioblastoma cell survival

Glioblastoma multiform (GBM), the most common and aggressive primary brain tumor, is characterized by a high degree of hypoxia and resistance to therapy because of its adaptation capacities, including autophagy and growth factors signaling. In this study, we show an efficient hypoxia-induced survival autophagy in four different GBM cell lines (U87MG, M059K, M059J and LN-18) and an activation of a particular neurotrophin signaling pathway. Indeed, the enhancement of both TrkC and NT-3 was followed by downstream p38MAPK phosphorylation, suggesting the occurrence of a survival autocrine loop. Autophagy inhibition increased the hypoxia-induced expression of TrkC and its phosphorylated form as well as the phosphorylation of p38, suggesting a complementary effect of the two processes, leading to cell survival. Alone, autophagy inhibition reduced cellular growth without inducing cell death. However, the double inhibition of autophagy and TrkC signaling was necessary to bring cells to death as shown by PARP cleavage, particularly important in hypoxia. Moreover, a very high expression of TrkC and NT-3 was found in tumor sections from GBM patients, highlighting the importance of neurotrophic signaling in GBM tumor cell survival. These data suggest that a combined treatment targeting these two pathways could be considered in order to induce the death of GBM cells.

P75 neurotrophin receptor is sequestered in the Golgi apparatus of the U-87 MG human glioblastoma cell line

The P75 neurotrophin receptor (p75NTR) is a cell surface receptor that can induce apoptosis in many cell types. This receptor plays a major role in the development of the central nervous system and is expressed in some adult brain cells. Its implication in cell apoptosis or survival is probably of major importance in cellular homeostasis and thus p75NTR could be implicated in tumor resistance to death. In this study, we investigated the intracellular expression of p75NTR in a human glioblastoma cell line. Detection of p75NTR receptor in Golgi apparatus by immunofluorescence microscopy, or after Golgi apparatus extraction, could be correlated with a decrease of cell apoptosis leading cells to become tumorous. This hypothesis is supported by a loss of ligand-induced apoptosis in this cell line. Our observations show that p75NTR can be sequestered in the Golgi complex and could then be, in part, responsible for the cell resistance to apoptosis and for brain tumor formation.

Cancer Stem-Like Cells in Glioblastoma

Glioblastoma is currently described as the worst brain tumor because of its aggressiveness and poor prognosis. Chemotherapy and irradiation are not cura-tive, and the average survival for patients with glioblastoma is around 15 months. The cellular heterogeneity and infiltrative capability of glioblastoma make complete surgical resection almost impossible. Moreover, the presence of cancer stem-like cells in this tumor leads to therapeutic resistance and tumor recurrence after surgery. Numerous studies have explored the physiology of these cancer stem cells, and attempts have been made to develop devices aimed at isolating this rare population of cells. This chapter describes the complexity of cancer stem cells in glioblastoma. Their role in autophagy, gene regulation by epigenetic modifications, and the challenges in isolating these cells are addressed. This knowledge may pave the way for a better understanding of cancer stem cells in glioblastoma, and the potential development of new therapeutic strategies for this deadly disease.

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.

BiCMOS microfluidic sensor for single cell label-free monitoring through microwave intermodulation

A novel microfluidic biosensing platform based on Bipolar-Complementary Oxide Semiconductor (BiCMOS) technology is presented. The device relies on a quadruple electrode system and a microfluidic channel that are directly integrated into the back-end-of-line (BEOL) of the BiCMOS stack. For proof of concept repeatable electrical trapping of single SW620 (colon cancer) cells in the quadruple electrode system is initially demonstrated. Additionally, for the first time a microwave intermodulation technique is used for high sensitivity dielectric spectroscopy, which could pave the way to label-free monitoring of intracellular processes and manipulation such as electroporation.

Biological cell discrimination based on their high frequency dielectropheretic signatures at UHF frequencies

This paper reports on the application of dielectrophoresis techniques in the radiofrequency range, in order to probe inner dielectric specificities and therefore characterize individual biological cells. The novelty of this work consists in exploring the capability of UHF signals to generate DEP-driven motion effects on flowing biological cells in a microfluidic micro-device. Additionally, with applied signals above 50MHz, distinct cross-over frequencies can be identified as function of both the cell type and the difference in the intracellular dielectric features, and between in-tracellular and extracellular media. Several experimental campaigns were led on three distinct cell lines by thoroughly scanning the UHF spectrum and specifically measuring the resulting second cross-over frequency for each cell type. The experimental results suggest that significant cross-over frequency differences can be observed from one cell line to the other and confirm that their high frequency DEP characteristics can be a relevant cell signature for discriminating them. This work is a first step towards the development of a UHF-DEP cytometer.