CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors

<p>Abstract</p> <p>Background</p> <p>Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.</p> <p>Methods</p> <p>We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas.</p> <p>Results</p> <p>A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide.</p> <p>Conclusions</p> <p>Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.</p

Description d'une nouvelle stratégie de criblage moléculaire par transfert de fluorescence intracellulaire à l'aide d'une chimiothèque de composés fluorescents et de protéines de fusion étiquetées par la protéine GFP (synthèse d'un modulateur de PEA-15)

ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Contribution à l'analyse des fonctions de la protéine PEA-15 (caractérisation d'un inhibiteur par criblage moléculaire et rôle dans la motilité astrocytaire)

PEA-15 (Phosphoprotein Enriched in Astrocytes-15 kDa) était connue pour inhiber l'apoptose et la prolifération. A l'aide de souris PEA-15 -/- et sauvages, nous démontrons que PEA-15 inhibe également la migration astrocytaire par un mécanisme qui dépend de la protéine kinase C delta et plus particulièrement du fragment catalytique de celle-ci. La faible expression de PEA-15 dans des cellules tumorales ayant migré à distance de glioblastomes maintenus en culture organotypiques suggère que cet effet inhibiteur de PEA-15 sur la migration peut également intervenir dans un cadre pathologique. Par ailleurs, la mise en oeuvre d'une nouvelle stratégie de criblage basée sur le transfert de fluorescence intracellulaire entre la protéine étiquetée par la GFP et des composés de synthèse couplés à un fluorophore a permis de découvrir un composé de synthèse, 6D6-1, qui interagit avec PEA-15 et inhibe l'interaction entre PEA-15 et la kinase ERK qui sous-tend l'effet anti-prolifératif de PEA-15PARIS-BIUP (751062107) / SudocSudocFranceF

Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo

International audienceTCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4+ T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4+ T cells. TCR-stimulated PEA-15-deficient CD4+ T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4+ T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4+ CD62L+ PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response

Activated PEA-15^-/- T cells have impaired proliferation, and reduced IL-2 & IFNγ production.

<p><b>(A & B)</b> Purified lymph node (LN) CD4⁺ T cells of <i>PEA-15</i>^-/—and <i>PEA-15</i>^+/+ mice were stimulated with 0.1μg/ml or 2μg/ml (when indicated) of anti-CD3, with or without anti-CD28 (2μg/ml), in the presence or not of recombinant IL-2 (50U/mL). Proliferation was analyzed <b>(A)</b> by flow cytometry analysis of CFSE-labeled cells (numbers indicate the percentage of cells in each division at 72H, data are representative of 4 independent experiments<b>)</b> and <b>(B)</b> by enumeration of cells at the indicated time by trypan blue exclusion test. Data shown are means +/- SEM of 3 independent experiments (<i>* p< 0</i>.<i>05</i>). <b>(C)</b><i>PEA-15</i>^-/—and <i>PEA-15</i>^+/+ T cells treated without (control) or with ConA mitogen for 18H, were analysed for CD25 and CD122 expression by flow cytometry at 12H and 24H after stimulation, respectively. The data shown were obtained using PE-labeled mAbs specific for CD25 and CD122 and FITC-labeled mAbs specific for CD4, and are gated on CD4-FITC positive cells. The percentage of cells in each quadrant is indicated. These values are representative of 6 experiments. <b>(D)</b> LN T cells from <i>PEA-15</i>^-/- mice and <i>PEA-15</i>^+/+ littermates were treated as in (A & B) for 72H. Flow cytometry of cells stained with propidium iodide (PI) gated on CD4+ cells was performed for cell cycle analysis. Histograms of the percentage of cells in the S and G2/M phases of the cell cycle of a representative experiment (<b>D/</b>upper panel) and cumulative data of 4 independents experiments +/-SD (<i>* p< 0</i>.<i>05</i>) (<b>D/</b>lower panel) are shown. <b>(E)</b> Purified T cells from <i>PEA-15</i>^-/- mice and <i>PEA-15</i>^+/+ littermates were stimulated as in (C). IL2 production in the culture supernatant was quantified by CTLL2 bioassay (n = 8; 3 independent experiments). Quantification of IL-4(Th2) and IFNγ (Th1) (n = 6; 3 independent experiments<b>)</b> in the culture supernatant was determined by ELISA (*<i>p<0</i>.<i>05)</i>.</p

Dysregulation of ERK signaling dependent—targets in TCR-stimulated PEA-15^-/- T cells.

<p>(A) Negatively sorted CD4+ T lymphocytes from <i>PEA-15</i>^-/- mice and <i>PEA-15</i>^+/+ littermates were preincubated or not with the MEK/ERK inhibitor PD98059 (30μM) for 30 minutes, and then stimulated with cross-linked anti-CD3 mAbs (0,1μg/ml), with or without anti-CD28 mAbs (2μg/ml) for 30 min. Indicated genes expression was quantified by real-time quantitative PCR. Means +/- SEM from 5 separate experiments are shown, and expressed as percentage of the “CD3 (0.1μg/ml)-stimulated-<i>PEA-15</i>^+/+-CD4⁺-T-cells” condition taken as positive control. Statistical significance is indicated *<i>p<0</i>.<i>05</i>: for comparison between PEA-15^+/+ or PEA-15^-/-; + <i>p<0</i>.<i>05</i>: for pairwise comparison of different culture condition groups; <i># p<0</i>.<i>05</i>: between cells treated without or with PD98059; (Mann-Whitney <i>U</i> test). (B) Negatively sorted CD4+ T lymphocytes from <i>PEA-15</i>^-/- mice and <i>PEA-15</i>^+/+ littermates were stimulated for 30 min with anti-CD3 (0.1–1μg/ml), with or without anti-CD28 (5μg/ml) mAbs, or with or without rIL-2 (100U/ml). Whole cell lysates were prepared, and equal amounts of proteins were resolved by SDS-PAGE and blotted with anti-cyclin E mAbs, or with anti-pRb mAbs. A representative experiment out of 3 is shown. Arbitrary densitometric units for the bands were analysed by computing densitometry. Means +/-SEM (n = 3) for pRb and cyclin E expression are represented below the immunoblot.</p

Lymphocytes composition of spleen, lymph nodes, and thymus in <i>PEA-15</i>^-/—mice.

<p>The cellular composition of spleen, lymph nodes, and thymus for <i>PEA-15</i>^-/- mice and their WT littermates was determined by cell counting and/or flow cytometry after staining with mAbs specific for lymphocyte subsets. (N = 6; <b>**</b>: P< 0.05).</p

Resistance of PEA-15^-/- mice to HEL+RBC alloimmunization.

<p>Three days before the red blood cell (RBC) transfusion, <i>PEA-15</i>^-/—or <i>PEA-15</i>^+/+ mice were depleted in Treg with an anti-CD25 mAbs. At day 0, mice were transfused with a leukoreduced blood from HEL⁺ HOD mice, 4 hours after injection of the adjuvant poly (I:C),. (A) Representative Dot plots showing % of CD25+ Foxp3+ Treg among CD4+ T-cells before and after <i>in vivo</i> Treg depletion with anti-CD25 Abs; (B) Histograms showing representative data of anti-HEL IgG in sera from <i>PEA-15</i>^-/—or <i>PEA-15</i>^+/+ mice using flow cytometry-based mHEL crossmatch. HEL⁺ HOD RBCs (blue line) or HEK-negative FVB RBCs (red line; negative control) were incubated with a 1/10 dilution of sera from transfused <i>PEA-15</i>^-/- or <i>PEA-15</i>^+/+ mice (n = 10; 2 independent experiments). (C) For each group of mice, the % of the positive mean fluorescence intensity was defined as the mean fluorescence of the serum crossmatched with HEL-negative FVB RBCs substracted from the mean fluorescence of the serum crossmatched with HEL⁺ HOD RBCs. Statistical analysis were performed with the Mann-Whitney test. * p<0.05; **p<0.01.</p

PEA-15 regulates ERK1/2 and phosphoERK1/2 subcellular compartmentalization.

<p>(A) Negatively sorted <i>PEA-15</i>^+/+ or <i>PEA-15</i>^-/- CD4⁺ T lymphocytes were stimulated with cross-linked anti-CD3 mAbs (1μg/ml) for the indicated times. Total cell lysates (50μg/line) were resolved by SDS-PAGE followed by western-blotting. Total ERK-1/2 was detected by the mean of anti-p42/p44 antibodies; ERK-1/2 phosphorylation was assessed with anti-phospho-ERK1/2 antibodies. Quantitative densitometric analysis of phospho-p42 and phospho-p44 expression out of 4 experiments is presented below a representative immunoblot. Results are expressed as means +/-SEM (n = 4). (B & C) ERK1/2 localization was assessed by immunofluorescence in resting cells (NS) or cells stimulated with PMA (200nM) for 15 minutes, using an anti-P42/P44 antibody. A representative experiment is shown in (B); histograms represent the % of enumerated cells in which the ERK1/2 staining was cytoplasmic and/or nuclear. Values are means +/- SD of 4 independent experiments (* <i>p<0</i>.<i>05</i>). (C) Phospho-ERK1/2 localization was assessed in the same stimulation conditions as in (B) using an anti-phospho ERK-1/2 antibody. Nuclei were stained with TOPRO3. (D) Expression of phospho-Akt and Akt was assessed in sorted CD4+ T lymphocytes from <i>PEA-15</i>^+/+ or <i>PEA-15</i>^-/- mice stimulated or not with cross-linked anti-CD28 mAb (5μg/ml) for 5 or 15 min. 50μg of total cell lysate proteins were resolved by 12% SDS-PAGE followed by western-blotting. Representative results out of 4 independent experiments are shown. Histograms representing densitometric analysis of phospho-Akt expression are shown on the right of the panel. Results are expressed as means +/- SEM of 4 independent experiments.</p