Clinical Relevance of Tumor Cells with Stem-Like Properties in Pediatric Brain Tumors

BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors

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

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

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

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

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

Activated PEA-15^-/- T cells have decreased IL-4 production, and increased activity of IL4I1.

<p>(A, B & C) Sorted naïve CD4⁺ CD62L⁺ T lymphocytes from <i>PEA-15</i>^-/- mice (■) and <i>PEA-15</i>^+/+ (□) mice were stimulated with anti-CD3 (0.1–1 μg/ml) with or without anti-CD28 (2μg/ml), for 5 days. (A) Cytokines production was quantified in the culture supernatants by Luminex assay. (B) GATA3, Tbet, FOXP3 and RORc, and IL4I1 (C left panel) genes expression was analyzed by real-time quantitative PCR in total mRNA extracts of the cultures. Means +/- SEM from n = 6 out of 2 independent experiments are presented. (C right panel) IL4I1 activity was measured in 10⁵ CD4⁺ CD62L⁺ T lymphocytes stimulated or not with plate-bound anti-CD3<i>-</i> (1 μg/ml) and soluble anti-CD28 (2μg/ml) mAbs for 5 days. Means +/- SEM from four separate experiments are presented. Statistical significance is indicated for comparison between <i>PEA-15</i>^+/+- and <i>PEA-15</i>^-/- T cells, *p<0.05 (Mann-Whitney U test).</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

Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

International audienceEpithelial-to-mesenchymal transition-like (EMT-like) is a critical process allowing initiation of metastases during tumour progression. Here, to investigate its role in intestinal cancer, we combine computational network-based and experimental approaches to create a mouse model with high metastatic potential. Construction and analysis of this network map depicting molecular mechanisms of EMT regulation based on the literature suggests that Notch activation and p53 deletion have a synergistic effect in activating EMT-like processes. To confirm this prediction, we generate transgenic mice by conditionally activating the Notch1 receptor and deleting p53 in the digestive epithelium (NICD/p53(-/-)). These mice develop metastatic tumours with high penetrance. Using GFP lineage tracing, we identify single malignant cells with mesenchymal features in primary and metastatic tumours in vivo. The development of such a model that recapitulates the cellular features observed in invasive human colorectal tumours is appealing for innovative drug discovery

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42·4% vs 44·2%; absolute difference -1·69 [-9·58 to 6·11] p=0·67; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5-8] vs 6 [5-8] cm H2O; p=0·0011). ICU mortality was higher in MICs than in HICs (30·5% vs 19·9%; p=0·0004; adjusted effect 16·41% [95% CI 9·52-23·52]; p&lt;0·0001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0·80 [95% CI 0·75-0·86]; p&lt;0·0001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status