Identification of germline cancer predisposition variants in pediatric sarcoma patients from somatic tumor testing

Genetic predisposition is an important risk factor for cancer in children and adolescents but detailed associations of individual genetic mutations to childhood cancer are still under intense investigation. Among pediatric cancers, sarcomas can arise in the setting of cancer predisposition syndromes. The association of sarcomas with these syndromes is often missed, due to the rarity and heterogeneity of sarcomas and the limited search of cancer genetic syndromes. This study included 43 pediatric and young adult patients with different sarcoma subtypes. Tumor profiling was undertaken using the Oncomine Childhood Cancer Research Assay (Thermo Fisher Scientific). Sequencing results were reviewed for potential germline alterations in clinically relevant genes associated with cancer predisposition syndromes. Jongmans¿ criteria were taken into consideration for the patient selection. Fifteen patients were selected as having potential pathogenic germline variants due to tumor sequencing that identified variants in the following genes: CDKN2A, NF1, NF2, RB1, SMARCA4, SMARCB1 and TP53. The variants found in NF1 and CDKN2A in two different patients were detected in the germline, confirming the diagnosis of a cancer predisposition syndrome. We have shown that the results of somatic testing can be used to identify those at risk of an underlying cancer predisposition syndrome

Correction: Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling

Correction de l'article DOI: 10.1371/journal.pbio.100246

Neuronal hyperactivity disturbs ATP microgradients, impairs microglial motility, and reduces phagocytic receptor expression triggering apoptosis/microglial phagocytosis uncoupling

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders

Immunogenicity of human embryonic stem cell-derived beta cells

Therapeutic cell differentiatio

Microglial phagocytic response during in vivo acute and chronic inflammatory challenge.

<p>(<b>A</b>) Experimental design and apoptosis in the DG of c57BL/6 fms-EGFP 1-mo mice injected systemically with LPS (1mg/kg; <i>n</i> = 5) or vehicle (saline; <i>n</i> = 4) 8 h prior to sacrifice. Apoptotic cells were identified by pyknosis/karryorhexis. <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.g002" target="_blank">Fig 2A</a></b> was generated from data that was originally published as part of [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.ref009" target="_blank">9</a>]. (<b>B</b>) Weighted Ph capacity of microglia (in parts per unit, ppu) in control and LPS mice. (<b>C</b>) Number of microglial cells in control and LPS mice. (<b>D</b>) Ph/A coupling in the 1-mo mouse hippocampus (in fold change) during acute inflammatory challenge. (<b>E</b>) Experimental design and representative confocal z-stacks of the DG of PND21 Swiss mice fed during gestation and lactation with a diet balanced (Ω3 bal; <i>n</i> = 7) or deficient (Ω3 def; <i>n</i> = 7) in the omega 3 polyunsaturated fatty acid, a diet that induces chronic inflammation in the hippocampus. Microglia were labeled with Iba1 (cyan) and apoptotic nuclei were detected by pyknosis/karyorrhexis (white, DAPI). Arrows point to apoptotic cells engulfed by microglia (M). Scale bars = 50 μm; z = 22.5μm. (<b>F</b>) Number of apoptotic (pyknotic/karyorrhectic) cells in mice fed with Ω3 balanced and deficient diets. (<b>G</b>) Ph index in the PND21 hippocampus (in % of apoptotic cells) in mice fed with Ω3 balanced and deficient diets. (<b>H</b>) Weighted Ph capacity of microglia (in ppu) in PND21 mice. (<b>I</b>) Histogram showing the Ph capacity distribution of microglia (in % of cells) in PND21 mice. (<b>J</b>) Total number of microglial cells (Iba1⁺) in PND21 mice. (<b>K</b>) Ph/A coupling in PND21 mice. Bars represent mean ± SEM. * indicates <i>p</i> < 0.05 and ** indicates <i>p</i> < 0.01 by one-tail Student´s <i>t</i> test. Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p

ATP impairs microglial phagocytosis in vivo.

<p>(<b>A</b>) Representative confocal z-stacks of saline, 100 mM ATP and 100 mM ATPγS (2 hpi) DG labeled with DAPI (nuclear morphology, white), activated caspase 3 (act-casp3⁺, red, for apoptotic cells), and fms-EGFP (cyan, microglia). Arrow points to a phagocytosed apoptotic cell, whereas arrowheads point to nonphagocytosed apoptotic cells. Activated-caspase 3 puncta within microglia are labeled with a round-ended arrow. (<b>B, H</b>) Experimental designs (<b>B</b>, 100 mM of ATP and ATPγS, 2 h; <b>H</b>, 10 and 100 mM ATP, 4 h; <i>n</i> = 3–4 per group) and number of apoptotic (pyknotic/karyorrhectic and act-casp3⁺) in the septal DG (<i>n</i> = 3–4 per group). No changes in the volume of the DG were found in either experiment (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s024" target="_blank">S11C Fig</a></b>). (<b>C, I</b>) Ph index in the septal DG (in % of apoptotic cells). (<b>D, J</b>) Weighted Ph capacity of hippocampal microglia (in ppu). (<b>E, K</b>) Histogram showing the Ph capacity distribution of microglia (in % of cells) in the septal DG. (<b>F, L</b>) Total number of microglial cells (fms-EGFP⁺) in the septal DG. (<b>G, M</b>) Ph/A coupling (in fold change) in the septal DG. Bars represent mean ± SEM, * indicates <i>p</i> < 0.05, ** indicates <i>p</i> < 0.01, and *** indicates <i>p</i> < 0.001 by Holm-Sidak posthoc test after one-way ANOVA were significant at <i>p</i> < 0.05. Scale bars = 50 μm, z = 11.9 μm (control, ATP), 9.8 μm (ATPγs). Inserts are single plane images of the corresponding confocal z-stacks. Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p

Microglial phagocytosis impairment is unrelated to monocytes.

<p>(<b>A</b>) CD45 staining in saline- and KA-injected mice at 3 dpi. Cell nuclei are shown in white (DAPI), microglia in cyan (fms-EGFP), and CD45 in red. In control mice, the expression of CD45 was dim, showing diffuse cytoplasmic inclusions within microglia. A CD45⁺ cell is shown engulfing an apoptotic cell (arrow, enlarged). In KA mice, CD45 had a higher and more widespread expression in all microglial cells, including a dividing cell (arrowhead, enlarged). A clear distinction between CD45^high and CD45^low cells was not evident. (<b>B</b>) Flow cytometry analysis of the expression of CD45 in fms-EGFP⁺ hippocampal cells from control and KA-treated mice. Gates for CD45^low (cyan) and CD45^high (red) were defined based on the distribution of the fms-EGFP⁺ cells in control (not injected) mice. 3 dpi after the KA injection, more cells were found in the CD45^high gate, although the fms-EGFP⁺ cells were in fact distributed along a continuum of CD45 expression, all of them with higher expression than control mice. At 7 dpi, the expression of CD45 returned to basal levels. The gating strategy is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s017" target="_blank">S4B Fig</a></b>. (<b>C</b>) Percentage of fms-EGFP⁺ cells that expressed low or high levels of CD45 in control or KA-treated mice determined by flow cytometry (<i>n</i> = 4 per group). (<b>D</b>) Experimental design and representative confocal z-stacks of the hippocampus of CCR2^-/- (CCR2 KO) mice and control WTs (C57BL/6) injected with KA (3 dpi). No obvious differences in the status epilepticus, neuronal damage, microglial morphology, nor in the DG volume (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s017" target="_blank">S4C Fig</a></b>), or neutrophil infiltration were found (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s017" target="_blank">S4D and S4E Fig</a></b>). (<b>E</b>) Number of apoptotic (pyknotic/karyorrhectic) in the septal DG in WT and CCR2 KO mice 3 dpi after KA (<i>n</i> = 4 per group). (<b>F</b>) Ph index in the septal DG (in % of apoptotic cells) in WT and CCR2^-/- mice 3 dpi after KA. (<b>G</b>) Multinuclearity in WT and CCR2^-/- mice. (<b>H</b>) Size of multinucleated cells in WT and CCR2^-/- mice. (<b>I</b>) Weighted Ph capacity in WT and CCR2^-/- mice. Note that the Ph capacity is higher than in our previous time course (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.g004" target="_blank">Fig 4H</a></b>), reflecting an increased number of apoptotic cells in this experiment compared to the previous one, possibly because it was performed in different animal facilities. (<b>J</b>) Weighted PhP (phagoptosis) capacity in the septal DG in WT and CCR2^-/- mice. Data are shown as mean ± SEM. * indicates <i>p</i> < 0.05, ** indicates <i>p</i> < 0.01, and *** indicates <i>p</i> < 0.001 by Holm-Sidak posthoc test, after one-way ANOVA was significant at <i>p</i> < 0.05; only significant interactions are shown. Scale bars = 20 μm (A), 50 μm (D); z = 14.7 μm (A), 12.6 μm (D). Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p

Early phagocytic impairment is related to reduced expression of phagocytosis receptors and reduced motility.

<p>(<b>A</b>) Experimental design and expression of phagocytosis and purinergic receptors by RTqPCR in FACS-sorted microglia from control and KA mice at 1 dpi (<i>n</i> = 3 from 8 pooled hippocampi). HPRT was used as a reference gene. (<b>B</b>) Experimental design and representative projections of 2-photon microscopy images of microglia at t0 (cyan) and 15 min later (magenta) from the DG of controls and KA-treated mice (1 dpi). (<b>C</b>) Motility of microglial processes by 2-photon microscopy in acute slices from CX3CR1^GFP/+ mice after in vivo injection of KA (1 dpi; <i>n</i> = 4–5 cells from 3–4 mice per group). (<b>D</b>) Retraction and protraction of microglial processes by 2-photon microscopy in acute slices from CX3CR1^GFP/+ mice after in vivo injection of KA (1 dpi). (<b>E</b>) Experimental design and representative projections of 2-photon images of microglia at t0 (cyan) and 13.5 min (magenta) in the cortex of controls and KA-treated mice (1 dpi). (<b>F</b>) Motility of microglial processes by 2-photon microscopy in the living cortex of CX3CR1^GFP/+ mice after the injection of KA (1 dpi; <i>n</i> = 6 cells from 3 mice per group). (<b>G</b>) Retraction and protraction of microglial processes by 2-photon microscopy in the living cortex of CX3CR1^GFP/+ mice after the injection of KA. Bars represent mean ± SEM. * indicates <i>p</i> < 0.05, ** indicates <i>p</i> < 0.01, and *** indicates <i>p</i> < 0.001 by Student´s <i>t</i> test (A, C, D). Scale bars = 20 μm (B), 50 mm (E). z = 50 μm (A), 40 μm (B). Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p

Microglial phagocytosis is impaired early (1 dpi) due to MTLE seizures in vivo.

<p>(<b>A</b>) Hippocampal electroencephalographic recordings of mice injected in the ipsilateral side (I) with KA (50 nL, 20 mM) during status epilepticus (0 dpi) and during a spontaneous seizure occurring in the chronic phase of MTLE (49 dpi). The contralateral hippocampus (C) is shown for comparison purposes. (<b>B</b>) Representative confocal z-stacks of saline and KA (1 dpi) hippocampi labeled with DAPI (nuclear morphology, white), activated caspase 3 (act-casp3⁺, red, for apoptotic cells), and fms-EGFP (cyan, microglia). (<b>C</b>) Number of apoptotic cells (pyknotic/karyorrhectic and act-casp3⁺) in the septal DG (<i>n</i> = 3−9 per time point and treatment). The volume of the septal DG is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s016" target="_blank">S3B Fig</a></b>. (<b>D</b>) Representative confocal image of a nonphagocytosed apoptotic (pyknotic and act-casp3⁺, arrowhead) cell in the SGZ (orthogonal projection, left; and 3-D-rendered image, right). M, microglial cell body. (<b>E</b>) Representative 3-D-rendered confocal z-stack of apoptotic (pyknotic and act-casp3⁺) cells, phagocytosed (arrow) or not (arrowheads) in the septal DG of mice treated with KA at 1 dpi. M, microglial cell body. (<b>F</b>) Representative 3-D-rendered confocal z-stack of an apoptotic (pyknotic), nonphagocytosed cells (arrowhead) in the DG of mice treated with KA at 1 dpi. The arrow points to a semiengulfed apoptotic cell. M, microglial cell body. (<b>G</b>) Ph index in the septal DG (in % of apoptotic cells) after KA. Phagocytosis by astrocytes and neuroblasts is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s016" target="_blank">S3C and S3E Fig</a></b>. (<b>H</b>) Weighted Ph capacity of DG microglia (in ppu). (<b>I</b>) Histogram showing the Ph capacity distribution of microglia (in % of cells) in the DG. (<b>J</b>) Total number of microglial cells (fms-EGFP⁺) in the septal DG. Microglial density is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s016" target="_blank">S3A Fig</a></b>. (<b>K</b>) Ph/A coupling (in fold change) in the septal DG. (<b>L</b>) Histogram showing the distribution of the distance (in μm) of apoptotic cells (in %) to microglial processes. The average distance of apoptotic cells to microglia is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s016" target="_blank">S3F Fig</a></b>. Bars represent mean ± SEM except in L, where they indicate the sum of cells in each distance slot. * indicates <i>p</i> < 0.05, ** indicates <i>p</i> < 0.01, and *** indicates <i>p</i> < 0.001 by Holm-Sidak posthoc test after two-way ANOVA (H–K) or one-way ANOVA (C, G, where a significant interaction time x treatment was found) were significant at <i>p</i> < 0.05. Scale bars = 50 μm (B), 10 μm (D–F). z = 25 μm (B), 13.9 μm (D), 14.1 μm (E), 8.4 μm (F). Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p