Tumor cell network integration in glioma represents a stemness feature

BACKGROUND: Malignant gliomas including glioblastomas are characterized by a striking cellular heterogeneity, which includes a subpopulation of glioma cells that becomes highly resistant by integration into tumor microtube (TM)-connected multicellular networks. METHODS: A novel functional approach to detect, isolate, and characterize glioma cell subpopulations with respect to in vivo network integration is established, combining a dye staining method with intravital two-photon microscopy, Fluorescence-Activated Cell Sorting (FACS), molecular profiling, and gene reporter studies. RESULTS: Glioblastoma cells that are part of the TM-connected tumor network show activated neurodevelopmental and glioma progression gene expression pathways. Importantly, many of them revealed profiles indicative of increased cellular stemness, including high expression of nestin. TM-connected glioblastoma cells also had a higher potential for reinitiation of brain tumor growth. Long-term tracking of tumor cell nestin expression in vivo revealed a stronger TM network integration and higher radioresistance of the nestin-high subpopulation. Glioblastoma cells that were both nestin-high and network-integrated were particularly able to adapt to radiotherapy with increased TM formation. CONCLUSION: Multiple stem-like features are strongly enriched in a fraction of network-integrated glioma cells, explaining their particular resilience

Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS

Differential Effects of Ang-2/VEGF-A Inhibiting Antibodies in Combination with Radio- or Chemotherapy in Glioma

Antiangiogenic strategies have not shown striking antitumor activities in the majority of glioma patients so far. It is unclear which antiangiogenic combination regimen with standard therapy is most effective. Therefore, we compared anti-VEGF-A, anti-Ang2, and bispecific anti-Ang-2/VEGF-A antibody treatments, alone and in combination with radio- or temozolomide (TMZ) chemotherapy, in a malignant glioma model using multiparameter two-photon in vivo microscopy in mice. We demonstrate that anti-Ang-2/VEGF-A lead to the strongest vascular changes, including vascular normalization, both as monotherapy and when combined with chemotherapy. The latter was accompanied by the most effective chemotherapy-induced death of cancer cells and diminished tumor growth. This was most probably due to a better tumor distribution of the drug, decreased tumor cell motility, and decreased formation of resistance-associated tumor microtubes. Remarkably, all these parameters where reverted when radiotherapy was chosen as combination partner for anti-Ang-2/VEGF-A. In contrast, the best combination partner for radiotherapy was anti-VEGF-A. In conclusion, while TMZ chemotherapy benefits most from combination with anti-Ang-2/VEGF-A, radiotherapy does from anti-VEGF-A. The findings imply that uninformed combination regimens of antiangiogenic and cytotoxic therapies should be avoided

Inhibition of MMP-3 does not prevent NMDA receptor removal from synapses induced by PKC activation.

<p>(A) At DIV26, cultured spinal neurons were fixed and stained with an NR1-S2-specific antibody (green) and a synaptophysin-specific antibody (red), revealing a large number of postsynaptic NR1 puncta (yellow). Scalebar is 10 µm. (B) At DIV26, cultured spinal neurons were stimulated with 100 nM PMA for 30 min in medium supplemented with NNGH to inhibit MMP-3 activity, fixed and stained with an NR1-S2-specific antibody (green) and a synaptophysin-specific antibody (red), revealing a strong reduction of postsynaptic NR-S2 puncta (yellow) upon PKC activation. Scalebar is 10 µm. (C) At DIV26, cultured spinal neurons were stimulated with 100 nM PMA in medium supplemented with NNGH to inhibit MMP-3 activity, fixed and stained with an NR1-C2-specific antibody (green) and a synaptophysin-specific antibody (red), revealing also a strong reduction of postsynaptic NR1-C2-specific puncta (yellow) upon PKC activation. Scalebar is 10 µm.</p

Analysis of MMP-3 cleavage sites by site-directed mutagenesis.

<p>(A) Immunoblot of S1S2 and mutant S1S2Ser542 protein fragments. S1S2 and mutant S1S2Ser542 proteins were purified, incubated with active MMP-3 and separated by SDS-PAGE. Immunoblotting with anti-S1S2 antibody revealed that the mutations abolished both the appearence of the 20 kDa and 18 kDa fragments (indicated by arrows), suggesting that both fragments are generated from the same <i>N</i>-terminal MMP-3 cleavage site. (B) Immunoblot of separated protein fragments generated upon MMP-3 digestion of S1S2- and mutant S1S2Leu790 proteins. Note the presence of three protein double bands of 40/38 kDa, 26/24 kDa and 20/18 kDa in the S1S2 protein digestion pattern (left), which are absent in the digestion pattern with mutant S1S2Leu790 protein (indicated by arrows). Note that the cleavage site generating the 20 kDa protein fragment (indicated by star) is not effected by this mutation, lower panel: mutated MMP-3 cleavage site of the S1S2Leu790 protein.</p

Expression of MMP-3 in cultured spinal cord neurons.

<p>(A) At DIV20, cultured spinal cord were fixed and stained with an MMP-3-specific antibody, revealing MMP-3 expression in soma, nucleus and neurites of spinal cord neurons. Scalebar is 15 µm. (B) Control experiments without first antibody using Cy3-conjugated anti-rabbit antibody. (C) Control experiments with anti-MMP-3 antibody which was preabsorbed with MMP-3 protein. Pictures (A–C) were taken under the same conditions (D) PSD-95-immunofluorescence of spinal cord neurons. (E) MMP-3-immunoreactivity of neurons shown in D. (F) Overlay of PSD-95 immunoreactivity (green) and MMP-3 immunoreactivity (red). Boxed region is shown as enlargement in G. (G) Enlargement of dendritic region shown in F. Arrowheads indicate accumulations of MMP-3 immunoreactivities within a dendrite. Arrow indicates overlapping PSD-95 and MMP-3-immunoreactivities. Scalebar is 15 µm (H) Control experiments using anti-mouse antibody conjugated with Cy2. Scalebar is 15 µm.</p

Analysis of MMP-3 and NR1 protein expression and digestion in cultured spinal cord neurons.

<p>(A) Upper panel: Solubilized proteins from rat brain membranes were incubated with MMP-3 proprotein and activated MMP-3 in the absence or presence of MMP-3 inhibitor NNGH and analysed by immunoblotting with a NR1-C2′-specific antibody. MMP-3 was activated by aminophenylmercuric acetate (APMA). Digestion of NR1 was prevented by the MMP-inhibitor NNGH. Lower panel: Immunoblot of the same probes with a synaptophysin-specific antibody (Syn.) revealing no strong degradation of this protein. (B) Immunoblot of S1S2M digestion assays (15h) with an MMP-3-specific antibody, demonstrating the presence of the active form of MMP-3 (45/44 kDa). (C) MMP-3 immunoblot of total protein extracts of cultured spinal cord neurons (DIV26) of control cells (lane 1) and cells treated with NMDA (4 days, 10 µM) (lane 2); for comparison, MMP-3 pro-protein (10 ng) was separated in parallel on the gel (lane 3). Lower panel: Immunoblot of the same gel as above probed with a β-tubulin antibody (loading control). (D) Detection of a 60 kDa proteolytic NR1 polypeptide on NR1 immunoblots (left panel, lanes 1–5; and right panel lanes 6–8). NR1 proteins were detected after separation of membrane proteins with SDS-PAGE and blotting using isolated membranes from spinal cord neurons cultured under control conditions (lane 3 and lane 6), from neurons treated with NMDA in the absence (lane 4 and lane 7), and presence of MMP-3 inhibitor NNGH (lane 5 and lane 8). The left panel was blotted with Na₂-tetraborate buffer (Materials). Notice the clear detection of a protein band of about 60 kDa and the incomplete transfer of marker proteins larger than 50 kDalton (Magic Marker, Invitrogen, containing a protein domain detected by the secondary antibody). The right panel was blotted with methanol-containing buffer. Notice the clearer detection of an undigested NR1 protein band under these blotting conditions. Lanes 1 and 2 are loaded with protein molecular mass standards.</p

Supplemental Material for Ratliff et al., 2018

Supplemental Figures S1-S5<br>Table S1: mib-1 mutations<br>Table S2: Primers Used for PCR and CRISPR Genome Engineering<br>Movies S1-S4<br

CDKN2A/B homozygous deletion is associated with early recurrence in meningiomas

Soon after birth, the regenerative capacity of the mammalian heart is lost, cardiomyocytes withdraw from the cell cycle and demonstrate a minimal proliferation rate. Despite improved treatment and reperfusion strategies, the uncompensated cardiomyocyte loss during injury and disease results in cardiac remodeling and subsequent heart failure. The promising field of regenerative medicine aims to restore both the structure and function of damaged tissue through modulation of cellular processes and regulatory mechanisms involved in cardiac cell cycle arrest to boost cardiomyocyte proliferation. Non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are functional RNA molecules with no protein-coding function that have been reported to engage in cardiac regeneration and repair. In this review, we summarize the current understanding of both the biological functions and molecular mechanisms of ncRNAs involved in cardiomyocyte proliferation. Furthermore, we discuss their impact on the structure and contractile function of the heart in health and disease and their application for therapeutic interventions

PerSurge (NOA-30) phase II trial of perampanel treatment around surgery in patients with progressive glioblastoma

Abstract Background Glioblastoma is the most frequent and a particularly malignant primary brain tumor with no efficacy-proven standard therapy for recurrence. It has recently been discovered that excitatory synapses of the AMPA-receptor subtype form between non-malignant brain neurons and tumor cells. This neuron-tumor network connectivity contributed to glioma progression and could be efficiently targeted with the EMA/FDA approved antiepileptic AMPA receptor inhibitor perampanel in preclinical studies. The PerSurge trial was designed to test the clinical potential of perampanel to reduce tumor cell network connectivity and tumor growth with an extended window-of-opportunity concept. Methods PerSurge is a phase IIa clinical and translational treatment study around surgical resection of progressive or recurrent glioblastoma. In this multicenter, 2-arm parallel-group, double-blind superiority trial, patients are 1:1 randomized to either receive placebo or perampanel (n = 66 in total). It consists of a treatment and observation period of 60 days per patient, starting 30 days before a planned surgical resection, which itself is not part of the study interventions. Only patients with an expected safe waiting interval are included, and a safety MRI is performed. Tumor cell network connectivity from resected tumor tissue on single cell transcriptome level as well as AI-based assessment of tumor growth dynamics in T2/FLAIR MRI scans before resection will be analyzed as the co-primary endpoints. Secondary endpoints will include further imaging parameters such as pre- and postsurgical contrast enhanced MRI scans, postsurgical T2/FLAIR MRI scans, quality of life, cognitive testing, overall and progression-free survival as well as frequency of epileptic seizures. Further translational research will focus on additional biological aspects of neuron-tumor connectivity. Discussion This trial is set up to assess first indications of clinical efficacy and tolerability of perampanel in recurrent glioblastoma, a repurposed drug which inhibits neuron-glioma synapses and thereby glioblastoma growth in preclinical models. If perampanel proved to be successful in the clinical setting, it would provide the first evidence that interference with neuron-cancer interactions may indeed lead to a benefit for patients, which would lay the foundation for a larger confirmatory trial in the future. Trial registration EU-CT number: 2023-503938-52-00 30.11.2023