Meclofenamate causes loss of cellular tethering and decoupling of functional networks in glioblastoma Matthias Schneider , Anna-Laura Pottho , Lea Vollmer, Vidhya Ravi, Andreas Dolf, Erdem Güresir , Patrick Schuss , Jürgen Beck, Torsten Pietsch, Mike-Andrew Westho, Hartmut Vatter , Oliver Schnell, Andreas Waha, Ulrich Herrlinger †, Dieter Henrik Heiland †

Introduction 2 MFA causes reduction of NCAM and NETRIN 4 Reduced connectivity due to MFA treatment signaling results in profound TMZ sensitivity Glioblastoma cells assemble to a syncytial communicating network based on ultra-long RNA-sequencing of three primary glioblastoma cell populations revealed 163 genes that A tumor cell exposed to gap junction-targeted therapies - therefore deprived of crucial in- membrane protrusions - so-called tumor microtubes (TMs). On the microscopical level, are commonly differentially expressed (Fig. 2A). A gene-set enrichment analysis identified tercellular microtubes as well as functional networking to neighboring cells - is supposed these TMs are supported by intercellular connexin-43 (Cx-43)-based gap junctions. Inhibi- a significant reduction of two pathways that are involved in neural development and to be more susceptible to chemotherapy-induced antitumoral effects. Specific DNA-frag- tion of intercellular networking via TMs is considered a promising therapeutic approach re- cell-cell connections: the neural cell adhesion molecule (NCAM) pathway and the NET- mentation of propidium iodide-stained nuclei was assessed as readout for cell death using sulting in functional and morphological isolation of tumor cells within the communication RIN-signaling pathway that is involved in axon guidance (Fig. 2B). NCAM1, a major import- flow cytometric analyses (Fig. 4A). Additional administration of MFA profoundly increased syncytium. However, up to date, no clinical feasibale drug has reached clinical practice. ant adhesion molecule in the brain was significantly downregulated as well as SEMA3A, a DNA-fragmentation rates seen for TMZ single treatment (Fig. 4B,C). Analysis of morpholo- Meclofenamate (MFA) as a FDA-approved NSAID has been reported to inhibit intercellular protein which is required for proper motor neuron and sensory neuron circuit organization gical tumor networks in the neocortical glioblastoma slice model (Fig. 4D) confirmed a bre- communication via gap junctions in several physiological cell entities. Here we analyzed (Fig. 2C). akdown of tumor microtube length (Fig. 4D,F) as well as a significant reduction of tumor p=0.02 MFA for potential effects on the TM-based malignant glioblastoma network. volumes for combination versus temozolomide single treatment (Fig 4G). A BTSC#233 B 0.6 C NCAM p<0.01 0.4 Ctr MFA TMZ TMZ+MFA BTSC#G35 n=452 NETRIN A treatment B C 0.3 4.0 80 **** p-adj<0.01 0.0

Score 60 Genes n=163 -0.2 n=301 log SSC-A log SSC-A log SSC-A log SSC-A Enrichment 2.0 Results 40 n=346 PI staining -0.4 log PI-A 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 20

Differential Expresses 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 -0.6 FACS ** 0.0 Specific analysis 0 BTSC#G35 BTSC#168 NCAM1 SEMA3A 2% 4% 34% -20 1 DNA-fragmentation (%) MFA inhibits intercellular gap-junction- -40 Counts Counts Counts Counts 65% 3 MFA demolishes tumor microtube-based A Ctr Laser MF TMZ TMZ mediated cytosolic traffic +MFA intercellular network morphology Cytosol-traffic within tumor networks was quantified by measuring the fluorescence-guided D E BTSC#233 F **** G In order to address the question to what extent MFA might impair tumor microtube formati- 300 1.0 cell-to-cell cytoplasm transfer of calcein that is only able to spread through Cx43 coupled

Primary olu me 0.8 on and therefore alter spatial glioblastoma network architecture morphology, glioblastoma TMZ Tumor Cells **** **** cells (Fig. 1A). MFA, as a potent gap junction inhibitor, significantly reduced the redistributi- Lentiviral Infection 200 0.6

cell populations were injected into human cortical brain slices (Fig. 3A). MFA treatment re- ZsGreen umor V on of calcein from „donor“ cells to „receiver“ cells after up to 150 minutes (Fig. 1B). 0.4 ive T sulted in a breakdown of intercellular network architectures (Fig. 3A,B), which was reflec- A 100 0.2

A B ted by a reduction in the number of tumor microtube-based intercellular connections per Rela t Microtube Length (µm) 0.0 0min +30min +60min +100min +160min Flourescence Ctr 1.8 TMZ+M F 0 Intensity cell (Fig. 3C). Meclofenamate Tumor Injection TMZ TMZ Ctr 1.0 *** Ctr MFA TMZ TMZ+MFA +MFA 1.6 Ctr MFA 0.8 *** Ctr A B C 1.4 ** 0.5 Ctr MFA 0.6 ** 50 µM 0.5 0.4 0.4 0.4 1.2 Ctr (Fold Change) 0.3 0.3 10 Conclusions 100 µM **

0.2 Z-Axis 0.2 0.2 Z-Axis 8 Increase of Receiver Cells 1.0 0.1 0.1 6 Meclofenamate 0 30 60 90 120 150 MFA effectively demolishes functional and morphological TM-based syncytial network ar- 100µm 0.0 0.0 0.0 Time (min)

Average 4

0.3 0.3 Connection chitectures. With regard to MFA as a clinically-approved drug, MFA might harbour the po- 0.2 0.2 2 MFA 0.1 −0.1 −0.1 0.1 tential of bridging the idea of a TM-targeted therapeutic approach from bench-to-bedside. 0.0 0.0 0.0 0.0 0 0.1 −0.1 Y-Axis 0.1 −0.1 Y-Axis X-Axis 0.20.3 −0.2 X-Axis 0.20.3 −0.2 Day 1 Day 7 A german-wide phase I/II clinical trial is currently being initiated.

Department of Neurosurgery, Rheinische Friedrich-Wilhelms-University Hospital, Bonn, Germany, Department of Neuropathology, Rheinische Friedrich-Wilhelms-University Hospital, Bonn, Germany, Translational NeuroOncology Research Group, University of Freiburg, Freiburg im Breisgau, Germany, Institute of Experimental Immunology, Rheinische Friedrich-Wilhelms-University Hospital, Bonn, Germany, Department of Neurosurgery, University of Freiburg, Freiburg im Breisgau, Germany, Department of Pediatrics and Adolescent Medicine, University Medical Center Ulm, Ulm, Germany, Division of Clinical Neurooncology, Department of Neurology, Rheinische Friedrich-Wil- helms-University Hospital, Bonn, Germany † contributed equally March 2021