Protein kinases control most cellular processes and aberrant kinase activity is involved in numerous diseases. Here we introduce molecular recorders of kinase activities for later analysis to investigate the link between specific kinase activities and cellular phenotypes in heterogeneous cell populations and in vivo. Based on split-HaloTag and a phosphorylation-dependent molecular switch, our recorders become rapidly labeled in the presence of a specific kinase activity and a fluorescent HaloTag substrate. The kinase activity in a given cell controls the degree of fluorescent labeling, whereas the recording window is set by the presence of the fluorescent substrate. We designed specific recorders for four protein kinases, including protein kinase A. We apply our protein kinase A recorder to sort heterogeneous cell populations for subsequent transcriptome analysis, in genome-wide CRISPR screens to discover regulators of PKA activity and to track neuromodulation in freely moving mice.

Abstract BACKGROUND Aggressive ependymoma (EPN) exhibit primary and secondary radio- and chemotherapy resistance with the poorest prognosis in the pediatric groups PF-EPN-A and ST-EPN-ZFTA. Recently, tumor cell networks formed by membrane protrusions, such as tunneling nanotubes (TNT) or tumor microtubes (TM), have been identified in other glial brain tumors. These structures facilitate intercellular hierarchical Ca2+-communication and contribute to therapy resistance. Previous data from our group suggested enhancer-regulated Ca2+-communication to be essential in EPN. This study aims to further characterize intratumoral interactions and to understand potential correlations between tumor cell connections and treatment resistance in EPN. METHODS Intercellular connections in EPN patients and model systems were characterized by scanning electron microscopy, immunofluorescence and ultra-high content imaging. Transcriptomic and proteomic data were analyzed to identify potential targetable vulnerabilities within network dynamics. Candidates were functionally validated in cell culture models applying Ca2+ live cell imaging. RESULTS Connectivity-related terms such as microtube-based movement or microtube bundle formation showed increased expression in EPN transcriptome data compared to healthy controls. EPN networks were found to be build of TM- and TNT-like structures with a higher abundance of TNT-like structures in PF-EPN-A compared to ST-EPN-ZFTA. All networks showed strong Nestin-positivity in cells, mouse models and human tumor tissue of ST-EPN-ZFTA and PF-EPN-A. Thrombospondin-1, a matricellular protein enhancing TMs in glioma cells, was strongly enriched in ST-EPN-ZFTA patients. The gap junction channel connexin 43, connecting glioma cells, was highly upregulated in PF-EPN-A. Active electrochemical communication was observed between ST-EPN-ZFTA cells performing Ca2+ imaging. Moreover, inhibition of T-type Ca2+ channels impaired intercellular signaling, leading to network perturbation in vitro. CONCLUSIONS Our study revealed the importance of gap junction-coupled networks for communication of aggressive EPN cells. Targeted disconnection of the EPN-specific network as demonstrated for Ca2+ channel inhibition suggests a new avenue for therapeutic strategies in EPN to overcome resistance.

This study investigates the biological effect of Tumor Treating Fields (TTFields) on key drivers of glioblastoma’s malignancy—tumor microtube (TM) formation—and on the function and overall integrity of the tumor cell network. Using a two-dimensional monoculture GB cell network model (2DTM) of primary glioblastoma cell (GBC) cultures (S24, BG5 or T269), we evaluated the effects of TTFields on cell density, interconnectivity and structural integrity of the tumor network. We also analyzed calcium (Ca2+) transient dynamics and network morphology, validating findings in patient-derived tumoroids and brain tumor organoids. In the 2DTM assay, TTFields reduced cell density by 85–88

Tumor microtubes (TMs) connect glioma cells to a network with considerable relevance for tumor progression and therapy resistance. However, the determination of TM-interconnectivity in individual tumors is challenging and the impact on patient survival unresolved. Here, we establish a connectivity signature from single-cell RNA-sequenced (scRNA-Seq) xenografted primary glioblastoma (GB) cells using a dye uptake methodology, and validate it with recording of cellular calcium epochs and clinical correlations. Astrocyte-like and mesenchymal-like GB cells have the highest connectivity signature scores in scRNA-sequenced patient-derived xenografts and patient samples. In large GB cohorts, TM-network connectivity correlates with the mesenchymal subtype and dismal patient survival. CHI3L1 gene expression serves as a robust molecular marker of connectivity and functionally influences TM networks. The connectivity signature allows insights into brain tumor biology, provides a proof-of-principle that tumor cell TM-connectivity is relevant for patients’ prognosis, and serves as a robust prognostic biomarker.

Background Gliomas are highly invasive brain neoplasms. MRI is the most important tool to diagnose and monitor glioma but has shortcomings. In particular, the assessment of tumor cell invasion is insufficient. This is a clinical dilemma, as recurrence can arise from MRI‐occult glioma cell invasion. Hypothesis Tumor cell invasion, tumor growth and radiotherapy alter the brain parenchymal microstructure and thus are assessable by diffusion tensor imaging (DTI) and MR elastography (MRE). Study Type Experimental, animal model. Animal Model Twenty‐three male NMRI nude mice orthotopically implanted with S24 patient‐derived glioma cells (experimental mice) and 9 NMRI nude mice stereotactically injected with 1 μL PBS (sham‐injected mice). Field Strength/Sequence 2D and 3D T2‐weighted rapid acquisition with refocused echoes (RARE), 2D echo planar imaging (EPI) DTI, 2D multi‐slice multi‐echo (MSME) T2 relaxometry, 3D MSME MRE at 900 Hz acquired at 9.4 T (675 mT/m gradient strength). Assessment Longitudinal 4‐weekly imaging was performed for up to 4 months. Tumor volume was assessed in experimental mice (n = 10 treatment‐control, n = 13 radiotherapy). The radiotherapy subgroup and 5 sham‐injected mice underwent irradiation (3 × 6 Gy) 9 weeks post‐implantation/sham injection. MRI‐/MRE‐parameters were assessed in the corpus callosum and tumor core/injection tract. Imaging data were correlated to light sheet microscopy (LSM) and histology. Statistical Tests Paired and unpaired t ‐tests, a P ‐value ≤0.05 was considered significant. Results From week 4 to 8, a significant callosal stiffening (4.44 ± 0.22 vs. 5.31 ± 0.29 kPa) was detected correlating with LSM‐proven tumor cell invasion. This was occult to all other imaging metrics. Histologically proven tissue destruction in the tumor core led to an increased T2 relaxation time (41.65 ± 0.34 vs. 44.83 ± 0.66 msec) and ADC (610.2 ± 12.27 vs. 711.2 ± 13.42 × 10 −6 mm 2 /s) and a softening (5.51 ± 0.30 vs. 4.24 ± 0.29 kPa) from week 8 to 12. Radiotherapy slowed tumor progression. Data Conclusion MRE is promising for the assessment of key glioma characteristics. Evidence Level NA Technical Efficacy Stage 2

Diffuse infiltrating gliomas, especially glioblastomas, are the most common incurable primary brain tumors in adults. The aggressive growth and high resistance against cytotoxic agents are mediated by ultra-long tubular membrane protrusions, Tumor Microtubes (TMs), that interconnect single glioblastoma cells via gap junctions to multicellular communicating and resistant networks. Early studies showed that targeting these malignant networks increases response to therapy. Thus, the disconnection of such networks has emerged as a new therapeutic strategy to decrease radioresistance. However, the development of drugs that interfere with TM- and network formation is compromised by the lack of established drug screening pipelines to study a compound‘s specific anti-TM and network-inhibiting activity in combination with radiotherapy. To identify compounds with anti-TM activity, we first developed an in vitro medium-throughput imaging-based drug screening. Human-derived primary glioblastoma cells were seeded in a newly developed 2D monolayer where tumor cells form TMs and networks. The cells were treated with compounds with potential anti-TM activity. Using high-resolution laser scanning microscopy, morphological readouts were obtained and analyzed. The most promising compounds were then evaluated in a xenograft chronic cranial window mouse model. Treatments were administered with concomitant radiotherapy (6Gy per day for 3 days). By observing TM and tumor cell network changes via longitudinal in vivo 2-photon microscopy, tumor cell count, growth, and TM parameters were measured in the live mouse over up to 12 weeks. Among the 87 compounds tested in vitro, 42 significantly inhibited TMs. Two of these hits showed a higher count of dead cells in combination with radiotherapy as opposed to glioblastoma cells treated with radiotherapy alone and were therefore studied in the in vivo pipeline. One of these two agents showed reductions of TM length and striking responses in a combinatorial treatment regime with radiotherapy in individual mice that were not observed in control animals treated with radiotherapy only; however, the responses observed were heterogeneous between regions and mice. Further studies revealed this compound directly phosphorylates the gap junction protein Connexin 43 on S368, which contributes to a decrease in intercellular communication of glioblastoma cells. By applying different imaging techniques and AI algorithms we successfully established a novel in-vitro-to-in-vivo drug screening pipeline for the development of disconnecting agents. Most importantly, our data shows that disconnecting multicellular brain tumor networks increases response to radiotherapy. Further studies will be needed to develop reliable anti-TM agents as novel co-treatment options to sensitize glioblastomas to cytotoxic therapy. Citation Format: Daniel D. Azorín, Sophie Weil, Dirk C. Hoffmann, David Hausmann, Erik Jung, Matthias Osswald, Jill Reckless, Nigel Ramsden, Simon Thompson, David Grainger, Wolfgang Wick, Frank Winkler. AI-aided drug development for disconnecting glioma tumor microtube networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1084.

Diffuse gliomas, particularly glioblastomas, are incurable brain tumours 1 . They are characterized by networks of interconnected brain tumour cells that communicate via Ca 2+ transients 2 – 6 . However, the networks’ architecture and communication strategy and how these influence tumour biology remain unknown. Here we describe how glioblastoma cell networks include a small, plastic population of highly active glioblastoma cells that display rhythmic Ca 2+ oscillations and are particularly connected to others. Their autonomous periodic Ca 2+ transients preceded Ca 2+ transients of other network-connected cells, activating the frequency-dependent MAPK and NF-κB pathways. Mathematical network analysis revealed that glioblastoma network topology follows scale-free and small-world properties, with periodic tumour cells frequently located in network hubs. This network design enabled resistance against random damage but was vulnerable to losing its key hubs. Targeting of autonomous rhythmic activity by selective physical ablation of periodic tumour cells or by genetic or pharmacological interference with the potassium channel KCa3.1 (also known as IK1, SK4 or KCNN4) strongly compromised global network communication. This led to a marked reduction of tumour cell viability within the entire network, reduced tumour growth in mice and extended animal survival. The dependency of glioblastoma networks on periodic Ca 2+ activity generates a vulnerability 7 that can be exploited for the development of novel therapies, such as with KCa3.1-inhibiting drugs.

Glioblastoma is a particularly challenging disease characterized by the connection of tumor cells to functional multicellular networks that effectively resist therapies. In this issue of Biochemical Journal, Pinto et al. report the discovery of two distinct classes of intercellular membrane tube connections, tunneling nanotubes and tumor microtubes, in the same state-of-the-art culture model of patient-derived glioblastoma material. These findings contribute to our understanding of the heterogeneity of intercellular membrane tubes in health and disease, and pave the way for future functional studies on their various roles for disease progression and tumor resistance.

Background. Glioma is sensitive to microtubule-targeting agents (MTAs), but most MTAs do not cross the blood brain barrier (BBB). To address this limitation, we developed the new chemical entity, ST-401, a brain-penetrant MTA. Methods. Synthesis of ST-401. Measures of MT assembly and dynamics. Cell proliferation and viability of patient-derived (PD) glioma in culture. Measure of tumor microtube (TM) parameters using immunofluorescence analysis and machine learning-based workflow. Pharmacokinetics (PK) and experimental toxicity in mice. In vivo antitumor activity in the RCAS/tv-a PDGFB-driven glioma (PDGFB-glioma) mouse model. Results. We discovered that ST-401 disrupts microtubule (MT) function through gentle and reverisible reduction in MT assembly that triggers mitotic delay and cell death in interphase. ST-401 inhibits the formation ofTMs, MT-rich structures that connect glioma to a network that promotes resistance to DNA damage. PK analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft flank tumor mouse model demonstrates significant antitumor activity and no over toxicity in mice. In the PDGFB-glioma mouse model, ST-401 enhances the therapeutic efficacies of temozolomide (TMZ) and radiation therapy (RT). Conclusion. Our study identifies hallmarks of glioma tumorigenesis that are sensitive to MTAs and reports ST-401 as a promising chemical scaffold to develop brain-penetrant MTAs.

Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that the loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by the inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, the drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type (WT) cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.