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Updated: Jan 19, 2026
A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons
Published on: August 6, 2020
Chemically induced neurite-like outgrowth reveals a multicellular network function in patient-derived glioblastoma
Barbara da Silva1, Bronwyn K Irving1, Euan S Polson1
1School of Medicine, University of Leeds, Leeds, LS2 9JT, UK.
Abstract:
Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of glioblastoma multiforme (GBM), the most aggressive type of brain cancer in adults. Here, we show that small-molecule inhibition of RHO-associated serine/threonine kinase proteins (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed de novo-induced cellular network (iNet) 'webs', which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range Ca2+ signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs ex vivo, and may thereby accelerate future studies into the biology of GBM cellular networks.
Insights
Small-molecule inhibition of RHO-associated kinase (ROCK) proteins promotes radiation-resistant glioblastoma networks. These networks, termed induced cellular networks (iNets), form reversible webs of connected cells, offering new research avenues for brain cancer therapy.
Area of Science:
- Neuroscience
- Cancer Biology
- Cellular Biology
Background:
- Glioblastoma multiforme (GBM) exhibits therapeutic resistance, linked to tumor stem cells and multicellular networks.
- Understanding GBM resistance mechanisms is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the effect of RHO-associated serine/threonine kinase inhibition (ROCKi) on GBM stem-like cell networks.
- To determine if ROCKi influences the formation of radiation-resistant cellular networks.
Main Methods:
- Utilized patient-derived GBM stem-like cells in culture.
- Administered small-molecule inhibitors of RHO-associated serine/threonine kinase proteins (ROCKi).
- Assessed cell projection dynamics, network formation (iNets), calcium signaling, trafficking, and radiation resistance.
Main Results:
- ROCKi promoted neurite-like projections and formed reversible, de novo-induced cellular network (iNet) 'webs'.
- Connected iNet cells showed long-range calcium signaling and significant lysosomal/mitochondrial trafficking.
- iNet cells demonstrated enhanced viability and proliferation after high-dose radiation compared to controls.
Conclusions:
- ROCKi regulates cell projection dynamics, leading to the formation of radiation-resistant multicellular networks in GBM.
- ROCKi provides a method to reversibly induce iNet webs ex vivo, facilitating GBM network biology research.
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