Experimental Framework to Investigate Glioma in Organotypic Human Cortex

Woltheus, L. R.; Baudouin, L.; van Lingen, M.; Verburg, N.; Idema, S.; Best, M. G.; de Witt Hamer, P.; Noske, D.; Douw, L.; Maas, D.

doi:10.64898/2026.09.21.753089

bioRxiv · 10.64898/2026.09.21.753089

Experimental Framework to Investigate Glioma in Organotypic Human Cortex

Woltheus, L. R.·Baudouin, L.·van Lingen, M.·Verburg, N.·Idema, S.·Best, M. G.·de Witt Hamer, P.·Noske, D.·Douw, L.·Maas, D.

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Abstract

Background Gliomas are primary brain tumors that integrate into the surrounding brain via neuron-glioma synapses and disrupt normal neuronal functioning. Recent work links tumor-brain connectivity to clinical measures such as patients' functional status. However, few experimental systems allow the visualization of tumor and resident brain cell architecture together in human tissue at the scale needed to connect cellular organization to clinical outcomes. To address this gap, we developed an experimental framework for investigating the neuron-glioma network in organotypic human cortex from glioma patients, designed to support future correlation of cellular architecture to clinical outcomes such as functional status and survival. Methods Brain tissue samples containing cortex from seven glioma patients were collected during tumor resection and cultured for seven days as organotypic slices. We established several multiplexed immunohistochemistry panels to stain for glioma cells, various types of resident brain cells including neurons and cells from the oligodendrocyte lineage, as well as axonal networks and myelination patterns. Results We demonstrated the presence of tumor cells in all samples using SOX2, a tumor cell marker. NeuN+ neuronal cells were also successfully identified, as were cells from the oligodendrocyte lineage using SOX10 (pan-lineage marker), BCAS1 (pre-myelinating oligodendrocyte marker), and ASPA (mature oligodendrocyte marker). The neuronal network and myelin were successfully visualized using MAP2 (neurons and dendrites), SMI312 (axons), and PLP1 (myelin). Conclusions High-quality visualization of tumor cells, resident brain cells and the (myelinated) neuronal network in organotypic human cortex from glioma patients is possible after seven days of culturing. Future studies could further optimize culture conditions and should study stability of all cellular components in longer term slice cultures. Preliminary analyses indicate that cellular measures derived from human organotypic slices can be linked to patients' functional status, demonstrating the capacity to generate data suitable for large-scale investigation of the effects of glioma-brain network architecture on clinical outcomes.

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Woltheus, L. R., Baudouin, L., van Lingen, M., Verburg, N., Idema, S., Best, M. G., de Witt Hamer, P., Noske, D., Douw, L., Maas, D.. 2026-09-25. Experimental Framework to Investigate Glioma in Organotypic Human Cortex. https://doi.org/10.64898/2026.09.21.753089

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