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. 2026 May 6;6(3):623–628. doi: 10.1021/acsmeasuresciau.6c00051

Observation of Acetylcholine Secretion on Living Glioblastoma Cells

Conrad B Milton †, Mahmuda Akter †, Olivia M Kawakita †, Siao-Han Huang †, Mei Shen †,‡,*
PMCID: PMC13281179  PMID: 42326849

Abstract

Glioblastoma, as the most common malignant brain tumor in humans, remains a big societal challenge; identifying new signaling molecules in glioblastoma opens avenues for alternative treatment strategies. Emerging evidence suggests that neurochemical signaling is involved in glioblastoma pathology. However, it is unknown if the neurotransmitter acetylcholine can be secreted by glioblastoma cells despite the expression of the acetylcholine synthesis enzyme. Here, we tested our hypothesis that glioblastoma cells secreted acetylcholine using a nanoelectrochemistry platform: scanning electrochemical microscopy (SECM) and nanoelectrodes. Linear and selective acetylcholine detection was achieved on the nano interface between two immiscible electrolyte solutions (nanoITIES, also called liquid/liquid interface). Using SECM, we positioned the acetylcholine-sensing nanoITIES electrodes very close (i.e., hundreds of nanometers) to the living T98G glioblastoma cells. We observed real-time secretion of acetylcholine from glioblastoma cells. We quantified the number of secreted acetylcholine molecules. Our discovery that glioblastoma cells secrete acetylcholine reveals a novel signaling axis in glioblastoma biology, highlighting a potential therapeutic target for glioblastoma treatment.

Keywords: Glioblastoma, Scanning electrochemical microscopy (SECM), Nanoelectrochemistry, Acetylcholine, Interface between two immiscible electrolyte solutions (ITIES), Immunocytochemistry, Single cell

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Introduction

Signaling molecules play vital roles in glioblastoma multiforme (GBM) biology; identifying new signaling molecules opens avenues for new strategies to intervene and treat GBM. Expression of acetylcholine receptors (AChRs) and the functionality of these receptors have been demonstrated in GBM cells; AChRs were upregulated in active infiltration zones in GBMs. In addition, choline acetyltransferase, the enzyme for acetylcholine synthesis , was expressed in GBMs, suggesting that these tumors may synthesize acetylcholine. Furthermore, vesicular acetylcholine transporter, which neurons use to package acetylcholine into vesicles for exocytosis, was expressed in GBMs. , The ability of GBM cells to acquire neuron-like properties while retaining some glial features has been suggested. For instance, action potential-generating cells were found in human glioblastomas by electrophysiological analysis of cells from surgically resected human GBM. While these suggest GBMs may be able to secrete acetylcholine, it remains unknown whether GBM cells secrete acetylcholine due to technical challenges of measuring acetylcholine secretion in real time from living GBM cells. These challenges arise from the small size of GBM cells (<100 μm, Figure S1A), the need for rapid sensing platforms, and the fact that acetylcholine is redox-inactive.

Elucidating acetylcholine signaling on living GBM cells is vital to understanding this most aggressive brain tumor. Emerging acetylcholine sensors, e.g., fiber photometry/genetically encoded fluorescence sensors, typically have submillimeter spatial resolution, making them unsuitable for high spatial resolution measurements from a single GBM cell. Advances in nano- and microscale electrochemical measurements have enabled a better understanding of biological processes. − Interface between two immiscible electrolyte solutions (ITIES) electrodes have emerged as a unique approach in chemical analysis due to their sensitivity to both redox-active and redox-inactive analytes. − The current response at the ITIES is generated from the transfer of ions across a polarized/electrified interface. ,− The generated Faradaic current is used for quantitation, while the identity of ions determines the transfer potential that needs to be applied. While nanoITIES electrodes have been employed by us to study acetylcholine release in neuronal systems, here we introduce nanoITIES electrodes for probing acetylcholine signaling in non-neuronal systems for the first time.

Scanning electrochemical microscopy (SECM) is a well-established technique for positioning ultramicroelectrodes with high spatial resolution down to nanometers for studies of biological and other systems. − Coupling nanoITIES-based detection with SECM enables probing of cellular processes with enhanced signal-to-noise ratios, detection of real-time spatiotemporal dynamics, etc. ,, To test our hypothesis of acetylcholine secretion from living T98G GBM cells, we introduce a novel nanoelectrochemistry approach coupling SECM and nanoITIES electrodes to the field of cancer-biology. We first positioned the nanoITIES electrode within hundreds of nanometers of the living T98G GBM cell using SECM, allowing acetylcholine secretion to be measured at a high signal-to-noise ratio before dilution due to diffusion away from secretion sites. − After that, we used chronoamperometry (current–time trace) to quantify the real-time secretion of acetylcholine. Our measurements suggest that GBM cells secrete acetylcholine.

Results and Discussion

It is unknown whether GBM cells secrete acetylcholine. Here, we employed nanoITIES electrodes to measure real-time acetylcholine secretion dynamics from living GBM cells for the first time. The fabrication and characterization of nanoITIES electrodes were detailed in the Supporting Information (SI). We used T98G cells, commonly used in cancer research, as a GBM model for this study. After culturing, T98G cells were characterized by optical imaging of morphology (Figures S1 and S2) and immunocytochemistry staining for cholinergic markers (Figure A,B, Figure S3). After that, nano-resolved SECM coupled with a side-view optical microscope (detailed in the SI) was used to position the nanoITIES electrode for acetylcholine detection within hundreds of nanometers of a living T98G GBM cell. We used the electrochemical signal from the transfer of cations in the cellular medium as a noninvasive mediator to do SECM approach curves, as reported previously. Following that, we used chronoamperometry (current–time trace) at a potential selective for acetylcholine detection (E ACh) to measure the real-time secretion of acetylcholine from T98G GBM cells in response to high-concentration K+ stimulation, commonly used in neuroscience to stimulate the release of neurotransmitters. ,, From the chronoamperometry data, we quantified the number of secreted acetylcholine molecules.

1.

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Cholinergic marker expression in T98G glioblastoma (GBM) cells and scanning electrochemical microscopy (SECM) instrumentation for positioning a nanoelectrode over a T98G GBM cell with nanometer spatial resolution. Confocal fluorescent images showing immunocytochemical labeling of (A) choline acetyltransferase (ChAT, Red) and (B) vesicular acetylcholine transporter (VAChT, Red) expression in T98G GBM cells. Blue color: cell nuclei. (C) Schematic of the SECM system for measuring acetylcholine secretion from living GBM cells. The LED light plus camera served as a side-view optical microscope to position the nanoelectrode near a T98G GBM cell. SECM was used to place the nanoelectrode over the cell with nanometer spatial resolution. Inset: scanning electron microscope image of a representative nanopipet used to make nanoITIES electrodes. Scale bar: 300 nm. (D) Schematic showing the nanoscale interface between two immiscible electrolyte solutions (nanoITIES) electrodes after nanopositioning over a living GBM cell. With the nanoITIES electrode in this position, cellular acetylcholine secretion was detected following high-concentration K+ chemical stimulation.

Immunocytochemical staining of T98G GBM cells shows that these cells express markers of acetylcholine, namely ChAT and VAChT (Red color, Figure A,B, Figure S3). Control experiments showed virtually no red fluorescence (Figures S4 and S5), confirming that the observed red fluorescence originated from the expression of ChAT and VAChT. These suggest that T98G GBM cells can both synthesize and package acetylcholine into vesicles. Overlaid images of the nuclear staining (Blue color, Figure A,B, Figure S3C,F) and ChAT/VAChT staining (Red color, Figure A,B, Figure S3C,F) show that ChAT and VAChT were localized around cell nuclei.

We employed nanoITIES electrodes to quantitatively detect acetylcholine based on the charge transfer across the polarized nano-liquid/liquid interface. Linear detection of acetylcholine in T98G cellular medium was achieved using both cyclic voltammetry and chronoamperometry (Figure S6).

We identified potential neurotransmitter interferents in T98G glioblastoma cell culture medium and lysed T98G cell pellets using liquid chromatography-tandem mass spectrometry, with the results listed in Table S1. After measuring background cyclic voltammograms of the cellular medium, we added the potential interferents1 mM GABA, 1 mM dopamine, 1 mM norepinephrine, 1 mM histamine, 1 mM serotonin, 1 mM tyramine, and levodopain order. Cyclic voltammograms were recorded after adding each interferent. Finally, we added acetylcholine and recorded cyclic voltammograms. At E ACh, the current levels after adding the potential interferents were similar to the background cellular medium (Figures S7 and S8). Likewise, the current level was similar to the background cellular medium after adding glutamate (Figure S7). In contrast, measurements after adding acetylcholine showed a clear and significant increase in current (Figures S7 and S8). These confirm that the nanoITIES electrode selectively detected acetylcholine versus potential interferents. We also confirmed that the added KCl chemical stimulation solution did not produce a current increase at E ACh (Figure S9), with additional discussion included in the Supporting Information.

To test our hypothesis that acetylcholine is secreted from living T98G GBM cells, we first positioned the nanoITIES electrode hundreds of nanometers from a living T98G GBM cell using an integrated side-view optical imaging and SECM platform (Figure C). , A nanoelectrode was optically placed over a cell and then precisely positioned hundreds of nanometers from the cell using feedback-mode SECM (Figures D and A). This close proximity to the cell enables detection of acetylcholine molecules immediately upon secretion, thereby minimizing signal loss caused by dilution as the signaling molecules diffuse away from the secretion site. −

2.

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Chemically stimulated acetylcholine secretion from living T98G glioblastoma (GBM) cells measured by chronoamperometry at a potential selective for acetylcholine detection (E ACh). (A) Side-view optical microscope image showing the nanoelectrode positioned with nm spatial resolution over the target T98G GBM cell during the on-cell chronoamperometry recording. (B) Chronoamperogram (i-t curves) at E ACh with the nanoelectrode over a living T98G cell. A current increase was observed in response to high-concentration K+ stimulation (blue curve); in contrast, for the control experiment, no current increase was observed after addition of cellular medium (black curve). This finding suggests that acetylcholine was secreted from the T98G cell. Cellular medium and high-concentration K+ were added at the red arrow. The size of the orifice of the pair of the nanopipete used to fabricate the nanoITIES electrode measured using SEM (Figure C inset) is 380 nm in diameter (longer axis) and 312 nm in diameter (shorter axis).

With the nanoelectrode over the cell, chronoamperometry measurements at E ACh showed a clear current increase in response to the addition of a high concentration of K+ (Figure B, blue curve). There was a delay between solution addition and the observed current increase, which was related to the distance between the source of the stimulant and the stimulated cell. A current increase was also observed after a second addition of high-concentration K+ (Figure S10, blue curve). In contrast, no current increase was observed after adding cellular medium (Figure B, black curve and Figure S10, blue curve), indicating that the solution addition process did not produce a current response. Similar results were observed for multiple T98G glioblastoma cells, with results from a second cell (Figure S11A) shown in Figures S11B and S12A. These results suggest that exposing T98G cells to elevated extracellular K+ stimulated acetylcholine secretion, as further confirmed by the additional control studies detailed below.

We performed control experiments to confirm that current increase following high-concentration K+ stimulation measured acetylcholine secretion by T98G glioblastoma cells. Chronoamperometry measurements at E No ACh (a nonacetylcholine-detecting potential) showed no current increase in response to high-concentration K+ or cellular medium additions, regardless of nanoelectrode position, i.e., over or off the cell (Figures S10 (black curve), S13D, S14D, and S15B). In addition, with the nanoelectrode off the cell, chronoamperometry measurements at E ACh did not show a current increase in response to high-concentration K+ additions (Figures S13C and S14C, blue curves and Figure S15A, red and blue curves). These control experiments were replicated for multiple T98G cells, with results from a second cell included in Figures S12B and S16–S18. Taken together, these controls demonstrate that a current increase occurred only when (1) chronoamperometry data were collected at E ACh, (2) KCl was added, and (3) the nanoelectrode was positioned over the cell. These findings suggest that T98G cells secrete acetylcholine in response to high concentration K+ chemical stimulation.

We determined the number of acetylcholine molecules secreted in response to high-concentration K+ stimulation by relating the integrated chronoamperometric charge (Q) to moles of acetylcholine according to Faraday’s law (Figure ). Following high-concentration K+ stimulation, 3.38 × 10–15 mol of acetylcholine (2.04 × 109 molecules) was secreted (Figure ). After repeating the high-concentration K+ stimulation, an additional 9.77 × 10–16 mol of acetylcholine (5.88 × 108 molecules) was secreted (Figure S19). A different T98G cell secreted 4.96 × 10–15 mol (2.99 × 109 molecules) and 3.57 × 10–15 mol (2.15 × 109 molecules) in response to the first and second high-concentration K+ stimulations, respectively (Figures S20 and S21). Notably, fewer acetylcholine molecules were secreted after the second stimulation, suggesting partial depletion of the releasable intracellular acetylcholine pool upon the first stimulation.

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The number of moles (n) and molecules (N) of acetylcholine secreted in response to K + chemical stimulation. The gray shaded region represents the integrated area following K+ chemical stimulation (the total charge, Q). The table shows the number of moles and molecules of acetylcholine secreted after K+ chemical stimulation. Figure S19 shows the quantitation of the number of moles and molecules of acetylcholine for a repeat K+ stimulation of the same cell. In addition, Figures S20 and S21 present the quantitation of acetylcholine secretion from a second cell.

Our findings suggest that T98G GBM cells secrete acetylcholine upon high-concentration K+ chemical stimulation, revealing a previously unrecognized cholinergic signaling axis in GBM. This finding aligns with transcriptomic analyses identifying neuronal-like GBM subtypes enriched for functions such as neurotransmitter exocytosis, synaptic transmission, and axonogenesis. Chemically stimulated acetylcholine secretion reported here suggests neuron-like excitability, consistent with reports that subsets of GBM cells express voltage-gated Na+ and K+ channels and can generate action potentials. ,, Our nano-resolved studies provide the first direct measurement of acetylcholine chemical transmitter secretion from living glioblastoma cells.

Acetylcholine secretion from living GBM cells revealed here suggests autocrine and paracrine cholinergic signaling, which could be involved in GBM proliferation and invasion. This proposed mechanism is analogous to glioma cell invasion promoted by self-secreted glutamate. Our findings fill a missing gap in the acetylcholine-GBM signaling axis by demonstrating that, in addition to expressing cholinergic machinery (such as cholinergic receptors), GBM cells secrete acetylcholine.

Conclusion

In summary, we achieved real-time detection and quantification of acetylcholine secretion from single living T98G GBM cells using nano-resolved amperometric measurements with nano-liquid/liquid interface electrodes coupled with SECM. Immunocytochemistry results showed that T98G GBM cells expressed cholinergic markers for acetylcholine synthesis and packaging into vesicles, i.e., ChAT and VAChT. This work is the first study reporting real-time and quantitative detection of acetylcholine secretion from living human GBM cells. Acetylcholine secretion from living GBM cells revealed here suggests autocrine and paracrine cholinergic signaling, highlighting cholinergic signaling pathways as potential therapeutic targets for GBM. Current work is in progress to study heterogeneity in acetylcholine secretion across GBM models and investigate the molecular mechanism governing acetylcholine secretion from living GBM cells.

Supplementary Material

tg6c00051_si_001.pdf (2.1MB, pdf)

Acknowledgments

The authors appreciate the support for this research from the University of Illinois Urbana–Champaign and an Alfred P. Sloan Research Fellowship (FG-2023-20536 to Mei Shen) from the Alfred P. Sloan Foundation. Mei Shen is a Biohub Investigator and an Alfred P. Sloan Research Fellow. Conrad Milton appreciates the support from the Robert C. and Carolyn J. Springborn Graduate Fellowship and the He and Chen Graduate Fellowship from the Department of Chemistry at University of Illinois Urbana–Champaign. The authors also appreciate the help of Duncan Nall and the Core Facilities at the Carl R. Woese Institute for Genomic Biology at the University of Illinois Urbana–Champaign for training on the Zeiss LSM 880 confocal microscope. The authors also appreciate the help of Hui Xu and Huimin Zhang in the Tumor Engineering and Phenotyping Shared Resource (TEP) at the Cancer Center at Illinois for glioblastoma culture training and assistance with mycoplasma testing. The SEM measurements were carried out in part in the Materials Research Laboratory Central Research Facilities, University of Illinois. The authors also appreciate the help of Xiaoyue Shen with cell imaging and culture.

.The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.6c00051.

  • Experimental section, T98G cell size and morphology, fluorescent imaging for immunocytochemical staining of ChAT and VAChT expression in T98G cells, fluorescent imaging controls for immunocytochemical staining of ChAT and VAChT expression in T98G cells, linear detection of acetylcholine by cyclic voltammetry and chronoamperometry, selectivity of acetylcholine detection versus K+ by cyclic voltammetry, selectivity of acetylcholine detection versus potential neurotransmitter interferents by cyclic voltammetry, chronoamperometry controls for detection of acetylcholine secretion from the cell in Figure B, additional quantitation for detection of acetylcholine secretion from the cell in Figure B, chronoamperometry detection of acetylcholine secretion from a second T98G cell, with chronoamperometry controls and quantitation, and references (PDF)

CRediT: Conrad Benjamin Milton, experimental design and methods, data collection, data curation, formal analysis, investigation, validation, visualization, writing - original draft, writing - review and editing; Mahmuda Akter, experimental design and methods, data collection, formal analysis, writing - experimental section of original draft, writing - review and editing; Olivia Maren Kawakita, data collection, formal analysis, writing - review and editing; Siao-Han Huang, data collection, formal analysis, writing - review and editing; Mei Shen, conceptualization, experimental design and methods, data curation, formal analysis, investigation, funding acquisition, project administration, resources, supervision, validation, visualization, writing - original draft, writing - review and editing.

The authors declare no competing financial interest.

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