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Glioma‑associated microglia and macrophages as a potential target for mTOR inhibition in glioblastoma

  • Authors:
    • Pia S. Zeiner
    • Michael Schulz
    • Jana Schomber
    • Jan-Béla Weinem
    • Nadja I. Lorenz
    • Benedikt Sauer
    • Bastian Roller
    • Katharina J. Weber
    • Anna-Luisa Luger
    • Annemarie Berger
    • Karl H. Plate
    • Lisa Sevenich
    • Joachim P. Steinbach
    • Mohammed H. Mosa
    • Patrick N. Harter
    • Michael W. Ronellenfitsch
  • View Affiliations / Copyright

    Affiliations: Dr. Senckenberg Institute of Neurooncology, University Hospital, Goethe University Frankfurt, D‑60596 Frankfurt, Germany, Georg‑Speyer‑Haus, Institute for Tumor Biology and Experimental Therapy, D‑60596 Frankfurt, Germany, Frankfurt Cancer Institute, Goethe University Frankfurt, D‑60596 Frankfurt am Main, Germany, Institute for Medical Virology, University Hospital, Goethe University Frankfurt, D‑60596 Frankfurt, Germany, Institute of Neurology (Edinger‑Institute), University Hospital, Goethe University Frankfurt, D‑60528 Frankfurt, Germany
    Copyright: © Zeiner et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 343
    |
    Published online on: October 6, 2025
       https://doi.org/10.3892/mmr.2025.13708
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Abstract

Glioma‑associated microglia/macrophages (GAM) constitute the predominant immune cell population in glioblastoma (GB). Both GB cells and GAM exhibit upregulated mTOR signaling. The present study aimed to investigate the effects of pharmacological mTOR inhibition (mTORi) specifically on GAM. The effects of mTORi on signal transduction, cell growth and viability were analyzed in immortalized microglia cell lines. Additionally, a comprehensive analysis of the GAM phenotype was conducted, including whole transcriptome analyses and cytokine profiling. Effects were investigated in a tumor cell/GAM co‑culture model under mTORi with rapamycin or torin2 or treatment with temozolomide, the standard chemotherapy agent for patients with GB. In the in vitro model, mTORi had significant effects on central biological functions of GAM, resulting in reduced proliferation and oxygen consumption. Additionally, treatment with mTORi induced a pro‑inflammatory phenotype in microglia cell lines. These findings demonstrate the relevance of mTOR signaling on GAM biology. Moreover, they provide rationales for therapeutic interventions targeting mTOR signaling specifically in GAM as a potential novel treatment strategy. 
View Figures

Figure 1

Impact of pharmacological mTOR
inhibition on basal functions of human microglia cell lines. (A)
Overview of the mTOR pathway and targets of the mTOR inhibitors
investigated in this study. Scheme adapted from (33). (B) C20 cells were incubated with
100 nM rapamycin, 100 nM torin2 and vehicle control for 24 h.
Protein lysates were analyzed by immunoblotting with antibodies for
NDRG1, P-NDRG1 (Thr346), S6RP, P-S6RP (Ser240/244), 4E-BP1,
P-4E-BP1 (Thr37/46) and actin as well as by immunocytochemistry
stainings of P-4E-BP1 (scale bar, 100 µm) of C20 cell pellets
incubated with 100 nM rapamycin, 100 nM torin2 and vehicle control
for 24 h. (C-E) C20 microglia cells were treated with 100 nM
rapamycin (red), 100 nM, torin2 (green) or vehicle control (grey).
(C) Crystal violet staining was used to quantify cell density at
baseline (day 0, after a 24 h attachment period prior to any
treatment intervention, black) and after 72 h of exposure to the
respective treatment conditions. Data are presented as mean ± SD
(n=4; one-way ANOVA, Tukey's multiple comparisons test). (D) Cell
death was quantified by PI FACS after 72 h. Data represent mean ±
SD (n=3; one-way ANOVA, Tukey's multiple comparisons test).
Histograms were depicted. (E) Quantification of oxygen by a
fluorescence-based assay was performed in C20 cells during 24 h
treatment. Oxygen consumption is shown relative to the start of the
experiment as mean (n=3) every hour; treatment groups were then
compared at timepoint 24 h (n=3; one-way ANOVA, Tukey's multiple
comparisons test). *P<0.05, **P<0.01 and ***P<0.001. ns,
not significant; PI, propidium iodide; SD, standard deviation;
NDRG1, N-myc downstream-regulated gene 1; P-, phosphorylated; S6RP,
S6 ribosomal protein; 4E-BP1, 4E-binding protein 1.

Figure 2

Impact of rapamycin compared with
temozolomide on the GAM transcriptome. (A) Overview on all
treatment conditions used throughout the experimental setup
(created in BioRender, Strecker, M. (2025); http://BioRender.com/wgg6ezp). Temozolomide effects
were only analyzed by RNA sequencing (transcriptome), torin2
co-culture experiments were only analyzed in the cytokine assay.
(B) Gene expression was analyzed in the whole transcriptome dataset
by PCA across all C20 samples including triplicates of the
different treatments (rapamycin, temozolomide and vehicle control)
in the co-culture (C20 microglia with LNT-229 glioma cells) or the
C20 mono-culture condition (bright colors, GAM co-culture of C20
with LNT-229; light colors, MG mono-culture control condition; red,
treatment with 100 nM rapamycin; blue, 400 µM temozolomide; grey,
vehicle control) (biological replicates were labelled 1 to 3). PCA,
principal component analysis; GAM, glioma-associated
microglia/macrophages; MG, microglia.

Figure 3

Differential expression of genes in
GAM following rapamycin and temozolomide treatment. Heatmap depicts
hierarchical clustering of the top 100 PCA gene loadings in C20 GAM
(co-cultivated) or C20 MG (C20 monoculture control condition)
following pharmacological mTORi with 100 nM rapamycin versus
vehicle control or treatment with 400 µM temozolomide versus
vehicle control. Variance-stabilized transformed data values are
shown. Genes involved in inflammatory processes were highlighted in
red. GAM, glioma-associated microglia/macrophages; PCA, principal
component analysis; MG, microglia; mTORi, mTOR inhibition.

Figure 4

Impact of rapamycin and temozolomide
on key gene programs in GAM. Gene set enrichment analysis was
performed to decipher gene programs most relevant to the respective
culture and treatment conditions. Heatmap of normalized enrichment
score of the hallmark gene sets (v7.4) in C20 GAM (co-cultivated)
or C20 MG (C20 monoculture control condition) following
pharmacological mTORi with 100 nM rapamycin versus vehicle control
or treatment with 400 µM temozolomide versus vehicle control.
Normalized enrichment scores are shown. A significance level of
*P<0.05 was depicted. Hallmark gene sets involved in key
immunogenic pathways were highlighted in red and for mTORC1
signaling in green. Hallmark gene sets displaying the same
regulation pattern under rapamycin in C20 and HMC3 (Fig. S6) were highlighted with squares.
GAM, glioma-associated microglia/macrophages; MG, microglia;
mTORC1, mTOR complex 1; mTORi, mTOR inhibition.

Figure 5

Impact of pharmacological mTOR
inhibition on cytokine expression profiles of GAM. (A)
Immunodetection of 36 different cytokines (each in duplicate) per
pooled sample (pool of n=3) incubated with rapamycin, torin2 or
vehicle. (B) Quantification by relative mean spot pixel density for
each cytokine was depicted. (A and B) Bright colors, GAM co-culture
of C20 with LNT-229; light colors, MG mono-culture control
condition; red, treatment with 100 nM rapamycin; green, 100 nM
torin2; grey, vehicle control. (C) Overview of protein expression
in the treatment conditions rapamycin and torin2 compared to
vehicle control in C20 GAM and C20 MG, respectively. Green,
increased; grey, similar; red, decreased; no color, not
analyzable/detectable. GAM, glioma-associated
microglia/macrophages; MG, microglia.
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Copy and paste a formatted citation
Spandidos Publications style
Zeiner PS, Schulz M, Schomber J, Weinem J, Lorenz NI, Sauer B, Roller B, Weber KJ, Luger A, Berger A, Berger A, et al: Glioma‑associated microglia and macrophages as a potential target for mTOR inhibition in glioblastoma. Mol Med Rep 32: 343, 2025.
APA
Zeiner, P.S., Schulz, M., Schomber, J., Weinem, J., Lorenz, N.I., Sauer, B. ... Ronellenfitsch, M.W. (2025). Glioma‑associated microglia and macrophages as a potential target for mTOR inhibition in glioblastoma. Molecular Medicine Reports, 32, 343. https://doi.org/10.3892/mmr.2025.13708
MLA
Zeiner, P. S., Schulz, M., Schomber, J., Weinem, J., Lorenz, N. I., Sauer, B., Roller, B., Weber, K. J., Luger, A., Berger, A., Plate, K. H., Sevenich, L., Steinbach, J. P., Mosa, M. H., Harter, P. N., Ronellenfitsch, M. W."Glioma‑associated microglia and macrophages as a potential target for mTOR inhibition in glioblastoma". Molecular Medicine Reports 32.6 (2025): 343.
Chicago
Zeiner, P. S., Schulz, M., Schomber, J., Weinem, J., Lorenz, N. I., Sauer, B., Roller, B., Weber, K. J., Luger, A., Berger, A., Plate, K. H., Sevenich, L., Steinbach, J. P., Mosa, M. H., Harter, P. N., Ronellenfitsch, M. W."Glioma‑associated microglia and macrophages as a potential target for mTOR inhibition in glioblastoma". Molecular Medicine Reports 32, no. 6 (2025): 343. https://doi.org/10.3892/mmr.2025.13708
Copy and paste a formatted citation
x
Spandidos Publications style
Zeiner PS, Schulz M, Schomber J, Weinem J, Lorenz NI, Sauer B, Roller B, Weber KJ, Luger A, Berger A, Berger A, et al: Glioma‑associated microglia and macrophages as a potential target for mTOR inhibition in glioblastoma. Mol Med Rep 32: 343, 2025.
APA
Zeiner, P.S., Schulz, M., Schomber, J., Weinem, J., Lorenz, N.I., Sauer, B. ... Ronellenfitsch, M.W. (2025). Glioma‑associated microglia and macrophages as a potential target for mTOR inhibition in glioblastoma. Molecular Medicine Reports, 32, 343. https://doi.org/10.3892/mmr.2025.13708
MLA
Zeiner, P. S., Schulz, M., Schomber, J., Weinem, J., Lorenz, N. I., Sauer, B., Roller, B., Weber, K. J., Luger, A., Berger, A., Plate, K. H., Sevenich, L., Steinbach, J. P., Mosa, M. H., Harter, P. N., Ronellenfitsch, M. W."Glioma‑associated microglia and macrophages as a potential target for mTOR inhibition in glioblastoma". Molecular Medicine Reports 32.6 (2025): 343.
Chicago
Zeiner, P. S., Schulz, M., Schomber, J., Weinem, J., Lorenz, N. I., Sauer, B., Roller, B., Weber, K. J., Luger, A., Berger, A., Plate, K. H., Sevenich, L., Steinbach, J. P., Mosa, M. H., Harter, P. N., Ronellenfitsch, M. W."Glioma‑associated microglia and macrophages as a potential target for mTOR inhibition in glioblastoma". Molecular Medicine Reports 32, no. 6 (2025): 343. https://doi.org/10.3892/mmr.2025.13708
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