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Article Open Access

Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during in vitro cultivation

  • Authors:
    • Jessica Nojszewski
    • Nils Oliver Schröder
    • Jacqueline Clüver
    • Jonna Holler
    • Hajrullah Ahmeti
    • Michael Synowitz
    • Janka Held-Feindt
  • View Affiliations / Copyright

    Affiliations: Department of Neurosurgery, University Medical Center Schleswig‑Holstein (UKSH), Campus Kiel, D‑24105 Kiel, Germany
    Copyright: © Nojszewski et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 85
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    Published online on: March 2, 2026
       https://doi.org/10.3892/or.2026.9090
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Abstract

Glioblastomas are characterized by extensive intra‑ and intertumoral heterogeneity, which drives their rapid invasive growth and frequent recurrence. As a result, prognosis remains poor, and developing effective treatments continues to be a major challenge. The survival and growth of glioblastomas are supported by a specific, protective tumor microenvironment (TME) that influences tumor morphology, growth rate, therapy resistance and recurrence. To better represent the complex TME with its immune cells and inflammatory mediators and the tumor heterogeneity, including glioma stem‑like cells (GSCs), realistic in vitro preclinical models are required. Patient‑derived glioblastoma organoids (GBOs), directly derived from tumor tissue obtained after surgical resection, were generated in the present study. Preserving the original tumor state in organoids, particularly regarding the TME and heterogeneity, is crucial. The present study evaluates the long‑term preservation of key tumor properties in cultivated GBOs, including glial structural proteins, GSCs, immune cells, endothelial cells, proliferation marker, and inflammatory mediators such as cytokines and chemokines. Expression levels of these components were measured at five defined time points in 10 independent GBO preparations, at both the mRNA and protein level, and statistical correlation analyses were performed. The findings of the present study indicate that the key components were generally maintained over a one‑month cultivation period. However, some markers showed reduced expression at later time points or recovered after an initial drop at early cultivation time points. Therefore, the optimal time window for employing patient‑derived organoids as an advanced preclinical model for glioblastoma research appears to be between 7‑14 days of cultivation.
View Figures

Figure 1

GBO preparation based on Jakob et
al (26). The process is shown
from day 0 to day 28. At day 0, the resected tumor tissue is
displayed on the left, with smaller dissected pieces in the center
of the image. GBO morphology was observed on days 4, 7, 14, and 28
using light microscopy. The scale bar at day 0 represents 1 cm;
scale bars for days 4, 7, 14, and 28 represent 1 mm. GBO,
glioblastoma organoid.

Figure 2

Reverse transcription-quantitative
PCR and correlation analysis of glial structural proteins,
stemness, vessel, and immune cell markers. Gene expression levels
are shown across the entire cultivation period for 10 independent
GBO preparations (n=10) at five time points: days 0, 3–5, 7–8,
14–15, and 26–28. The x-axis represents days, the left y-axis shows
∆CT values, and the right y-axis displays the
logarithmic scale of expression relative to GAPDH as the
housekeeping gene (GAPDH expression=1). (A) Glial structural
proteins. (B) Stemness markers. (C) Vessel marker. (D) Immune cell
markers. (E) Correlation analysis. Correlations are indicated by a
color gradient: Dark green represents a high positive correlation,
light green a low positive correlation, dark blue a high negative
correlation, and light blue a low negative correlation. Statistical
analysis was performed using one-way ANOVA with Dunnett's multiple
comparisons post hoc test. *P<0.05, **P<0.01 and
***P<0.001. GBO, glioblastoma organoid; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary
acidic protein; S100B, S100 calcium-binding protein B; OCT4,
octamer-binding transcription factor 4; MSI1, Musashi RNA-binding
protein 1; SOX2, SRY-box transcription factor 2; KLF4, Krüppel-like
factor 4; CD133, cluster of differentiation 133; CD31, cluster of
differentiation 31; CD11b, cluster of differentiation 11b; Iba1,
ionized calcium-binding adapter molecule 1; CD68, cluster of
differentiation 68; CD3, cluster of differentiation 3.

Figure 3

H&E staining and
immunofluorescence double-staining images of an exemplary GBO
preparation at five time points: days 0, 4, 7, 14, and 28. Selected
insets were taken from two additional GBO preparations (total n=3).
(A) H&E staining images with low (×2,5)- and high
(×40)-magnification; black arrows indicate blood vessels. (B and C)
Immune cell markers. (D) Vessel marker. (E) Areas with cell death
in one GBO preparation during the cultivation period. Scale bars: 1
mm for whole-organism H&E images, 50 µm for the corresponding
H&E-insets, 20 µm for immunofluorescence images. GBO,
glioblastoma organoid; DAPI, 4′,6-diamidino-2-phenylindole; vWF,
von Willebrand factor; CD11b, cluster of differentiation 11b; Iba1,
ionized calcium-binding adapter molecule 1; CD68, cluster of
differentiation 68; CD3, cluster of differentiation 3; S100B, S100
calcium-binding protein B.

Figure 4

Immunofluorescence double-staining
images of an exemplary GBO preparation at five time points: Days 0,
4, 7, 14, and 28. Selected insets were taken from two additional
GBO preparations (total n=3). (A-D) Stemness markers. (E) Exemplary
Apotome image of KLF4 at day 28. GBO, glioblastoma organoid; GFAP,
glial fibrillary acidic protein; S100B, S100 calcium-binding
protein B; OCT4, octamer-binding transcription factor 4; MSI1,
Musashi RNA-binding protein 1; SOX2, SRY-box transcription factor
2; CD133, cluster of differentiation 133.

Figure 5

Reverse transcription-quantitative
PCR and correlation analysis of chemokines and cytokines with their
respective receptors. Gene expression levels are shown across the
entire cultivation period for 10 different glioblastoma organoid
preparations (n=10) at five time points: days 0, 3–5, 7–8, 14–15,
and 26–28. The x-axis represents days, the left y-axis shows
∆CT values, and the right y-axis displays the
logarithmic scale of expression relative to GAPDH as the
housekeeping gene (GAPDH expression=1). (A) Chemokines and
chemokine receptors. (B) Cytokines and cytokine receptors. (C)
Correlation analysis. Correlations are indicated by a color
gradient: Dark green represents a high positive correlation, light
green a low positive correlation, dark blue a high negative
correlation, and light blue a low negative correlation. Statistical
analysis was performed using one-way ANOVA with Dunnett's multiple
comparisons post hoc test. *P<0.05, **P<0.01 and
***P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
CX3CL1, C-X3-C motif chemokine ligand 1; CX3CR1, C-X3-C motif
chemokine receptor 1; CXCL12, C-X-C motif chemokine ligand 12;
CXCR4, C-X-C motif chemokine receptor 4; CXCR7, C-X-C motif
chemokine receptor 7; CXCL16, C-X-C motif chemokine ligand 16;
CXCR6, C-X-C motif chemokine receptor 6; IL-1β, interleukin-1 beta;
IL-1βR, interleukin-1 beta receptor; IL-6, interleukin-6; IL-6R,
interleukin-6 receptor.

Figure 6

Immunofluorescence double-staining
images of three glioblastoma organoid preparations at three time
points (days 0, 4, and 28). (A-G) Chemokines and chemokine
receptors. (A-C) CXCL12-CXCR4-CXCR7 axis. (D and E) CXCL16-CXCR6
axis. (F and G) CX3CL1-CX3CR1 axis. Scale bars: 20 µm. CXCL12,
C-X-C motif chemokine ligand 12; CXCR4, C-X-C motif chemokine
receptor 4; CXCR7, C-X-C motif chemokine receptor 7; CXCL16, C-X-C
motif chemokine ligand 16; CXCR6, C-X-C motif chemokine receptor 6;
CX3CL1, C-X3-C motif chemokine ligand 1; CX3CR1, C-X3-C motif
chemokine receptor 1; GFAP, glial fibrillary acidic protein; S100B,
S100 calcium-binding protein B; MSI1, Musashi RNA-binding protein
1; SOX2, SRY-box transcription factor 2; Iba1, ionized
calcium-binding adapter molecule 1.

Figure 7

Cytokines and cytokine receptors were
visualized using immunofluorescence double-staining at three time
points (days 0, 4, and 28), exemplified for one glioblastoma
organoid preparation. (A and B) IL-1β and IL-1βR. (C and D) IL-6
and IL-6R. Scale bars, 20 µm. IL-1β, interleukin-1 beta; IL-1βR,
IL-1β receptor; IL-6, interleukin-6; IL-6R, IL-6 receptor; Iba1,
ionized calcium-binding adapter molecule 1; CD11b, cluster of
differentiation 11b.
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Copy and paste a formatted citation
Spandidos Publications style
Nojszewski J, Schröder NO, Clüver J, Holler J, Ahmeti H, Synowitz M and Held-Feindt J: Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during <em>in vitro</em> cultivation. Oncol Rep 55: 85, 2026.
APA
Nojszewski, J., Schröder, N.O., Clüver, J., Holler, J., Ahmeti, H., Synowitz, M., & Held-Feindt, J. (2026). Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during <em>in vitro</em> cultivation. Oncology Reports, 55, 85. https://doi.org/10.3892/or.2026.9090
MLA
Nojszewski, J., Schröder, N. O., Clüver, J., Holler, J., Ahmeti, H., Synowitz, M., Held-Feindt, J."Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during <em>in vitro</em> cultivation". Oncology Reports 55.5 (2026): 85.
Chicago
Nojszewski, J., Schröder, N. O., Clüver, J., Holler, J., Ahmeti, H., Synowitz, M., Held-Feindt, J."Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during <em>in vitro</em> cultivation". Oncology Reports 55, no. 5 (2026): 85. https://doi.org/10.3892/or.2026.9090
Copy and paste a formatted citation
x
Spandidos Publications style
Nojszewski J, Schröder NO, Clüver J, Holler J, Ahmeti H, Synowitz M and Held-Feindt J: Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during <em>in vitro</em> cultivation. Oncol Rep 55: 85, 2026.
APA
Nojszewski, J., Schröder, N.O., Clüver, J., Holler, J., Ahmeti, H., Synowitz, M., & Held-Feindt, J. (2026). Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during <em>in vitro</em> cultivation. Oncology Reports, 55, 85. https://doi.org/10.3892/or.2026.9090
MLA
Nojszewski, J., Schröder, N. O., Clüver, J., Holler, J., Ahmeti, H., Synowitz, M., Held-Feindt, J."Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during <em>in vitro</em> cultivation". Oncology Reports 55.5 (2026): 85.
Chicago
Nojszewski, J., Schröder, N. O., Clüver, J., Holler, J., Ahmeti, H., Synowitz, M., Held-Feindt, J."Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during <em>in vitro</em> cultivation". Oncology Reports 55, no. 5 (2026): 85. https://doi.org/10.3892/or.2026.9090
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