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. 2023 May 10;15(10):2698.
doi: 10.3390/cancers15102698.

Characterization and Optimization of the Tumor Microenvironment in Patient-Derived Organotypic Slices and Organoid Models of Glioblastoma

Vera Nickl  1 , Juliana Eck  2 , Nicolas Goedert  1 , Julian Hübner  3 , Thomas Nerreter  3 , Carsten Hagemann  1 , Ralf-Ingo Ernestus  1 , Tim Schulz  1 , Robert Carl Nickl  1 , Almuth Friederike Keßler  1 , Mario Löhr  1 , Andreas Rosenwald  2 , Maria Breun  1 , Camelia Maria Monoranu  4
Affiliations

Characterization and Optimization of the Tumor Microenvironment in Patient-Derived Organotypic Slices and Organoid Models of Glioblastoma

Vera Nickl et al. Cancers (Basel). .

Abstract

While glioblastoma (GBM) is still challenging to treat, novel immunotherapeutic approaches have shown promising effects in preclinical settings. However, their clinical breakthrough is hampered by complex interactions of GBM with the tumor microenvironment (TME). Here, we present an analysis of TME composition in a patient-derived organoid model (PDO) as well as in organotypic slice cultures (OSC). To obtain a more realistic model for immunotherapeutic testing, we introduce an enhanced PDO model. We manufactured PDOs and OSCs from fresh tissue of GBM patients and analyzed the TME. Enhanced PDOs (ePDOs) were obtained via co-culture with PBMCs (peripheral blood mononuclear cells) and compared to normal PDOs (nPDOs) and PT (primary tissue). At first, we showed that TME was not sustained in PDOs after a short time of culture. In contrast, TME was largely maintained in OSCs. Unfortunately, OSCs can only be cultured for up to 9 days. Thus, we enhanced the TME in PDOs by co-culturing PDOs and PBMCs from healthy donors. These cellular TME patterns could be preserved until day 21. The ePDO approach could mirror the interaction of GBM, TME and immunotherapeutic agents and may consequently represent a realistic model for individual immunotherapeutic drug testing in the future.

Keywords: glioblastoma; organoids; slice culture; tumormicroenvironment.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Relative expression of tumor microenvironment (TME) cells in primary tissue (PT). n = 10 patients. The relative expression of antigen patterns in GBM is given in [%]; PT is displayed in bar plots; whiskers indicate standard deviation.
Figure 2
Figure 2
Alteration of TME cell presence over time in PT and in patient-derived organoids (PDOs) at day 7 and 14 of culture. n = 9 patients. The relative expression of cellular TME components [%] was quantified in PT (blue) and at day 7 (violet) and day 14 (green) of PDO culture: (A) CD45, (B) CD7, (C) CD4, (D) CD8, (E) CD14, (F) CD68, (G) iNOS, (H) CD163, (I) CCR2, (J) CD25, (K) PD1, (L) FOXP3, (M) TIM3, (N) LAG3; Whiskers indicate standard deviation; significant differences are indicated.
Figure 3
Figure 3
Example of representative immunohistochemical images for the TME composition in PT and in PDO day 7 and PDO day 14 (patient 6, Scale bar: 50 µm, magnification 40×).
Figure 3
Figure 3
Example of representative immunohistochemical images for the TME composition in PT and in PDO day 7 and PDO day 14 (patient 6, Scale bar: 50 µm, magnification 40×).
Figure 4
Figure 4
Alteration of TME cell marker abundance over time in organotypic slice culture (OSC) on day 1 to day 9 of culture. n = 5 patients. The relative expression of cellular TME components [%] was quantified from day 0 to day 9 of culture: (A) CD45, (B) CD7, (C) CD4, (D) CD8, (E) CD14, (F) CD68, (G) iNOS, (H) CD163, (I) CCR2, (J) CD25, (K) PD1, (L) FOXP3, (M) TIM3, (N) LAG3. No significant differences between day 0 and day 9 could be shown in antigen expression; Whiskers indicate standard deviation; significant differences are indicated.
Figure 5
Figure 5
Example of representative immunohistochemical images for the TME composition in OSCs on day 1, day 2, day 4 and day 7 of culture (patient 5, Scale bar: 50 µm, magnification 40×).
Figure 5
Figure 5
Example of representative immunohistochemical images for the TME composition in OSCs on day 1, day 2, day 4 and day 7 of culture (patient 5, Scale bar: 50 µm, magnification 40×).
Figure 6
Figure 6
Expression of cellular TME markers in normal PDOs (nPDOs) and enhanced PDOs (ePDOs) at day 0, 7, 14 and 21 compared to PT. n = 3 patients. The relative expression of cellular TME components [%] was quantified in PT (blue) and at day 7 (green), day 14 (orange) and day 21 (red) of PDO culture in nPDOs and ePDOs: (A) CD45, (B) CD7, (C) CD4, (D) CD8, (E) CD14, (F) CD68, (G) iNOS, (H) CD163; Whiskers indicate standard deviation; significant differences are indicated.
Figure 7
Figure 7
Example of representative immunohistochemical images for the TME in PT, in ePDO and nPDO (patient 13, Scale bar: 50 µm, magnification 40×).
Figure 7
Figure 7
Example of representative immunohistochemical images for the TME in PT, in ePDO and nPDO (patient 13, Scale bar: 50 µm, magnification 40×).
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