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. 2020 Jun 16;12(6):1589.
doi: 10.3390/cancers12061589.

Applying Tissue Slice Culture in Cancer Research-Insights from Preclinical Proton Radiotherapy

Theresa Suckert  1   2 , Treewut Rassamegevanon  1   2 , Johannes Müller  2   3 , Antje Dietrich  1   2 , Antonia Graja  1   2 , Michael Reiche  2   4 , Steffen Löck  1   2   5 , Mechthild Krause  1   2   3   4   5 , Elke Beyreuther  2   6 , Cläre von Neubeck  1   2   7
Affiliations

Applying Tissue Slice Culture in Cancer Research-Insights from Preclinical Proton Radiotherapy

Theresa Suckert et al. Cancers (Basel). .

Abstract

A challenge in cancer research is the definition of reproducible, reliable, and practical models, which reflect the effects of complex treatment modalities and the heterogeneous response of patients. Proton beam radiotherapy (PBRT), relative to conventional photon-based radiotherapy, offers the potential for iso-effective tumor control, while protecting the normal tissue surrounding the tumor. However, the effects of PBRT on the tumor microenvironment and the interplay with newly developed chemo- and immunotherapeutic approaches are still open for investigation. This work evaluated thin-cut tumor slice cultures (TSC) of head and neck cancer and organotypic brain slice cultures (OBSC) of adult mice brain, regarding their relevance for translational radiooncology research. TSC and OBSC were treated with PBRT and investigated for cell survival with a lactate dehydrogenase (LDH) assay, DNA repair via the DNA double strand break marker γH2AX, as well as histology with regards to morphology. Adult OBSC failed to be an appropriate model for radiobiological research questions. However, histological analysis of TSC showed DNA damage and tumor morphological results, comparable to known in vivo and in vitro data, making them a promising model to study novel treatment approaches in patient-derived xenografts or primary tumor material.

Keywords: DNA damage; head and neck cancer; organotypic brain slice culture; proton beam radiotherapy; thin-cut tissue slices; tumor biology.

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Conflict of interest statement

In the past 5 years, M.K. received funding for her research projects by IBA (2016), Merck KGaA (2014–2018 for preclinical study; 2018–2020 for clinical study), Medipan GmbH (2014–2018). In the past 5 years, M.K. and S.L. have been involved in an ongoing publicly funded (German Federal Ministry of Education and Research) project with the companies Medipan, Attomol GmbH, GA Generic Assays GmbH, Gesellschaft für medizinische und wissenschaftliche genetische Analysen, Lipotype GmbH, and PolyAn GmbH (2019–2021). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Development and characterization of a proton irradiation setup for thin-cut tissue slices. (a) Exemplary tested angles using a Lego® rapid prototype. Membrane inserts tilted when angles >76° were used. Lower angels showed dose inhomogeneity. (b) Dose homogeneity for a 76° angle measured with EBT3 films on the plate bottom. (c) Adjustable milled setup at 76°. (d) Exemplary tumor (left) and brain slices (right) on the first day in culture.
Figure 2
Figure 2
Metabolism of Cal33 tumor slices measured via PrestoBlueTM. Values were normalized to the first measurement at day 1 in culture. Cell viability stayed stable over the observed cultivation period of six days (mean with standard deviation (SD), n = 7).
Figure 3
Figure 3
Histological analysis of tumor slice morphology and cell composition of a Cal33 squamous cell carcinoma of the head and neck (HNSCC) model. After two days in culture, tumor tissue contains (a) CD44 positive cells (brown), as well as (b) a necrotic fraction (light pink) located at the core of the tissue. (c) The center is hypoxic (red, pimonidazole), with proliferating cells (brown, bromodeoxyuridine (BrdU)) in the oxic rim. (d) Ki67 staining (brown) reveals cell proliferation across a culture period of six days. Counter staining with hematoxylin, blue cell nuclei.
Figure 4
Figure 4
Cell composition of tumor slices along the depth of the tissue. Cancer stem cells (CD44+), cell numbers (DAPI+), and the necrotic area were analyzed 24 h post irradiation with 4 Gy or control slices. Across the tumor a high variance was noted, showing that the morphology depends on the depth within the tissue. (a) Marker distribution across the tumor depth: locations further away from the nutrient-providing blood vessels in the muscle show a reduced cell number and an increased necrotic area. An inter-slice variability was noted, but no significant difference between the experimental conditions (full dots: control slices, open dots: irradiated slices). (b) Pearson correlation of tumor morphology. Significant positive and negative correlations were found between all markers (see text, nControl = 7, nTumor = 8).
Figure 5
Figure 5
Formation of γH2AX foci in tumor slices exposed to sham (0 Gy) or 4 Gy proton irradiation at 24 h post irradiation. (a) Representative immunofluorescent images of tumor slices treated with sham (left) or proton irradiation (right) show γH2AX foci. Apoptotic cells and endogenous DNA damages were observed in both groups; nevertheless, an increased number of foci could be detected in irradiated slices. (b) cfoci, nucleus area, and γH2AX foci numbers are significantly increased in slices that were irradiated with 4 Gy, compared to non-irradiated controls (linear mixed-effects model, *: p < 0.05, **: p < 0.01; nControl = 7, nTumor = 8).
Figure 6
Figure 6
Cell type composition of organotypic brain slice culture after cutting, and at day 5 in culture for irradiated (10 Gy) and non-irradiated slices (24 h post irradiation). Both (a) H&E and (b) staining of neuronal nuclei (NeuN) revealed a shrunken cell morphology. Distribution of neurons (NeuN) and myelin (OSP) was unchanged, but (c) dendrite density (MAP2) decreased during cultivation. (d) Some astrocytes (GFAP) started uncontrolled proliferation, creating cell clusters. (e), (f) Loss of function was observed for astrocytes lining vessels and microglia (Iba1) in the tissue.
Figure 7
Figure 7
Formation of γH2AX signal in organotypic brain slice cultures irradiated with 10 Gy protons. (a) Irradiated slices show a higher number of γH2AX positive cells. However, the typical spot-like foci formation is missing (blue—cell nuclei, orange—γH2AX). (b) The percentage of γH2AX positive nuclei (mean with SD) in non-irradiated and proton irradiated organotypic brain slices revealed a significant increase after proton irradiation (unpaired, two-tailed t-test, n = 3, *: p = 0.02).
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