Unless stated otherwise, all chemicals were purchased from Sigma Aldrich (Taufkirchen, Germany). Carnosine was kindly provided by Flamma (Flamma s.p.a. Chignolo d’Isola, Italy).
Cell lines and primary cell cultures
The GBM cell line T98G, negative for IDH1R132H-mutation and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, was obtained from the ATCC, genotyped using a PowerPlex 21 System (Promega; Mannheim; Germany) by the Genolytic GmbH (Leipzig, Germany) and authenticated by comparison to data at the ATCC and the DSMZ. T98G cells were used at passage 5–7 after genotyping and authentication. Both, primary GBM cultures and primary fibroblast cultures were established from tissue samples obtained during standard surgery performed at the Neurosurgery Department of the University Hospital Leipzig during 2015 and 2016. All patients provided written informed consent according to the German laws as confirmed by the local committee. When possible, one primary GBM cell culture and one primary fibroblast culture was established from tissue samples obtained from each patient (Additional file 1: Table S1). All GBM samples were diagnosed and have been approved by the Neuropathology Department of the Leipzig University Hospital. IDH 1 status has been determined using immunohistochemistry and pyrosequencing, MGMT promoter methylation status was determined using nucleic acid amplification followed by pyrosequencing.
For cultivation, tissue specimens from the tumor, from galea or from periost were cut into approximately 1 mm3 large pieces and then separately placed into 25 mm2 culture flasks (TPP, Trasadingen, Switzerland) until tumor cells or fibroblasts grew out. When more than 90% confluence was reached specimens were removed and primary cell cultures were transferred into 75 mm2 culture flasks (TPP) for further cultivation. Cell cultures were maintained in high glucose DMEM (4.5 g glucose/ml) supplemented with 2 mM Glutamax™, 1% penicillin/streptomycin (all from Gibco Life Technologies, now Thermo Fisher Scientific, Darmstadt, Germany) and 10% FBS (Biochrom GmbH, Berlin, Germany), further referred to as “standard medium”, and kept in incubators (37 °C, 5% CO2/95% air).
Cell viability assays
For cell viability assays, cells were counted and seeded into sterile 96-well plates (µClear, Greiner Bio One, Frickenhausen, Germany) at a density of 5000 cells/well in 200 µl standard medium. After 24 h of cultivation (37 °C, 5% CO2/95% air) the medium was aspirated and fresh medium supplemented with or without carnosine was added (100 µl/well) and the cells were incubated for additional 48 h. Then, the CellTiter-Glo Luminescent Cell Viability Assay (CTG, Promega, Mannheim, Germany) was employed to determine viable cells by measuring ATP in cell lysates and the CellTiter-Blue Cell Viability Assay (CTB, Promega) was used to quantify the cell’s metabolic capacity in living cells. All assays were carried out according to manufacturer’s protocols. Luminescence and fluorescence were measured using a SpectraMax M5 multilabel reader (Molecular Devices, Biberach, Germany).
Co-cultivation of GBM cells and fibroblasts (ring-cultures)
When cells reached more than 90% confluence in 75 cm2 cell culture flasks they were detached using accutase (Thermo Fisher), counted and diluted for co-cultivation. The ring-cultures were established in 12-well plates. Therefore, sterile cloning rings (steel, 6 mm inner; 8 mm outer diameter, Hartenstein, Würzburg, Germany) usually used for the isolation of clones, were placed in the middle of each well dividing it into an inner-ring and an outer-ring part. Then, 2500 tumor cells suspended in standard medium (112 µl) were seeded inside the ring. Afterwards, 50,000 fibroblasts (in 658 µl standard medium) were seeded outside of the ring (Additional file 2: Figure S1). Co-cultures with cloning rings were incubated for 4 h (37 °C, 5% CO2/95% air) before the rings were carefully removed using sterile forceps. Medium was exchanged immediately after ring removal containing various concentrations of carnosine. On the following days medium was exchanged twice a week.
Carnosine co-culture experiments
Carnosine was diluted in 0.7% NaCl solution and carnosine experiments were performed with concentrations of 0 mM, 10 mM, 25 mM, 50 mM and 75 mM. All ring-culture experiments were prepared as described above. Control experiments with T98G cells inside the ring and without fibroblasts in the outer part were kept for over 2 weeks. Ring co-cultures with T98G and fibroblasts (P0385) and with GBM cells and fibroblasts of the same patient (P0383 with P0385 and P0431 with P0433) were cultivated for 4 weeks. Throughout cultivation, cell growth and dissemination were monitored by bright field microscopy. After 4 weeks all co-cultures were fixated in 4.5% paraformaldehyde and stored in 1% sodium azide solution at 4 °C until microscopic analysis.
Immunofluorescent staining was carried out to discriminate between tumor cells and fibroblasts using anti-fibroblast TE-7 (CBL271, Merck Millipore, Darmstadt, Germany), anti-nestin (AB5922, Merck Millipore) and secondary antibodies (ab6563, ab150081, abcam, Cambridge, UK). Briefly, for the detection of TE-7 the fixated co-cultures were permeabilized with 0.1% TritonX-100 at room temperature (RT) for 5 min. After blocking with 10% goat serum for 15 min, samples were incubated with anti-fibroblast TE-7 primary antibodies (dilution 1:100) at 5 °C overnight, washed with TBS (20 mM Tris, 134 mM NaCl) and subsequently incubated with the secondary antibody (1:250; ab6563) for 45 min at RT. For the detection of nestin, fixated cell cultures were permeabilized with 0.1% TritonX-100 in TBS for 1 h at RT, blocked for 15 min with 10% goat serum and then incubated with an anti-nestin antibody (dilution 1:250) for 1 h at RT. Afterwards, cultures were washed with TBS and incubated with a dilution of secondary antibody (1:250; ab150081) for 45 min at RT. Finally, nuclei were counterstained with DAPI (4 µg/ml) and cell cultures were preserved in 10% sodium azide solution at 4 °C until microscopy.
For microscopic analysis a Zeiss Axiovert 200M microscope (Zeiss, Oberkochen, Germany) equipped with a motorized stage (Märzhäuser, Wetzlar, Germany) with MosaiX software and by means of a CCD camera (Zeiss MRC) connected to an AxioVision 4.8.2 image analysis system (Zeiss) was used to create tile pictures. Each tile picture is composed of 285 single microscopic images taken at a magnification of 50 and represents a whole well of a 12-well plate. As denoted in the figure legend to Fig. 2 ImageJ images in this figure have been graphically enhanced for representation purposes using the Corel Draw Graphics Suite 2017 (Corel Corporation, Ottawa, Canada).
Quantitative and statistical analysis
The number of colonies in ring co-culture experiments was determined using ImageJ after a color threshold and a common pixel size were defined. All pictures used for the analysis were taken at the same magnification and had the same size. Statistical analysis was performed using the algorithm for t-test implemented in Microsoft Excel 2010.