Abstract
Meningiomas are the most common non-malignant intracranial tumors. Like most tumors, meningiomas prefer anaerobic glycolysis for energy production (Warburg effect). This leads to an increased synthesis of the metabolite methylglyoxal (MGO). This metabolite is known to react with amino groups of proteins. This reaction is called glycation, thereby building advanced glycation endproducts (AGEs). In this study, we investigated the influence of glycation on two meningioma cell lines, representing the WHO grade I (BEN-MEN-1) and the WHO grade III (IOMM-Lee). Increasing MGO concentrations led to the formation of AGEs and decreased growth in both cell lines. When analyzing the influence of glycation on adhesion, chemotaxis and invasion, we could show that the glycation of meningioma cells resulted in increased invasive potential of the benign meningioma cell line, whereas the invasive potential of the malignant cell line was reduced. In addition, glycation increased the E-cadherin- and decreased the N-cadherin-expression in BEN-MEN-1 cells, but did not affect the cadherin-expression in IOMM-Lee cells.
Introduction
Meningioma represents the most common non-malignant intracranial tumor (Goldbrunner et al. 2016; Holleczek et al. 2019; Ostrom et al. 2018, 2019).
Like many tumors, meningiomas need large amounts of glucose as primary energy source, because they mainly metabolize glucose to lactate during anaerobic glycolysis (Warburg effect) (Bharadwaj et al. 2015), which generates only low amounts of adenosine triphosphate (ATP). This changed (anaerobic) energy metabolism is one of the “hallmarks of cancer” (Gill et al. 2016). In line with this, there is a correlation between serum glucose levels and meningioma risk (Edlinger et al. 2012; Michaud et al. 2011; Niedermaier et al. 2015; Wiedmann et al. 2013). However, there are many inconsistent data suggesting a positive (Schneider et al. 2005; Schwartzbaum et al. 2005) or inverse (Bernardo et al. 2016) relationship between diabetes and serum glucose levels and the risk of meningioma. Patients with type 2 diabetes have a decreased survival after surgical resection of a WHO grade I meningioma (Nayeri et al. 2016).
Methylglyoxal (MGO) has been discussed as a possible linker between diabetes and serum glucose levels and cancer (Bellier et al. 2019), since diabetic patients and aged individuals have elevated MGO concentrations (Rabbani and Thornalley 2015). Some authors even propose MGO as a tumor promoting agent (Antognelli et al. 2019; Bellahcène et al. 2018). Approximately 0.1–0.4% of the glucose is transformed to MGO during glycolysis as a regular side product from dihydroxyacetone phosphate or glyceraldehyde-3-phosphate (Allaman et al. 2015). Importantly, MGO is 20,000 times more reactive than glucose and reacts mainly with proteins (through arginine, lysine, and cysteine residues) or to a lower degree also with DNA or lipids, thereby forming advanced glycation endproducts (AGEs) (Falone et al. 2012; Kalapos 2008; Schalkwijk 2015). This non-enzymatic reaction between the carbonyl groups of dicarbonyls (like MGO or glyoxal) or sugars (like glucose or fructose) and the amino groups of proteins is called glycation (Ahmed 2005; Rabbani and Thornalley 2008). Electrophilic carbonyl groups of glucose or other reactive sugars react with free amino groups of amino acids and forming a non-stable Schiff base. This reaction is called classical Maillard reaction. Further rearrangement leads to formation of a more stable ketosamine (Amadori product). The formation of Schiff bases and Amadori products are reversible reactions. In later reactions, they form irreversible adducts or protein crosslinks (Ahmed 2005; Paul and Bailey 1996). The process of producing AGEs affects all proteins including cell adhesion molecules or receptors and proteins of the extracellular matrix (Pedchenko et al. 2005; Rabbani and Thornalley 2012).
It was previously suggested that application of MGO lead to altered adhesion and migration (Antognelli et al. 2019; Loarca et al. 2013; Nokin et al. 2019).
Cadherins represent cell adhesion molecules that mediate Ca2+-dependent homophilic interaction with cadherin molecules on the surface of neighboring cells. The cytoplasmic domain binds downstream to members of the catenin protein family and regulates functions like cell–cell interactions (Harrison et al. 2011; Mège and Ishiyama 2017). Cadherins also play an important role in the epithelial–mesenchymal-transition (EMT). The EMT describes the change of epithelial markers like E-cadherin to mesenchymal markers like N-cadherin or Vimentin and can promote migration of transformed cells (Gloushankova et al. 2017; Mendonsa et al. 2018). Decreasing E-cadherin expression is mostly associated with weakening of cell–cell adhesion in tumor progression (Rodriguez et al. 2012). Expression of N-cadherin is closely related to tumor invasion and metastasis (Cao et al. 2019).
In the present study, we analyzed differences between benign and malignant meningioma cell lines before and after glycation using MGO. We could show that physiological MGO concentrations had no effect on cell morphology, metabolic activity, chemotaxis and adhesion. However, the invasiveness of the benign meningioma cells was increased whereas the invasiveness of the malignant meningioma cells was reduced in the presence of MGO, which means that glycation resulted in a switch of benign tumor to a more aggressive tumor cell. Furthermore, glycation led to increased E-cadherin and reduced N-cadherin-expression in the benign meningioma cells.
Results
MGO-treatment of meningioma cells interferes with their morphology
First of all, we analyzed whether treatment with MGO affects meningioma cells by comparing their morphology. Therefore, we cultured both cell lines in absence or presence of MGO for 24 h. Representative micrographs for the benign BEN-MEN-1 cells are shown in Figure 1A and for the malignant IOMM-Lee cells in Figure 1B. When cells were cultured in the presence of 0.1 or 0.3 mM MGO, we could not observe any changes in the morphology compared with untreated control cells. BEN-MEN-1 cells had a typical meningothelial shape and grew in monolayers. IOMM-Lee cells were more epithelial-like and grew as multilayers. After treatment with 0.6 mM MGO, we observed less cells and morphological differences compared with the controls. When culturing cells in presence of 1.0 mM MGO, cells became round and we observed less cells and less spreading in both cultures.
Micrographs of meningioma cells after 24 h MGO-treatment.
Cells (A = BEN-MEN-1 and B = IOMM-Lee) were cultured in the absence (control) or presence of different concentrations (0.1, 0.3, 0.6 and 1.0 mM) of MGO. Scale bar in white: 50 µm.
High concentrations of MGO interfere with the cell viability of meningioma cells
Since morphology was changed in presence of 0.6 mM MGO or more, we examined the cell viability of both meningioma cell lines, after culturing in absence or presence of different concentrations of MGO (Figure 2). We measured decreased cell viability after incubation with high concentrations of MGO. For the benign cell line, we could not show significant differences at MGO concentrations up to 0.6 mM. Only at 1.0 mM MGO, cell viability was significantly reduced compared with untreated controls (BEN-MEN-1; Figure 2A). For the malignant cell line (IOMM-Lee; Figure 2B), we observed similar effects. Only at 1.0 mM MGO, there was a significant difference compared with control, which confirmed our microscopic observations presented in Figure 1A and B. Furthermore, we analyzed caspase 3 expression by Western blot analysis. Caspase 3 was only expressed in cultures treated with 1.0 mM MGO and not in cultures treated with 0.1, 0.3 or 0.6 mM MGO (data not shown), indicating that only very high concentrations of MGO induce apoptosis in our experiments.
Cell viability of meningioma cell lines after 24 h MGO-treatment.
Cell viability was analyzed using an MTT-assay for BEN-MEN-1 (A) and IOMM–Lee cells (B). Cells were seeded in absence (Ctrl) or presence of MGO (0.1, 0.3, 0.6 and 1.0 mM). Both cell lines showed a decreasing metabolic activity with increasing concentration of MGO. Statistical analysis was performed using t-test and error bars represent SD (n = 7; ***p < 0.005; (BEN-MEN-1: p = 0.0027; IOMM-Lee: p = 0.00045)).
Increased glycation with increasing concentrations of MGO
Next, we wanted to examine whether treatment of meningioma cells with MGO led to glycation. For this, we cultured both meningioma cell lines in the presence (0.1, 0.3, 0.6 and 1.0 mM) or absence of MGO for 24 h. To verify the effect of the treatment, we performed immunoblotting using anti-AGE antibodies. We could show that increasing MGO concentrations led to increasing AGE-signals, which is shown in Figure 3A and C for BEN-MEN-1 and in Figure 3B and D for IOMM-Lee cells. Glycation is quantified in Figure 3B and D. We found significant increase of glycation already at concentrations of 0.3 mM MGO (Figure 3E).
Glycation of meningioma cells.
(A) Ponceau S staining of the total proteins (left side) and immunoblot (right side) of BEN-MEN-1 with different MGO concentrations. Controls (Ctrl) were cells without MGO treatment. We used an anti-AGE antibody to verify cellular glycation. (B) Ponceau S staining of the total proteins (left side) and immunoblot (right side) of IOMM-Lee cells with different MGO concentrations. Controls (Ctrl) were cells without MGO treatment. We used an anti-AGE antibody to verify cellular glycation (C) Representative quantification of the blot of BEN-MEN-1 cells shown in panel (A). (D) Representative quantification of the blot of IOMM-Lee cells shown in panel (B). (E) Quantification of glycation of BEN-MEN-1 and IOMM-Lee after treatment with 0.3 mM MGO. Non-glycated cells were used as controls (Ctrl). Statistical analysis was performed using t-test and error bars represent SD (n = 4; (BEN-MEN-1: p = 0.0452; IOMM-Lee: p = 0.0143)).
MGO has no effect on the adhesion of meningioma cells to ECM components
We then analyzed the effect of glycation on adhesion of the two meningioma cell lines. Since 0.3 mM MGO had no effect on the cell viability of both meningioma cell lines and led to significant formation of AGEs, we decided to use 0.3 mM MGO for all further experiments. To quantify adhesion, we cultured both meningioma cell lines in E-plates. Cells were seeded in absence or presence of MGO on two different matrices (collagen IV or fibronectin) which were in addition preincubated with or without MGO. Figure 4A and B shows the adhesion of both meningioma cell lines on the two different matrices. No significant difference in adhesion could be detected in both cell lines after MGO treatment. Please note, that we observed that BEN-MEN-1 cells adhere much better on both substrates compared to IOMM-Lee cells and that both cancer cells prefer fibronectin to collagen IV as substrate.
Adhesion of meningioma cells with MGO-treatment.
BEN-MEN-1 (BM1) (A) and IOMM-Lee (IOMM) (B) cells were seeded in absence (Ctrl) or presence of 0.3 mM MGO on collagen IV (left) or fibronectin (right). Graphs display adhesion on collagen IV and fibronectin with or without treatment with untreated or treated cells after 2 h. Gray and black bars in (A) and (B) represent the untreated matrix with untreated cells as control. Untreated matrix and treated BEN-MEN-1 cells (BM1 Gly; light green bar) or treated IOMM cells (IOMM Gly; yellow bar). The green (BM1 + Mtx Gly) and orange (IOMM + Mtx Gly) bars show cell adhesion of treated matrix with untreated BEN-MEN-1 and IOMM-Lee cells. Dark green (BM1 Gly + Mtx Gly) and red (IOMM Gly + Mtx Gly) bars represent treated matrix with treated BEN-MEN-1 and IOMM-Lee cells. Error bars represent SD (n = 4), (Mtx = Matrix; Gly = MGO treatment).
MGO has no effect on chemotaxis of meningioma cells
Since MGO leads to glycation of almost all cellular proteins, we next wanted to analyze whether cell surface receptors are in general inactivated after glycation. We therefore investigated the impact of glycation on the chemotaxis, which is mediated by cell surface receptors. We cultured both meningioma cell lines in CIM-plates for 24 h. Cells were cultured again in the absence or presence of 0.3 mM MGO. Figure 5A and B shows the chemotactic cell migration after 12 h or 24 h. Treatment with MGO had no effect, neither on BEN-MEN-1 (Figure 5A) nor on IOMM-Lee cells (Figure 5B). Please note that chemotaxis of malignant IOMM-Lee cells is higher in contrast to benign BEN-MEN-1 cells.
Chemotaxis of meningioma cells.
BEN-MEN-1 (A) and IOMM-Lee (B) were cultured for 24 h in the absence (Ctrl) or presence of 0.3 mM MGO. Graphs display relative chemotaxis (presented as cell indices) with treatment (MGO) or without treatment (Ctrl) for 12 and 24 h. Error bars represent SD (n = 4).
Glycation increases invasiveness of the benign cell line and decreases the invasiveness of malignant cell line
Since invasiveness is the most important parameter in most tumors, we finally wanted to examine whether there are changes in invasiveness due to glycation. From our chemotaxis experiments, we knew already that glycation does not interfere with the function of cellular receptors in general. We therefore cultured meningioma cell lines in CIM-plates for 48 h on matrigel in absence or presence of 0.3 mM MGO. Figure 6A displays the invasion over 48 h for both cell lines. Untreated IOMM-Lee cells are more invasive compared with BEN-MEN-1 cells. BEN-MEN-1 (Figure 6B) cells had a significantly increased invasion after MGO-treatment after 24, 36 and 48 h compared with the untreated controls. This effect could be also observed in another benign meningioma cell line (HBL-52) (data not shown). Interestingly, the malignant cell line (IOMM-Lee, Figure 6C) showed a significant reduction in their invasiveness over 48 h.
Invasion of meningioma cells.
BEN-MEN-1 (A, B) and IOMM-Lee (A, C) were cultivated for 48 h in absence (Ctrl) or presence of 0.3 mM MGO. Graphs display invasion (presented as cell indices) with treatment (MGO) or without treatment (Ctrl) for 12 h, 24 h (BEN-MEN-1: p = 0.012; IOMM-Lee: p = 0.0347), 36 h (BEN-MEN-1: p = 0.0112; IOMM-Lee: p = 0.0377) and 48 h (BEN-MEN-1: p = 0.0324; IOMM-Lee: p = 0.0374). In the graphs (B) and (C) normalized cell indices were shown for both cell lines relative to the controls. Statistical analysis was performed using t-test. Error bars represent SD (n = 4; *p < 0.05).
Glycation influences the expression of E- and N-cadherin in benign meningioma cells
Finally, we wanted to investigate whether MGO treatment influences the expression of members of the cadherin family, which are involved in epithelial-mesenchymal or mesenchymal-epithelial-transition. Therefore, we cultured meningioma cells in absence or presence of 0.3 mM MGO for 24 h and performed immunoblotting using anti-E- and N-cadherin antibodies (Figure 7). The expression of E- and N-cadherin in IOMM-Lee was not changed after glycation (Figure 7A–D). However, glycation of BEN-MEN-1 cell line resulted in increasing expression of E-cadherin (Figure 7A and B) and decreasing expression of N-cadherin (Figure 7C and D).
Cadherin expression of meningioma cells with and without MGO-treatment.
(A) BEN-MEN-1 and IOMM-Lee were cultured in the absence or presence of 0.3 mM MGO and E-cadherin was analyzed by Western blotting. (B) Quantification of E-cadherin expression in BEN-MEN-1 (p = 0.0406) and IOMM-Lee cells before (Ctrl) and after treatment with 0.3 mM MGO (n = 3). (C) BEN-MEN-1 and IOMM-Lee were cultured in the absence or presence of 0.3 mM MGO and N-cadherin was analyzed by Western blotting. (D) Quantification of N-cadherin expression in BEN-MEN-1 (p = 0.0276) and IOMM-Lee cells before (Ctrl) and after treatment with 0.3 mM MGO (n = 7). All statistical analysis (B, D) were performed using t-test. Error bars represent SD (*p < 0.05).
Discussion
Little is known about the role of MGO and glycation on meningioma cells. In this study, we analyzed the effect of MGO-treatment and glycation on two meningioma cell lines, representing the WHO grade I (BEN-MEN1) and the WHO grade III (IOMM-Lee). We could show that treatment with MGO led to glycation and modulated the invasiveness of both meningioma cell lines. However, glycation did not affect cell adhesion and chemotaxis of these cell lines.
Up to 90–99% of cellular MGO is bound in vivo to macromolecules; however, cellular concentrations up to 0.3 mM have been reported (Chaplen 1998; Chaplen et al. 1998; Thornalley 1996). Our data on cell viability using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay showed that meningioma cells have no significantly reduced metabolism at physiological concentration of 0.3 mM MGO, but are influenced by MGO at high non-physiological concentrations. This could be confirmed via microscopy, where fewer cells and less cell spreading were observed and in immunoblotting in high concentrations, we could detect the cleaved- caspase-3 activity, which is one of the key player of programmed cell death (i.e. apoptosis) (data not shown). Similar observations have been shown in human glioblastoma multiforme T98G, U87MG cells and SH-SY5Y neuroblastoma cells, where MGO interfered with proliferation (Paul-Samojedny et al. 2016; Yin et al. 2012). Another study showed that MGO affects rat schwannoma RT4 cells, PC12 cells and U87 glioma cells in cell viability by decreasing of the key signaling pathway for cell survival (gp130/STAT3 signaling) and as a consequence promotes cytotoxicity (Lee et al. 2009).
MGO led in our hands to detectable glycation of meningioma cell proteins. The influence of glycation on adhesion, migration, invasion and apoptosis of cancer cells could be demonstrated in several recent studies (He et al. 2016; Loarca et al. 2013; Scheer et al. 2020). Our data show that cell adhesion of meningioma cells is not altered at physiological relevant MGO concentrations. Meningioma cells prefer fibronectin as substrate, what is in line with their integrin expression (α4, 5, 6 and β1, 3, 4) (preliminary data not shown), which are also known to be involved in proliferation, adhesion, migration and invasion in meningiomas (Bello et al. 2000; Chen et al. 2009; Gogineni et al. 2011; Nigim et al. 2019).
Our data on chemotaxis indicate that glycation of meningioma cells does not lead to a general loss of function. MGO-treatment seems to have cell-specific effects on behavior, since migration was reduced in liver and colon cancer cells and increased in neuroblastoma cells (He et al. 2016; Loarca et al. 2013; Scheer et al. 2020).
However, our results from the invasion of the meningioma cells indicate that MGO-treatment leads to a higher degree of invasiveness in benign meningioma cells, consequently to increased aggressiveness of meningioma cells. Antognelli et al. have shown that MGO treatment increases the invasion in anaplastic thyroid cancer (ATC) cells (Antognelli et al. 2019). This observation was also confirmed in neuroblastoma cells (Scheer et al. 2020). In liver cancer, it has been shown that invasion was inhibited by MGO treatment (Loarca et al. 2013). However, one has to keep in mind that the concentrations of MGO treatments were different in most studies. Low concentration of 5 µM MGO resulted in an increased invasion in ATC cells and a decreased invasion in liver cancer cells (Antognelli et al. 2019; Loarca et al. 2013). In neuroblastoma cells, increased invasiveness occurred after treatment with 0.1 mM MGO (Scheer et al. 2020).
Canel et al. proposed an E-cadherin–integrin crosstalk during cancer invasion and metastasis (Canel et al. 2013). Although Utsuki and colleagues have shown that low expression of E-cadherin is associated with invasive meningioma (Utsuki et al. 2005), we found high expression of E-cadherin in the malignant grade III IOMM-Lee cells and low expression in the benign grade I BEN-MEN-1 cells (see blots in Figure 7). Glycation led to increased expression of E-cadherin in BEN-MEN-1 cells, which fits nicely to the increased invasiveness after glycation. On the other hand, we found decreased expression of N-cadherin in BEN-MEN-1 cells. Asano and colleagues reported for gliomas, that an increased expression of N-cadherin correlated with a decreased invasiveness (Asano et al. 2004). Another study has shown that a decreased expression of N-cadherin resulted in a faster and less-directed migration of tumor cells (Camand et al. 2012). In our model, it appears that increased expression of E-cadherin and a decreased expression of N-cadherin are associated with a more invasive behavior. Many studies suggest a correlation between N-cadherin expression and matrix metalloproteinase-9 (MMP-9) (Hsu et al. 2016; Suyama et al. 2002; Walker et al. 2014). Preliminary data suggest an up-regulation of MMP-9 in BEN-MEN-1 cells after treatment with 0.3 mM MGO (data not shown). Although this has to be validated, one could speculate this as one reason for the increased invasion of this cell line.
Further studies should include expression analysis of the receptor of advanced glycation endproducts (RAGE), since Dai and colleagues showed that the AGE-induced RAGE-signaling pathways promotes development and progression of meningiomas (Dai et al. 2018) and several other studies suggested also an involvement of RAGE in tumor progression (Abe and Yamagishi 2008; Ahmad et al. 2018; Takino et al. 2010). In our hands, RAGE expression was increased in both cell lines after glycation (data not shown).
In summary, we propose that glycation has a specific effect on different cancer cells. Glycation promotes invasiveness of a WHO grade I meningioma cell line in vitro, but decreases invasive behavior in a WHO grade III meningioma cell line. Further studies are necessary, which could include strategies for “de-glycation”, which may be important for future cancer treatment.
Materials and methods
Cell culture
The human benign meningioma cell line BEN-MEN-1 was obtained from Leibniz-Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) and the human malignant meningioma cell line IOMM-Lee (ATCC® CRL-3370™) was obtained from American Type Culture Collection (ATCC, Manassas, USA). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 μg/mL of streptomycin, 100 U/mL of penicillin, 4 mM glutamine and 10% fetal bovine serum (FBS, Sigma Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO2 incubator. The cell lines were split every 2–3 days with 0.1% Trypsin-ethylenediaminetetraacetic acid (EDTA) solution for 2 min.
Cell viability and cell morphology assays
The cell viability of glycated BEN-MEN-1 and IOMM-Lee cells was measured using an MTT (Sigma Aldrich) assay. Both cell lines were seeded into 96-well plates at a density of 7.8 × 104/cm2 cells per well in DMEM with 1% FBS. After 2 h of attachment, cells were treated with different concentrations of MGO (Sigma Aldrich, 40% aqueous solution; diluted in 1× PBS; 0.1, 0.3, 0.6 and 1.0 mM). Controls (Ctrl) were cells (BEN-MEN-1, IOMM-Lee) without MGO treatment. Cell lines were cultivated for 24 h. Morphology of the cells was acquired with bright field microscopy (Axiovert 100, Carl Zeiss AG, Oberkochen, Germany). MTT was diluted to a final concentration of 0.5 mg/mL in normal growth medium and cells were incubated for 2 h with 100 μL MTT solution per well. After elimination of MTT-containing medium, residual formazan crystals were dissolved in 150 μL dimethyl sulfoxide (DMSO). The absorption values were measured (Plate-Reader, Clariostar, BMG Labtech GmbH, Ortenberg, Germany) at a wavelength of 570 nm (background 630 nm). The untreated control cells were set to 1 of cell viability. The changes in cell viability of the treated cells were calculated in relation to the untreated control.
Glycation and immunoblotting
Cells were seeded in 12-well plates at a density of 3.95 × 104/cm2 in DMEM with 1% FBS. After 2 h of attachment, the cells were treated with different concentrations of MGO (0.1, 0.3, 0.6 and 1.0 mM). Controls (Ctrl) were cells (BEN-MEN-1, IOMM-Lee) without MGO treatment. The cell lines were cultivated for 24 h. Cells were directly lysed in hot SDS-sample buffer (2.5% sodium dodecyl sulfate, 0.06 M TRIS (tris(hydroxymethyl)aminomethane) pH 6.8, 10% glycerin, 0.01% bromophenol blue, 10 mM dithiothreitol in TBS-T (TRIS-buffered saline/0.1% Tween)) to isolate the total protein. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 10%) and transferred to a nitrocellulose membrane using Western blot techniques. The monoclonal anti-AGE antibody Carboxymethyllysine (CML)-26 (0.05 μg/mL, Abcam, Cambridge, UK) together with the secondary peroxidase-coupled antibody (ImmunoResearch Inc., Eagan, USA) was used to detect the glycation. Detection of E- and N-cadherin expression were done with monoclonal anti-E-cadherin antibody (0.05 μg/mL, Abcam, Cambridge, UK) and with monoclonal anti-N-cadherin antibody (0.0483 μg/mL, Abcam, Cambridge, UK) and a secondary peroxidase-coupled antibody (ImmunoResearch Inc., Eagan, USA). Images were taken using Chemidoc MP imaging system (Bio-Rad Laboratories, Hercules, USA). Ponceau S staining (0.1% Ponceau S, 3% trichloroacetic acid and 3% sulfosalicylic acid) of total loaded protein and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (0.04 μg/mL, Santa Cruz Biotechnology Inc., Dallas, USA) were used as loading controls. Band intensity of proteins of interest were transformed into numeric values using Image lab software (Bio-Rad Laboratories, Hercules) and normalized to the corresponding ponceau S staining to quantify the results.
Examination of adhesion with real time cell analysis
Fibronectin and collagen IV at a concentration of 10 μg/mL were used for each experiment and were added to the wells of the 96X E-plates® (ACEA Biosciences, San Diego, USA) and incubated for 1 h at 37 °C. E-plates® have gold microelectrode biosensors in each well of ACEA’s electronic microtiter plates. After a washing step with PBS, the wells were incubated with 0.5% BSA solution in PBS for 20 min. Before the cells and media were added, the wells were washed with PBS. The cells were trypsinized and detached. The reaction was stopped and the cells were resuspended in media with 1% FBS. 50 µL serum-free media was added to every well in order to measure the background signal. Cells were added in density of 1.5625 × 104/cm2. The adhesion of cell lines was measured as changes in impedance with the Real Time cell electronic sensing (RT-CES®) system (ACEA Biosciences) and monitored every 15 min for a period of 4 h. The measurement was done with the Real Time Cell Analyzer dual purpose (RTCA DP) Analyzer (ACEA Biosciences,) and displayed with the Real Time Cell Analyzer (RTCA) program 2.0 (ACEA Biosciences,) as Cell Index (CI). The index is calculated as follows: CI = (impedance at time point n − impedance in the absence of cells)/nominal impedance value.
Examination of chemotaxis with real time cell analysis
Chemotaxis was analyzed in 96X Cellular invasion and migration (CIM)-plates (ACEA Biosciences). The CIM-plates are composed of an upper and a lower chamber. The bottom surface of the upper chamber consists of a microporous membrane where cells can migrate through. Underneath the membrane, a gold electrode detects the presence of adherent cells. 160 µL DMEM with 20% FBS were added in the lower chamber. 50 µL DMEM with 1% FBS were added in the upper chamber. CIM-plates were incubated for 1 h at 37 °C, followed by background measurement. Cells were trypsinized, detached, the reaction was stopped and the cells were resuspended in media with 1% FBS. Cells were added to the upper chamber in density of 7.8 × 104/cm2. The chemotaxis on every label was measured as changes in impedance with the RT-CES® system and monitored every 15 min for a period of 24 h. The measurement was conducted with the RTCA DP Analyzer (ACEA Biosciences) and displayed with the RTCA program 2.0 (ACEA Biosciences).
Examination of invasion with real time cell analysis
Invasion was analyzed in 96X CIM-plates (ACEA Biosciences). The CIM-plates are composed of an upper and a lower chamber. The bottom surface of the upper chamber consists of a microporous membrane where cells can migrate through. On the underside of this membrane a gold electrode detects the presence of adherent cells. To investigate the invasion, 800 μg/mL Basement Membrane Matrix, lactose dehydrogenase-elevating virus (LDEV)-free Matrigel® (Corning, Minneapolis, MN, USA) were added in the upper chamber. After an incubation for 4 h at 37 °C, 160 µL DMEM with 20% FBS were added in the lower chamber and 50 µL DMEM with 1% FBS were added in the upper chamber. The CIM-plates were incubated for 1 h at 37 °C. Afterwards, the background signal was measured. Cells were trypsinized and detached. The reaction was stopped by adding media with 1% FBS and the cells were resuspended. Cells were added to the upper chamber in density of 1.1 × 105/cm2. Invasion was measured as changes in impedance with the RT-CES® system and monitored every 15 min for a period of 48 h. The measurement was carried out with the RTCA DP Analyzer (ACEA Biosciences) and displayed with the RTCA program 2.0 (ACEA Biosciences).
Statistical analysis
All analyses and visualizations were performed using OriginPro 2019 software (OriginLab Corporation, Northampton, USA). Paired student’s t-test against the control group, both cell lines or a theoretical value of 1 (due to data normalization) were executed. Figures show the average mean with standard deviation (SD) and levels of significance are represented within the Figures.
Funding source: Wilhelm Roux -program
Award Identifier / Grant number: FKZ 31/21
Funding source: Deutsche Forschungsgemeinschaft (DFG)
Award Identifier / Grant number: ProMoAge RTG 2155
Acknowledgment
Special thanks to Dr. Heidi Olzscha for critical reading of the manuscript.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This research was funded by Wilhelm Roux-program, FKZ 31/21 and Deutsche Forschungsgemeinschaft (DFG, ProMoAge RTG 2155).
Conflict of interest statement: The authors declare no conflict of interest. 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.
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