This study included 10 malignant ascites specimens from patients undergoing ovarian cancer resection. By adjusting the pH of the cell culture medium, 3D spheroid cells and ovarian tumor cells were cultured, morphological changes were examined by scanning electron microscopy (SEM), and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, adhesion assay, flow cytometry, and scratch assay were used to evaluate metabolic activity, adhesion, anti-apoptosis, and migration abilities of ovarian tumors at various pH. The effect of ascites on tumor cells at different pH was assessed by adding 10% of malignant ascites to the test samples. Blood-gas analyzer for airflow analysis of ascites, 1H-NMR for metabolomic analysis, and flow cytometry for cytokine analysis were used to combine the results for in-depth phenotypic analysis of ascites.
This study was approved by the Ethics Committee, Faculty of Medicine, University of Tübingen, Germany (Ref. Nr. 696/2016BO2 and 117/2020BO1). All patients gave the written informed consent. Sampling did not influence patient treatment. All data were anonymized according to European Data Regulation Regulations (EDPR) and German law.
Collection and storage of patient specimens
Ascites samples were collected from patients undergoing surgery for ovarian cancer at the department of General and Transplant Surgery and at the Women's Hospital, University Hospital Tübingen, Germany. Ascites probes were sampled in the operating room, stored in tubes, and transferred to the laboratory using an ice box. The collected samples were divided into 1 mL tubes and frozen directly in a − 80 °C refrigerator for physical–chemical parameter measurements. The remaining ascites was first centrifuged at 4 °C for 30 min at 1200 rpm and stored in 2 mL tubes at − 80 °C until analysis. Patient information was collected, including standard demographics, cancer histology, and the extent of peritoneal disease. Samples and data were anonymized.
Cell culture and treatment
Human ovarian cancer cells (OAW42, German Cancer Research Center (DKFZ), Heidelberg, Germany) were cultured in DMEM (Thermo Fisher, New York, USA). Monolayered cells were cultured in 175 cm2 flasks (Falcon, Corning, New York, USA) supplemented with 10% fetal bovine serum and penicillin G 100 U/mL, streptomycin 100 μg/mL in a humidified atmosphere of 5% CO2, 95% air at 37 °C. For experimental purposes, cells were cultured in 24-well plates (1.5 mL of cell culture medium or 10% ascites/well) under different pH conditions (pH 6.0, 6.5, 7.0, 7.5) (using phosphate buffered saline (PBS) and phosphoric acid or sodium hydroxide (NaOH)). pH was monitored using a pH meter (SevenCompact™ Duo pH/Conductivity S213, Mettler-Toledo GmbH, Greifensee, Switzerland).
3D spheroid cell model
We first established 3D spheroid-matrigel-based models, in which cell aggregates are grown in suspension. OAW42 cell suspensions were mixed with Matrigel (A1413201, Thermo Fisher, USA), seeded into 96 U-Form plates, and centrifuged at 2,000 rpm for 10 min at 4 °C. Then, 3D spheroids were incubated in a standard cell culture medium for seven days. After 7 days, 3D spheroids were transported to glass plates in 24 well plates, and the cell culture medium was titrated to different pH conditions (6.0, 6.5, 7.0, 7.5) for seven days. The pH was titrated with phosphoric acid or NaOH to reach pre-specified extracellular pH conditions (6.0, 6.5, 7.0, 7.5). The cell culture medium was changed every two days. After seven days of culture in different pH ranges, samples were fixed in 4.5% formaldehyde/2.5% glutaraldehyde in 0.1 M PBS at 4 °C overnight, washed with PBS, post-fixed with 1% osmium tetroxide in PBS, and dehydrated by 30%, 50%, 70%, 95%, and 100% ethanol (EtOH). Then, samples were placed in a critical point dryer (EM CPD300, Leica Microsystems, Wetzlar, Germany) and coated with platinum in a sputter (EM ACE200, Leica Microsystems, Wetzlar, Germany). The dried and coated samples were then observed by SEM (Phenom G2pro and Software Phenom ProSuite, Phenom-World BV, Eindhoven, The Netherlands).
Cell metabolic activity assay
After seeding OAW42 cells, the pH of the cell culture medium was titrated daily to remain at a constant pH value which was measured by a conventional pH meter (SevenCompact™ Duo pH/Conductivity S213, Mettler-Toledo GmbH, Greifensee, Switzerland). The metabolic activity of OAW42 cells was determined by a MTT Assay (R&D Systems, 4890-050-k, Germany). Viable cancer cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce a tetrazolium salt MTT to formazan. The MTT assay is a colorimetric assay based on the yellow MTT changing to purple formazan crystals in metabolically active cells. After seeding every cell line to three 96-wells plates overnight. One plate was incubated for four hours at 37 °C with 20 μL MTT reagent (R&D Systems, 4890-25-01, Germany) until the purple dye was visible. 100 µL of detergent reagent (R&D Systems, 4890-25-02, Germany) was added, and the cells were kept at RT overnight. The following day, the absorbance in each well was measured at 560 nm using the NanoQuant Infinite M200 Pro microplate reader (Tecan Austria GmbH, Grödig, Austria). Half of the remaining two 96-well plates were supplemented with 10% human ascites. The MTT assay was repeated after 24 h and 48 h.
The influence of pH on the adhesion of OAW42 cells was determined in vitro by a colorimetric cell adhesion assay (The CytoSelect 48-Well Cell Adhesion Assay Kit, CBA-070, USA). The absorbance was determined with the NanoQuant Infinite M200 Pro microplate reader (Tecan Austria GmbH, Grödig, Austria). After titrating the pH of the cell culture medium and supplementing 50% of the samples with 10% human ascites, OAW42 cells were grown in the incubator for 12, respectively, 24 h. Cells were suspended in serum-free media, and the suspension was pipetted into 40 ECM protein-coated (Fibronectin, Collagen I, Collagen IV, Laminin I, Fibrinogen) wells and 8 Bovine serum albumin (BSA)-coated wells and then incubated in a cell culture incubator. After PBS-washing, cells were stained by Cell Stain Solution (The CytoSelect 48-Well Cell Adhesion Assay Kit, CBA-070, USA). Then, cells were washed with deionized water, the supernatant extracted, and cells were transferred to a 96-well plate. Optical density was then measured by NanoQuant Infinite M200 Pro microplate reader (Tecan Austria GmbH, Grödig, Austria) at a wavelength of 560 nm.
Flow cytometry assay
The effect of increasing or reducing the extracellular pH in ascites on cell resistance to apoptosis of OAW42 cells was measured in vitro by flow cytometry (FACS). Apoptosis was measured with the Annexin V- Fluorescein (FITC) Apoptosis detection kit (Thermo Fisher, BMS500FI, USA). OAW42 cells were kept in a cell culture medium at different pH for 12, respectively, 24 h. 50% of the wells were supplemented with 10% human ascites. Cold PBS and binding buffer washed resuspend cells, then mixed with Annexin V-FITC and PI at RT. According to the protocol of the manufacturer, cells were stained with propidium iodide (PI)/annexin V-FITC (annexin V-fluorescein isothiocyanate). Stained cells were acquired on flow cytometry (FACS Canto II, Becton Dickinson GmbH, Heidelberg, Germany).
Wound scratch assay
The migratory ability of cancer cells at different extracellular pH was measured using a scratch wound assay. OAW42 cells were seeded in a 24-well plate overnight. A standardized wound was scratched with a 10 ul pipette. Cells were washed twice with PBS, half of the wells were supplemented with 10% human ascites, and the cell culture medium was titrated to different pH (6.0 6.5 7.0 7.5). The plates were placed into an incubator at 37 °C, and cell migration was monitored in real-time by micro-cinematography (zen Cell-Owl, Bremen, Germany). The extent of cell migration at 6, 24 and 36 h was expressed as the wound width of the scratch relative to the initial scratch, expressed as %.
Gene expression omnibus (GEO) databases
The mRNA expression comparison with primary and ascites cells was done by downloading respective data sets from the GSE73064 database  and processing them by standard methods. To characterize the effect of ascites on ovarian cancer cells, we performed a differential analysis (Log2|FC|> 1, p < 0.05) by comparing ascites cancer cells to primary cancer cells by R language using the "limma" package. In the gene set enrichment analysis (GSEA) , the statistical significance was defined as p < 0.05, and the overrepresentation of indicated hallmark gene sets in the ranked gene lists presented by the normalized enrichment score (NES). Gene ontology (GO) analyses were conducted for the selected common differentially expressed genes (DEGs) using the R language, p < 0.05.
Physical–chemical characterization of ascites
150 µl of each ascites sample were taken for a measurement of an industry-standard blood gas analyzer (GEM PREMIER4000, Werfen, Germany). Outcome parameters were pH, pCO2, glucose, lactate, and electrolytes (Na+, K+, Ca2+, Cl−).
1H-NMR spectroscopy equipment and spectra acquisition
1H-NMR spectroscopy (Bruker Avance III HD) was operated at 600 MHz (14.1 Tesla) with a 3.0 mm probe at 298 K using Carr-Purcell-Meiboom-Gill pulse programs (CPMG). CPMG spectra were pre-processed by Bruker TopSpin 3.6.1 and quantified using Chenomx NMR suite 9.02 software.
Metabolite extraction of ascites for metabolomics analysis
All the ascites of OCs were thawed, and 2 mL of each ascites was pipetted to the Eppendorf tube for centrifugation at 30,000G for 10 min. 500 μL of each supernatant were transferred to a Covaris tube (Covaris, Woburn, USA) and placed in SpeedVac (Thermo Fisher, SPD300DDAA-230, USA) to evaporate unwanted solvents for 3 h. 300 μL of methanol and tert-butyl methyl ether (MTBE) were added to the Covaris tube using filter-containing pipette tips for total lipid extraction and vortexed into a homogeneous solution. A total of 5 min ultrasound extraction was used for each sample in the Covaris Ultrasonicator E220 Evolution instrument (Covaris, Woburn, USA). After ultrasonication, 250 μL of molecular biology water (ultra-pure grade) were added and tubes were centrifuged at 12,000G for 10 min to obtain polar and non-polar (lipid) phases. Lipid and polar phase were separated to glass vials and Polytetrafluorethylene (PTFE) tubes, respectively.
Metabolomics analysis of polar metabolites by 1H-NMR spectrometry
210 μL of deuterated 1 mM 3-(trimethylsilyl)propionic-2,2,3,3 acid sodium salt D4 (TSP-D4) buffer (K2HPO4 in D2O + 10 mM NaN3), adjusted to pH 7.44, was pipetted to each polar sample dried pellet in Eppendorf tube. The Eppendorf tubes were vortexed until the samples were dissolved and then bathed under ultrasound for seconds. Next, the PTFE tube was centrifuged at 30,000G for 30 min. The supernatants were pipetted to a 3 mm NMR spectrometer compatible tube (Bruker Biospin, Reinstatten, Germany) using two pipetting steps with 95 μL each. All the NMR tubes were then fixed with a cap sealing ball and wiped with fusel-free tissue right before placing them inside the NMR spectrometer autosampler unit. The polar samples spectra were recorded using Nuclear Overhauser Spectroscopy (NOESY 1D) and CPMG experiments; the duration of 7 min and 2 h, respectively. The CPMG spectra were chosen for analysis due to the better signal-to-noise ratio (SNR) and lipid signal suspression compared to 1D NOESY spectra.
Lipid metabolite sample preparation for metabolomics analysis
500 μL of each lipid aliquot in MTBE solvent was transferred to a glass high performance liquid chromatography (HPLC) vial and evaporated to dryness in a vacuum dryer 200 μL of deuterated chloroform solution with an internal 1 mM tetramethylsilane (TMS) standard was added to the dried lipid pellet in a glass vial, and thoroughly mixed vortexed. 200 μL of each sample was pipetted into a 3 mm NMR tube using a round solvent-safe tip; 100 μL of each lipid sample was pipetted twice. All the NMR tubes were then fixed with a cap sealing ball and wiped with fusel-free tissue right before placing them inside the NMR autosampler. Lipid samples were analyzed by 1H NMR using a 1'hour lasting simple proton experiment (zg30) and 1 h lasting J-coupling resolved spectroscopy (JRES) experiment. Proton experiments were chosen for spectral assignments of lipid metabolites.
Quantification of cytokines present in malignant ascites
To measure the cytokines levels, 25 μL of ascites of OCs was mixed with 25 μL of assay buffer. Then, 25 μL of 13-plex-beads were pipetted to a 96-well microplate (LEGENDplex™ Human Inflammation Panel 1 (13-plex) #740,809, BioLegend, USA). This assay can quantify a total of 13 cytokines/chemokines (the minimum detectable concentration (MDC) in brackets: IL-1β (1.5 + 0.6 pg/ml), IFN-α2 (2.1 + 0.2 pg/ml), IFN-γ (1.3 + 1.0 pg/ml), TNF-α(0.9 + 0.8 pg/ml), MCP-1 (1.1 + 1.2 pg/ml), IL6 (1.5 + 0.7 pg/ml), IL-8 (2.0 + 0.5 pg/ml), IL-10 (2.0 + 0.5 pg/ml), IL-12p70 (2.0 + 0.2 pg/ml), IL17A (0.5 + 0.pg/ml), IL-18 (2.0 + 0.5 pg/ml), IL-23 (1.8 + 0.1 pg/ml), IL-33 (4.4 + 1.5 pg/ml)). The microplate was then incubated and shaken for 2 h at room temperature in which the analytes (cytokines) bound to an antibody-conjugated capture bead. After washing, biotinylated detection antibodies (25 μL) were added and bound to the analytes. Streptavidin–phycoerythrin (25 μL) was subsequently added that bound to the antibodies and provided a fluorescent signal with intensities in proportion to the amount of the bound analyte. After 1 h incubation, beads were washed and flow cytometer was used to quantified the fluorescent signal, and concentrations of the analytes were determined based on a known standard curve using LEGENDplex™ data analysis software (BioLegend, USA).
All the data were normalized to a pooled sample from group probabilistic quotient normalization (PQN), and log-transformation was applied. Univariate, multivariate, and correlation analysis was performed using the MetaboAnalyst 5.0 toolbox (https://www.metaboanalyst.ca), where due to the low number of total samples the comparison was made based on combining clinical stage II-III versus stage IV.
The volcano plot was used to analyze the polar and lipid metabolites present in the OC with II-IV. A value of p < 0.05 and fold change (FC) cut-off > 1.2 was considered significant.
Principle component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) score plots were used to see how each OC stage samples clustered to another. PCA and OPLS-DA loading plots and PCA bi-plot were further analysed for specific metabolite involvement in OC progression. The correlation analysis was carried out to observe correlations between certain lipid metabolite species, polar metabolite, pCO2, pH, and cytokines. The selected cytokines and polar metabolites were IL-8, glutathione, acetate, 3-hydroxybutyrate, glycerol, and lactate, respectively.
This was an exploratory study without prior sample size calculation. Data were first visualized with pair plots (Seaborne, Anaconda, Berlin). Descriptive statistics and correlations were calculated with R, GSEA, Prism GraphPad, and SPSS software. Comparative statistics were performed using t-test, one-way analysis of variance (ANOVA) and two-way ANOVA for normally distributed data, and non-parametric tests for skewed data. Graphical abstract created with BioRender.com.