Cxcr4

Monoclonal antibodies to p65, as well as phospho-specific p65-NF-κB, MMP-9, CXCR4, cleaved-Caspase-3, β1-Integrin and PARP, were obtained from R&D Systems (Heidelberg, Germany). Antibodies to β-Actin were from Sigma-Aldrich (Taufkirchen, Germany). Secondary rhodamine-coupled antibodies for immunofluorescence and anti-Ki67 were from Dianova (Hamburg, Germany) and alkaline phosphatase-linked antibodies for Western blotting from EMD Millipore (Schwalbach, Germany). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DAPI, 5-Fluorouracil (5-FU), alginate, BMS-345541 and dithiothreitol (DTT) were from Sigma-Aldrich (Taufkirchen, Germany). Stock solutions of BMS-345541 (1000 µM) and of DTT (1000 mM) were prepared in PBS and further diluted in normal cell culture growth medium to obtain final concentrations. Then, 5-FU was diluted as a 1000 µM stock solution in ethanol and further diluted in the cell culture medium. Final concentrations of ethanol did not exceed 0.1% during treatment. TNF-β was purchased from eBiosciences (Frankfurt, Germany). Further, TNF-β was given as a kind gift by Genetech (South San Francisco, CA, USA). Calebin A (CA), with a purity of 99.65%, was a generous gift from Sabinsa Corporation (East Windsor, NJ, USA). Epon for electron microscopy was purchased from Plano (Marburg, Germany).

FITC-conjugated mouse antihuman CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR1, CXCR3, CXCR4, and CXCR5 or unconjugated monoclonal mouse-antihuman CCR1, CCR2, and CXCR6, as well as PE-conjugated rat antihuman CCR8, PE-conjugated rat antihuman CCR10, and PE-conjugated rat IgG2b, were obtained from R&D Systems Europe Ltd. (Abingdon, UK). FITC-conjugated mouse antihuman CX₃CR1 was purchased from Medical and Biological Laboratories Co. Ltd. (Nagoya, Japan). FITC-conjugated monoclonal mouse antihuman CD3, PE-conjugated monoclonal mouse antihuman CD56, and FITC-conjugated goat anti-mouse were purchased from Becton-Dickinson (San Diego, CA, USA). FITC-conjugated mouse IgG, PE-conjugated mouse IgG, unconjugated mouse IgG, and unconjugated rat IgG were obtained from either Becton-Dickinson or from R&D Systems. Pertussis toxin (PTX), MMF, and DMF were obtained from Sigma-Aldrich (Saint Louis, MO, USA). CCL1, CCL27, CCL28, and CXCL10 were purchased from PeproTech (London, UK).

Leukocytes were defined as CD45pos, and the different cell subtypes were defined as CD3pos (T cells), CD19pos (B cells) CD14pos (monocytes) and Cd56pos (NK cells). Cell viability was assessed by propidium iodide staining. Anti-isotype controls (Exbio, Praha, CZ) were performed.
1 × 10⁵ eNK cells were labeled with fluorophore-conjugated antibodies: CD3-PE-Cy7, CD16-PE, CD56-APC, NKG2D-PE, NKG2C-PE, NKG2A-PE, NKp30-PE, NKp44-PE, NKp46-PE, KIR2DL2/3-PE, KIR2DL1-PE (BD, Italy), KIR2DL4-PE (R&D Systems, Italy); CXCR1, CXCR2, CXCR3, CX3CR1, CXCR4, CCR1, CCR2, CCR3, CCR5, and CCR7 (R&D Systems, Italy). eNK cells were gated as CD56pos CD3neg. eNK cells activation status was determined by CD107a staining (R&D Systems, Italy), as previously reported (Rizzo et al., 2016 (link)).
5 × 10⁵ epithelial cells were stained specific Ab HLA-I (HLA-A,-B,-C)-PE (BD Biosciences, Italy), HLA-E (clone MEM-E/08, Exbio, Praha, CZ) or HLA-DR (BD Biosciences, Italy) and matched isotype controls.
The NKG2D-ligands were detected on epithelial cells by binding of NKG2D-Fc chimera (R&D Systems, Italy) and indirect labeling with the secondary Ab FITC-coupled mouse anti-human IgG1 (Abcam, Cambridge, United Kingdom).
Data were analyzed using FACS CantoII flow cytometer (BD, Milan, Italy) and FlowJo LLC analysis software (Ashland, OR, United States). Ten thousand events were acquired.

The proliferation of RL95-2, Ishikawa, and HEC-1A cells grown in 1:1 mix of EMSC-CM and self culture medium-Ctrl were determined using XTT assays on culturing days 0, 2, and 4. The culture medium was changed every 2 days. Additionally, the blocking assay was used with CXCR4 (5 μg/mL) blocking Abs (R&D). The assays were performed using the XTT reagent mixture (Roche, Germany) in accordance with the manufacturer’s protocol.

Cells at the 5th passage of culture in each oxygen tension were detached from flasks using Accutase (Sigma-Aldrich). For identification of stem cell surface markers, cells (1 × 10⁵) were labeled with antibodies against the surface markers CD105 (FITC, mouse, Abcam, Cambridge, UK), CXCR4 (FITC, mouse, R&D systems, USA), G-CSFR (FITC, mouse, R&D systems, USA), IgG1 isotype control (FITC, mouse, Biolegend, USA) and IgG2a isotype control (FITC, mouse, Santa Cruz, USA) for 1 hour, at 4 °C. Labeled cells were analyzed using FACSAria II flow cytometer (Becton Dickinson, USA). Only viable cells as determined by propidium iodide (PI) (Sigma-Aldrich) exclusion were gated and analyzed.