CEACAM1-3S Drives Melanoma Cells Into NK Cell-Mediated Cytolysis and Enhances Patient Survival

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CEACAM1 splice variants in melanoma

CEACAM1-3S drives melanoma cells into NK cell-mediated cytolysis and enhances patient survival

Supplementary information: detailed material information, methods and supplemental figure legends

Supplementary Material and Methods

Antibody information

The following antibodies were used for Western Blot and Immunofluorescence: mouse anti-human Actin (MP Biomedicals), mouse anti-human CEACAM1 (clone 4/3/17, Aldevron), goat anti-human MICA, goat anti-human ULBP2 (both from R&D Systems), mouse anti-human MICA (BioLegend), HRP-labeled goat anti-mouse (Southern biotech), HRP-labeled rabbit anti-goat (Southern biotech),TRITC-conjugated phalloidine (F-actin, Invitrogen), rabbit anti-human ULBP2 (Santa Cruz Biotechnology), goat anti-mouse Alexa Fluor 488 (Invitrogen), goat anti-rabbit Alexa Fluor 594 (Invitrogen).

For flow cytometric analyses were the following antibodies used:mouse anti-human CEACAM1 (clone 4/3/17),FITC-labeled goat anti-mouse F(ab')2 (Jackson ImmunoReseach), anti-MICA mAb AMO(Bamomab), anti-ULBP2 mAb BUMO1 (Bamomab), Cy5-conjugated goat anti-mouse F(ab´)2 antibody (Jackson ImmunoReseach), anti-human mAb, anti-CD3-PE-Cy-7 (BD Biosciences), and anti-CD56-APC ( BD Biosciences),mouse anti-human ULBP1 (R&D Systems), mouse anti-human ULBP3 (R&D Systems), mouse anti-human CD155-PE (BioLegend), mouse anti-human CD112-PE (BD Bioscineces), PE-conjungated mouse IgG1 isotype control (BD Bioscineces).

To block NK cell-receptors in cytotoxicity experiments the following antibodies were used: mouse anti-human NKG2D (R&D Systems), mouse IgG1 isotype control (R&D Systems), mouse anti-human DNAM-1 (BD Biosciences),

The xCELLigence Assay

The Real-Time Cell Analyzer System (RTCA, xCELLigence, Roche Applied Science) allows real-time in-vitro analyzes of cell proliferation, migration and invasion in the absence of interfering labels. These cellular events are measured by detecting alterations of electric impedance between micro-electrodes caused by attaching and detaching of cells, respectively. The relative changes in impedance are displayed as cell indices (CI) and monitors changes in cell viability, cell shape and adhesion. Cells were seeded 48h before starting the xCELLigence measurement to a confluence of 80%. For migration and invasion analyzes cells were starved with FCS free medium for 24h prior to the experiment. Cell proliferation was determined using E-Plate 16 (ACEA). Background impedance was measured using 50µl of culture medium per well. Afterwards, 2x104 cells were added in 100µl medium. CIM-Plate 16 (ACEA) was used for the measurement of cell migration and invasion. 160µl culture medium was used for the lower and 50µl of starvation medium, in presence or absence of 0.1mM Marimastat (R&D Systems) for the upper chamber. After 30 min of incubation, background impedance was measured and 1.2 x105 cells were added in 100µl starvation medium into the transwells. For the assessment of cell invasion the CIM-Plate 16 was prepared in correspondence to the migration assay, with the exception of using 20µl of growth factor reduced Matrigel (BD Bioscience) diluted with starvation medium (1:60) for the upper chamber, as recommended by the manufacturer. 4h after incubation, 30µl starvation medium was added to the Matrigel-dilution and the background impedance was measured. The cellular events were continuously monitored by serial impedance measurement.

RNA-Isolation and RT-PCR

Total RNA was isolated from cell lines and biopsies using the RNeasy Kit (Qiagen, Hilden, Germany) as described in the manufacturer’s instructions and included DNase I digestion (Fermentas) to avoid endogenous DNA contamination. Afterwards, 1µg RNA was reverse transcribed using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's recommendations. All RT-PCRs were performed in linear range of amplification. Following primers were used: Actin forward 5'-ACCCTGAAGTACCCCAT-3', reverse 5'-TAGAAGCATTTGCGGTG-3'; CEACAM1 Splice variants forward 5’-AACCAAAGCGACCCCATCA-3’, reverse 5’-RTGGGTCATTGGAGTGGTCC-3’.

Western Blot

Proteins were extracted by using Cell Lysis Buffer® (Cell Signaling) and separated by SDS-Page, electroblotted either onto nitrocellulose or PVDF membranes and probed with primary antibodies. Subsequent to washing, membranes were incubated with the adequate secondary antibody linked to horseradish peroxidase. Antibody binding was visualized by using Pierce ECL Western Blot Substrate (Thermo Fisher). For the analysis of soluble MICA and ULBP2, 1.75x105 cells were seeded in a 6-well culture plate. After 24h, culture media was replaced by 1ml starvation media (without FCS) and cells were incubated for additional 48h. Volumes of conditioned media were normalized to cell numbers.

Supporting Information Legends

Supplementary Figure S1.

CEACAM1-3S modulates melanoma cell sensitivity against NK cell-mediated cytolysis. (A) NK cell-mediated cytotoxicity at an E/T (effector to target) ratio of 1:1 measured by using the xCELLigence system two hours after NK cell addition. NK cells were either pre-incubated with an anti-NKG2D mAb (aNKG2D) or with IgG isotype control. Mean values of five independent experiments are presented. All statistically tests were run on unnormalized data. Mean ± SEM; *p<0.05 (B) One representative experiment is shown at various effector to target (E/T) ratios one (I), two (II), three (III) and four (IV) hours after addition of pre-treated NK cells.

Supplementary Figure S2.

CEACAM1 isoforms also modulate surface expression of DNMA-1 ligands in an isoform specific mode of action. (A) CD155 (I), CD112 (II), NKp30L (II) and NKp46L (IV) cell surface expression of control cells compared to CEACAM1 isoform transfectants by flow cytometry. Mean MFI values of four or five independent experiments are shown. All statistically tests were run on unnormalized data. Mean ± SEM; *p<0.05 by Student´s t-test. (B) xCELLigence system based cytotoxicity assay with Ma-Mel-86a cells transfected with empty vector control or CEACAM1-3S. DNAM-1, NKG2D or both receptors were blocked by incubating NK cells with specific monoclonal antibody (aDNAM-1, aNKG2D) or IgG control. Results of one represent experiment at an effector to target ratio (E/T) of 1:1 out of three independent is presented one, two, three and four hours after NK cell addition.