Journal of Molecular Medicine 2012
Electronic Supplementary Material
Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to
antibody-independent and ch14.18-mediated NK cell cytotoxicity
Anastasia Shibina1, Diana Seidel1,2, Srinivas S. Somanchi3, Dean A. Lee3, Alexander
Stermann2, Barry J. Maurer1, Holger N. Lode2, C. Patrick Reynolds1, Nicole Huebener1,2,4*
Supplementary Figures
Fig. S1: Fenretinide treatment of NB cells enhances their susceptibility to NK cells killing.
Overall cytotoxicity was analyzed with expanded NK cells plus ch14.18/CHO antibody
(1µg/ml) and human NB cells that were pre-treated with 10µM 4-HPR (gray bars) for 48 h at
an E:T ratio of 5:1 compared to vehicle-treated controls (black bars). Each bar represents the
mean value for 8 replicates. Results are presented as % cytotoxicity ± SD. All experiments
performed in our studies are shown.
Fig. S2: Fenretinide treatment of NB cells enhances their susceptibility to PBMC-mediated
killing.
Overall cytotoxicity was analyzed with PBMCs from healthy volunteers (donors A to E) plus
ch14.18/CHO antibody (1µg/ml) and human NB cells that were pre-treated with 10µM 4HPR (gray bars) for 48 h at an E:T ratio of 25:1 compared to vehicle-treated controls (black
bars). Each bar represents the mean value for 8 replicates. Results are presented as %
cytotoxicity ± SD. All experiments from our studies performed with CHLA-172, SK-NBE(2), CHLA-79 and CHLA-20 are shown. This graph emphasizes the donor-dependent
differences in cytotoxicity that can be seen independent of the target cell line, e.g. donor C
shows the highest cytotoxic ability towards all 4 target cell lines compared to the other
donors, while donor B’s PBMCs are relatively weak.
Fig. S3: Fenretinide treatment enhances ch14.18/CHO-mediated CDC towards 4-HPRresistant human NB cell lines, while the enhancing effect can be blocked with the GCS
inhibitor PPPP.
The effect of 4-HPR on ch14.18/CHO-mediated CDC by human complement from one
healthy donor was analyzed using 5 human NB target cells after exposure to 4-HPR. CDC
was analyzed with 10% human serum and NB target cells treated with vehicle (black bars), 4HPR (5µM, gray bars) or 4-HPR/PPPP (5µM/1µM, open bars) in the presence of
ch14.18/CHO (10μg/ml) or rituximab control (not shown). Each bar represents the mean
value for eight replicates. The results from all experiments performed are presented.
Fig. S4: Inhibition of GD2-specific
ADCC by expanded human NK cells
but not AICC by the GCS inhibitor
PPPP in cell line CHLA-136.
Human NB cell line CHLA-136 was
treated with 1µM PPPP/5µM 4-HPR
(right column), vehicle (ethanol, final
concentration 0.1%, left column) or
5µM 4-HPR alone for 48h (middle
column). Expanded NK cells were
employed as effector cells at an E:T
ratio of 5:1 in the absence or
presence of ch14.18/CHO antibody
(1µg/ml). Each bar represents the
mean value for 8 replicates. Results
from all experiments performed are
presented as % AICC by NK cells
(black part of stacked bars) and ch14.18/CHO-mediated lysis (gray part of stacked bars) ±
SD.
Fig. S5: Influence of 4-HPR on the expression of the inhibitor of DR-mediated apoptosis
FLICE-inhibitory protein cFLIP.
All 6 human NB cell lines in our panel were treated with 5µM 4-HPR for 48 h. Cells were
harvested and intracellular proteins isolated as described in Material and Methods. Protein
extracts were separated by SDS gel electrophoresis and then blotted onto a PVDF membrane.
Detection of FLIP was performed with a monoclonal anti-human FLIP antibody in blocking
solution over night at 4°C. As a control we employed a monoclonal mouse anti-β-actin
antibody. Blots show the three forms of FLIP, i.e. cFLIP long (55 kDa), p43 FLIPlong (43 kDa)
and FLIPshort (27 kDa).