1. Melanoma-Directed Activation of Apoptosis Using a Bispecific Antibody Directed at MCSP and TRAIL Receptor-2/Death Receptor-5 
Yuan He, Djoke Hendriks, Robert van Ginkel, Douwe Samplonius, Edwin Bremer, Wijnand Helfrich 
Journal of Investigative Dermatology 
Volume 136, Issue 2, Pages (February 2016)
DOI: /j.jid
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2. Figure 1
Melanoma-associated chondroitin sulfate proteoglycan (MCSP)-directed apoptotic activity of MCSPxDR5 toward melanoma cells. (a) Schematic diagram of the tetravalent bispecific antibody MCSPxDR5. (b) SK-MEL-28(MCSP+) and (c) DLD1(MCSP–) cancer cells were incubated with MCSPxDR5 (250 ng/ml) in the presence or absence of anti-MCSP antibody (mAb ; 10 μg/ml). Next, cells were stained using a polyclonal phycoerythrin (PE)-conjugated goat anti-mouse antibody and analyzed by flow cytometry. (d) MCSP+ melanoma cell lines and MCSP– colorectal cancer cell line DLD-1 were preincubated with MCSPxDR5 (2.5 μg/ml) or medium at 4 °C for 40 minutes, followed by 2 washes with excess cold phosphate buffered saline. Subsequently, cells were incubated for 18 hours at 37 °C/5% CO2, after which apoptosis was measured by flow cytometry using annexin V-FITC/propidium iodide (PI) staining. (e) MCSP+ and MCSP– cancer cell lines were treated with MCSPxDR5 (1.0 μg/ml) or left untreated, after which apoptosis was measured after 18 hours as in d. (f) MCSP+ and MCSP– cancer cell lines were treated with MCSPxDR5 (1.0 μg/ml) or left untreated, after which cell viability was determined by a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) viability assay after 72 hours. Viability after MCSPxDR5 treatment was calculated as percentage of medium control. (g) MCSP+/DR5+ cell lines MM-RU, SK-MEL-28, and A375M were treated with MCSPxDR5 (250 ng/ml) or left untreated in colony-forming agar assays for 72 hours, after which the number of colonies was determined by counting three fields of view per condition in triplicate. Number of colonies was represented as percentage of colonies in medium control. (h) Representative light microscopic pictures of colony size of A375M cells in medium control versus MCSPxDR5-treated conditions in colony-forming assay and dose-response curve of colony size upon MCSPxDR5 treatment. Bar = 100μm. (i) After approval of the University Medical Center Groningen ethics review board and written informed consent, primary patient-derived melanoma cells were obtained from surgical waste. Primary melanoma cells (n = 11, used before passage 4) were treated with MCSPxDR5 (1.0 μg/ml) or left untreated for 18 hours, after which apoptosis was analyzed by flow cytometry using annexin V-FITC staining. Statistical analysis was performed using two-sided unpaired Student t test. Statistical analysis of primary patient-derived cultures in i was performed using the Mann-Whitney U test. *P < 0.05; ***P < n.s., not significant.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions

3. Figure 2
Antitumor of MCSPxDR5 is augmented by Fc cross-linking. (a) Jurkat (MCSP–/DR5+) cells were treated with MCSPxDR5 in the presence or absence of goat anti-human IgG (cross-linker [CL]; 0.5 μg/ml). Apoptosis was measured after 18 hours by flow cytometry using annexin V-FITC. (b) Melanoma cell line MM-RU was treated with MCSPxDR5 (250 ng/ml) in the presence or absence of CL for 18 hours, after which apoptosis was measured by flow cytometry using annexin V-FITC. (c) Primary patient-derived melanoma cell cultures (n = 11) were treated by the combination of MCSPxDR5 (0.25 μg/ml) and CL. Apoptosis was measured after 18 hours. (d) MM-RU cells were treated with MCSPxDR5 in the presence or absence of pan-caspase inhibitor (z-VAD-fmk, 5 μM) or recombinant human DR5: Fc (5 μg/ml) for 18 hours, after which apoptosis was evaluated by flow cytometry using annexin V-FITC/propidium iodide (PI) staining. (e) RNA silencing was used to selectively knock down DR5 expression in SK-MEL-28 cells, which was confirmed by flow cytometry using an anti-DR5 mAb. (f) SK-MEL-28 cells were treated with either CL alone, MCSPxDR5 plus CL, or DR5 agonistic antibody HGS-ETR2 for 18 hours, after which apoptosis was evaluated by flow cytometry using annexin V-FITC/PI staining. (g) Pre-seeded MM-RU (MCSP+/DR5+) cells or M14 (MCSP–/DR5+) cells were cocultured with parental HEK293 cells or HEk293.CD64 cells and subsequently treated with either medium only, MCSPxDR5, or its IgG4 isotype variant MCSPxDR5-4 for 24 hours. Apoptosis in melanoma cells was assessed by flow cytometry using annexin V-PI staining. (h) 1,10-dioctadecyl-3,3,30,30–tetramethylindodicarbocyanine (DiD)-labeled A375M cells were treated with either culture medium or MCSPxDR5 (1.5μg/ml) in the presence or absence of anti-MCSP mAb Unbound antibodies were removed by repeated washing steps. Subsequently, parental HEK293 cells or HEK293.CD64 cells were added, after which apoptosis was assessed in DID-labeled cells by flow cytometry using annexin V/PI staining. (i) White blood cell (WBC) immune effector cells (E) of healthy control were obtained after written informed consent and added to preseeded MM-RU target cells (T) at an E:T ratio of 5:1 and treated with a dose range of MCSPxDR5 for 24 hours. Subsequently, the nonadherent immune cells were carefully removed, and the cell viability of MM-RU cells was assessed by an tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) viability assay. Statistical analysis was performed using two-sided unpaired Student t test. Statistical analysis of primary patient-derived cultures in c was performed using the Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < MCSP, melanoma-associated chondroitin sulfate proteoglycan.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions