Cetuximab

Cetuximab (Cat# HY-P9905) was purchased from MedChemExpress (Monmouth, NJ, USA). The human IgG1 isotype (Cat# BE0297) was purchased from BioXCell (Lebanon, NH, USA) and used as a control. The jetOPTIMUS transfection reagent for transient transfection in cells was purchased from Polyplus (Alsace, Grand Est, France). The bifunctional chelating agent [(R)-2-Amino-3-(4-isothiocyanatephenyl) propyl]-trans-(S, S)-cyclohexane-1,2-diamine-pentaacetic acid (p-SCN-Bn-CHX-A-DTPA, Cat# B-355) was purchased from Macrocyclics Inc. (Richardson, TX, USA).

All of the chemicals at the best grade available were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Scientific (Pittsburgh, PA, USA) unless otherwise stated. DNA oligonucleotides were acquired from MBiotech (Hanam, Republic of Korea) and restriction enzymes were purchased from Elpis Biotech (Daejeon, Republic of Korea) and New England Bio Labs (Ipswich, MA, USA). Cell culture reagents were procured from Invitrogen Life Technologies (Carlsbad, CA, USA) and Welgene (Daegu, Republic of Korea). Cetuximab was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and transforming growth factor-α (TGF-α) was purchased from Acro Biosystems (Cambridge, MA, USA).
Confocal fluorescence microscopy images were obtained using an Eclipse Ti (Nikon Instruments, Tokyo, Japan) with excitation wavelengths of 358, 488, and 594 nm using the corresponding filters. Fluorescence in the images was also visualized using a fluorescence microscope (Zeiss Axio Imager Z2, Jena, Germany); the fluorescence intensity was measured using Nikon NIS-Element BR 4.60 software and quantitative analysis was performed using Image J ver.1.53 (NIH, Bethesda, MD, USA).

CD14⁺ cells were purified from frozen PBMCs and CD14-positive selection (Miltenyi Biotec) according to the manufacturer’s protocols. MDMs were generated by seeding three million CD14⁺ cells into one six-well plate (Nunc™, Thermo Fisher Scientific) in IMDM (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific) and M-CSF (50 ng/mL; Miltenyi Biotec) and cultured for 7 to 9 days. Cells were detached from culture plates with Accutase® (Sigma-Aldrich). DLD-1 cells were labeled with the CFSE Cell Division Tracker Kit (BioLegend) according to manufacturer’s instructions. A total of 100,000 DLD-1 cells and 50,000 MDMs were incubated in U-bottom 96-well plates (Corning) with hSIRPα Nbs (1 µM) or KWAR23 (100 nM) and cetuximab (0.66 nM) (MedChemExpress) for 2 h at 37° C, followed by detachment of adherent cells from culture plates with Accutase® (Sigma-Aldrich). For flow cytometry, cells were incubated with CD206 Ab AF647 (clone 15–2, BioLegend) and dead cell marker Zombie Violet (BioLegend). Percent of phagocytosis indicates the percentage of viable CD206⁺CFSE⁺ macrophages.