Patient consent and study inclusion
The study was approved by the Institutional Review Board of Asan Medical Center (IRB; protocol No. 2017 − 0837) and performed according to the ethical principles of the Declaration of Helsinki. All patients gave written informed consent before being included in the study.
Animal study ethics
All animal studies were conducted in accordance with the regulations formulated by the Institutional Animal Care and Use Committee (IACUC; Approval No. 2018-12-151) at Asan Medical Center. All animal care and experimental procedures were carried out in accordance with relevant guidelines and regulations of the Institutional Animal Care and Use Committee (IACUC) at Asan Medical Center.
Generation of anti-CCSP-2 scFv antibody
Three White Leghorn chickens were immunized and boosted three times with 20 µg of CCSP-2-His protein. The spleen, bursa of Fabricius, and bone marrow were harvested for total RNA isolation using TRI Reagent (Invitrogen, CA, USA). Complementary DNA was synthesized using the Superscript® IV First-Strand Synthesis system (Invitrogen, CA, USA) to generate scFv-displaying phage libraries as described previously (C.F. Barbas, Phage Display: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). The libraries were subjected to five rounds of bio-panning with CCSP-2-conjugated magnetic beads. Individual phage clones were selected randomly from the output titration plate of the last round, and scFv-displaying phages were subjected to enzyme-linked immunosorbent assay (ELISA) as described previously 41.
Expression and purification of anti-CCSP-2 scFv-Cκ fusion protein
The gene encoding anti-CCSP-2 scFv was subcloned into modified pCEP4 mammalian expression vectors to express human constant kappa region (Cκ) fusion proteins as described previously 41. The C-terminal cysteine residue of Cκ was excluded to abolish the dimerization of scFv-Cκ fusion proteins through disulfide bond formation. The construct was transfected into HEK293F cells, and scFv-Cκ fusion proteins were purified using KappaSelect resin (GE Healthcare) as described previously 42. FITC conjugation was performed by BioActs (Incheon, Korea).
Cell lines
The HCT116 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in DMEM (HyClone, UT, USA) with 10% FBS (HyClone, UT, USA) and 1% Antibiotic-Antimycotic (AA; Gibco Laboratories, MD, USA). The FreeStyle 293-F cell line was purchased from Thermo Fisher Scientific (MA, USA). CCSP-2-overexpressing HCT116 cells (HCT116/CCSP-2) and control HCT116 (HCT116/pcDNA) cells were established from the transfection of CCSP-2-V5/His-tagged plasmid vectors with Lipofectamine 2000 (Invitrogen, CA, USA) 23. Transfected cells were selected with 200 µg/mL geneticin (G418; Gibco Laboratories, MD, USA) in DMEM/F12 supplemented with 10% FBS and 1% AA.
Immunoblot analysis
Colorectal cancer cell lysates were prepared in RIPA lysis buffer (Thermo Fisher Scientific, MA, USA) with protease inhibitor cocktail (Gendepot, Barker, TX, USA). Cell lysates and culture supernatant samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. Protein-transferred membranes were blocked with 5% skim milk in Tris-buffered saline Tween 20 (TBS-T) blocking buffer for 1 h at room temperature and incubated at 4 °C overnight with blocking buffer-diluted anti-CCSP-2 IgG (1:1000) and anti-CCSP-2 scFv (1:1000) as primary antibodies. Anti-mouse IgG-HRP (1:2000; Cell Signaling Technology, MA, USA) for anti-CCSP-2 IgG and anti-human kappa IgG-HRP (1:2000; Novus Biologicals, Littleton, CO, USA) for scFv antibodies were used as secondary antibodies. Anti-β-actin antibody (Sigma-Aldrich Co., MO, USA) was used as the loading control. Protein expression signals were detected with ECL substrate (Thermo Fisher Scientific, MA, USA) and visualized using Luminograph Ⅲ (ATTO Corporation, Tokyo, Japan).
Xenograft anti-CCSP-2 scFv-FITC imaging
HCT116/CCSP-2 and HCT116/pcDNA cell suspensions in 100 µL of PBS with Matrigel (Corning, NY, USA) were subcutaneously injected into 6- to 8-week-old balb/c nude mice (Orient Bio Inc., Seongnam, South Korea). After 20 days, the mice were examined by molecular imaging. The CCSP-2-targeted (n = 6) or control (n = 4) probe was intravenously injected into the tail vein of xenograft mice (1.5 µg/g body weight), and fluorescence images were acquired at 2, 4, 6, and 8 h after i.v. injection. For biodistribution, the molecular images of isolated organs (heart, liver, kidney, spleen, lung, and colon) were acquired immediately after the mice were sacrificed. The molecular imaging of xenograft tumors was performed using the Xenogen IVIS spectrum system (Caliper Life Sciences, Inc., MA, USA).
Immunofluorescence localization of anti-CCSP-2 scFv in tissues and cells
Immunofluorescence staining of CCSP-2 was performed by seeding HCT116/CCSP-2 and HCT116/pcDNA cells (30,000 cells/well) in 8-chamber slides (Thermo Fisher Scientific, MA, USA) and fixation with 4% paraformaldehyde. The cells were washed with PBS and blocked with 5% normal goat serum (Cell Signaling Technology, MA, USA) in 1% BSA in PBS-Tween 20 (PBS-T) as the dilution buffer for 60 min. FITC-conjugated anti-CCSP-2 scFv, and control scFv (5 µg/mL) in dilution buffer were added to each cell line for 60 min after washing with PBS. PBS-washed cells were incubated with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen, CA, USA) to stain the nucleus for 10 min, and the cells were visualized with a LSM880 confocal microscope (Carl Zeiss, Jena, Germany).
Molecular colonoscopic imaging of orthotopic colon cancer mice
Before orthotopic injection was performed, a combined needle was prepared for insertion through the working channel of the colonoscope (Vetcom; Karl Storz, Tuttlingen, Germany). A 30G needle was bound to a 23G needle with a flexible plastic pipe. Then, 1 × 107 HCT116/CCSP-2 cells in 50–100 µL of 10% Matrigel (Corning)/PBS were injected into 6- to 8-week-old balb/c mice (Orient Bio Inc., Seongnam, Korea). Colonic submucosal injection was carefully performed through the working channel of the endoscope. After injection, colonoscopic imaging was performed every week to confirm tumor formation (Video S1A). Colonoscopy was performed using the IMAGE1 H3-Z F1 THREE-CHIP FULL HD Camera system (part TH102), Image 1 HUB CCU (parts TC200EN, TC300), Modified D-Light P VET source (part 66100M3), AIDA HD control system, STRAIGHT FORWARD (part 64301AA), and fluorescent filters (Karl Storz, Tuttlingen, Germany). All acquired optical and fluorescence colonoscopy images were recorded as MP4 video files by the AIDA HD control system (Karl Storz, Tuttlingen, Germany). For i.v. administration, the FITC-conjugated anti-CCSP-2 scFv probe was intravenously injected into the tail vein of orthotopic mice (1.5 µg/g body weight). Molecular (FITC) imaging was performed at 0, 2, 4, and 6 h after injection by fluorescence colonoscopy (Vetcom; Karl Storz, Tuttlingen, Germany). In addition, the molecular images of isolated organs (colon, heart, liver, kidney, spleen, and lung) were acquired immediately after the mice were sacrificed at 6 h after injection using the Xenogen IVIS spectrum system (Caliper Life Sciences, Inc., MA, USA). For topical administration, the FITC-conjugated anti-CCSP-2 scFv probe (30 µg/mL) was intrarectally administered to the orthotopic mice. Fluorescence colonoscopy (Vetcom; Karl Storz, Tuttlingen, Germany) was performed at 10 ~ 15 min after administration.
Ex vivo molecular imaging of human colorectal tumors
Adenocarcinoma and adjacent normal tissues excised from colorectal cancer patients were washed with ice-cold PBS. Excised tissues were loaded using low-melting agarose gels at room temperature until solidified to prevent false positive detection from the dissected tissue surface by sprayed FITC-conjugated scFv antibodies. Incubation with FITC-conjugated scFv antibodies (30 µg/mL) diluted in 1% BSA in PBS-T was carried out at room temperature for 30 min. Tissues loaded on agarose were washed with PBS-T and detected with the Xenogen IVIS spectrum system (Caliper Life Sciences, Inc., MA, USA) at an excitation wavelength of 500 nm and an emission wavelength of 540 nm.
Immunohistochemistry of anti-CCSP-2 IgG in tissues
Immunohistochemistry was performed as described previously 23. In brief, patient colon tissues were fixed with 4% paraformaldehyde and embedded in paraffin. The BenchMark XT automatic immunostaining device (Ventana Medical Systems, Inc., AR, USA) and OptiView DAB IHC Detection Kit (Ventana Medical Systems, Inc., AR, USA) were used for immunostaining. Tissue sections (4 µm) were transferred to salinized, charged slides and incubated at room temperature and 65℃. After epitope retrieval for 64 min, the sections were incubated in an automatic immunostainer with anti-CCSP-2 IgG (1:100) for 32 min. The staining patterns of the slides were visualized with OptiView DAB IHC Detection Kit (Ventana Medical Systems, Inc., AR, USA).
Statistical methods
Data are presented as the mean ± standard error of the mean (SEM), which were analyzed by paired t-test to determine significant differences with GraphPad Prism (GraphPad Software, CA, USA).