Anti-carcinoembryonic antigen-related cell adhesion molecule antibody for fluorescence visualization of primary colon cancer and metastases in patient-derived orthotopic xenograft mouse models

Abstract

Background: Monoclonal antibody (mAb) 6G5j is a novel anti-CEACAM monoclonal antibody. Our aim was to investigate mAb 6G5j binding characteristics and to validate fluorescence targeting of colorectal tumors and metastases in patient derived orthotopic xenograft (PDOX) models with fluorescently labeled 6G5j. Materials/Methods: The MAb 6G5j binding profile was analyzed with ELISA, Western blot and immunohistochemistry. MAb 6G5j was conjugated to near-infrared dye IR800CW (LI-COR). Western blotting was performed with various colon cancer cell lysates to determine CEACAM expression. Nude mice received orthotopic implantation of patient-derived primary colon cancer and patient-derived colon cancer metastases. Mice were administered varying doses of 6G5j-IR800CW via tail vein injection and imaged 24 and 48 hours later. Results: MAb 6G5j bound to human CEACAM1, 3, 5, 6 and 8. Western blotting demonstrated varied expression of CEACAMs in 15 of 16 colon cancer lysates. Dose and time-response imaging demonstrated optimal imaging 48 hours after administration of 50 μg 6G5j-IR800CW (Tumor-to-liver ratio (TLR) 3.17, SEM ± 0.45). Primary cancers and multiple metastases were fluorescently visualized. Conclusions: Anti-CEACAM antibody 6G5j binds multiple CEACAMs which may lead to improved detection of tumor margins for tumors and metastases that have variable expression of CEA and other CEACAMs. 6G5j mAb may be useful for colon cancer detection for pre-surgical diagnosis and fluorescence-guided surgery.

Bacterial protein domains with a novel Ig-like fold target human CEACAM receptors

“…To ensure that the CEACAM-observed binding was not influenced by the background of the cell line, we assessed GBS adhesion to CEACAM-expressing CHO cells. (Hollandsworth et al, 2020) Also in this case, a higher percentage of the GBS inoculum was recovered from the CEACAM1-and CEACAM5-expressing CHO cells in comparison to all other CHO cell lines (Fig. 4C).…”

“…HeLa and Chinese Hamster Ovary (CHO) cell lines expressing CEACAMs were cultured as previously described (Bos et al, 1998;Hollandsworth et al, 2020;Daniel et al, 1993), and seeded into 24-well tissue culture plates. Tightly confluent monolayers were infected with midlogarithmic phase (absorbance at OD600 = 0.4) GBS strains at a multiplicity of infection (MOI) of 10.…”

Fluorophore-conjugated Helicobacter pylori recombinant membrane protein (HopQ) labels primary colon cancer and metastases in orthotopic mouse models by binding CEA-related cell adhesion molecules

“…Cell-culture media and media supplements were purchased from Gibco-Life Technology (Eggenstein, Germany) and the chemicals were obtained from Sigma (Taufkirchen, Germany), unless stated differently. Hybridoma secreting the mouse monoclonal antibody (mAb) 6G5j (specific for human CEACAM1/3/5/6/8) have been recently described [ 15 ]. The anti His mAb 2B6 and isotype matched IgG controls were purchased from LeukoCom (Germany) and c-Myc tag specific mAb 9E10 was purchased from AbD Serotec (Germany).…”

“…We have previously demonstrated that CEACAMs are ideal targets for imaging of patient-derived orthotopic xenograft (PDOX) nude mouse models using an anti-CEACAM antibody conjugated to a near infrared fluorophore [ 15 ]. In the present study, we utilized the fluorescence-labeled recombinant bacterial protein HopQ to test its suitability for fluorescence targeting of colorectal tumors in PDOX and orthotopic colon cancer cell-line models.…”

Harnessing Omics Approaches on Advanced Preclinical Models to Discovery Novel Therapeutic Targets for the Treatment of Metastatic Colorectal Cancer

“…In particular, the Hoffman’s group developed orthotopic models of mCRC via the implantation of human tumor tissues into the serosa of the cecum of immunosuppressed mice. The growth of primary tumors and spontaneous metastases were visualized by the use fluorophore-conjugated anti-CEA antibodies [ 48 ] or by transfecting cells with green or red fluorescence protein [ 49 , 50 ]. This technology represents a very potent tool for the identification of the anticancer activity of new therapeutic approaches.…”