Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the cell surface and present in colorectal cancer serum specimens

Abstract

Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the cell surface and present in colorectal cancer serum specimens, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10. 1016/j.jpba.2017.02.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HIGHLIGHTS 1. First report of an enzyme-linked immunosorbent assay (ELISA) to detect fragments of the protein CDCP1 in human serum 2. The ELISA has a wide working range of 0.68 to 26.5 ng/ml, and a low limit of detection of 0.25 ng/ml 3. The ELISA has high intra-assay (repeatability) and high inter-assay (reproducibility) precision with all coefficients of variation ≤ 7% 4. The ELISA displays high accuracy detecting ShE-CDCP1 levels at ≥ 94.8% of actual concentration 5. Because CDCP1 has potential as a biomarker for colorectal, prostate, breast, kidney and ovarian cancer, the findings will be relevant to investigators interested in clinical applications as well as those interested in the functions of CDCP1.ABSTRACT CUB domain containing protein 1 (CDCP1) is a transmembrane protein involved in progression of several cancers. When located on the plasma membrane, full-length 135 kDa CDCP1 can undergo proteolysis mediated by serine proteases that cleave after two adjacent amino acids (arginine 368 and lysine 369). This releases from the cell surface two 65 kDa fragments, collectively termed ShE-CDCP1, that differ by one carboxyl terminal residue. To evaluate the function of CDCP1 and its potential utility as a cancer biomarker, in this study we developed an enzyme-linked immunosorbent assay (ELISA) to reliably and easily measure the concentration of ShE-CDCP1 in biological samples. Using a reference standard we demonstrate that the developed ELISA has a working range of 0.68 to 26.5 ng/ml, and the limit of detection is 0.25 ng/ml. It displays high intra-assay (repeatability) and high inter-assay (reproducibility) precision 2 with all coefficients of variation ≤ 7%. The ELISA also displays high accuracy detecting ShE-CDCP1 levels at ≥ 94.8% of actual concentration using quality control samples. We employed the ELISA to measure the concentration of ShE-CDCP1 in human serum samples with our results suggesting that levels are significantly higher in serum of colorectal cancer patients compared with serum from individuals with benign conditions (p < 0.05). Our data also suggest that colorectal cancer patients with stage II-IV disease have at least 50% higher serum levels of ShE-CDCP1 compared with stage I cases (p < 0.05). We conclude that the developed ELISA is a suitable method to quantify ShE-CDCP1 concentration in ...

Mechanistic insights into CDCP1 clustering on non-small-cell lung cancer membranes revealed by super-resolution fluorescent imaging

“…The intracellular domain of CDCP1 has multiple tyrosine residues that can be phosphorylated by Src kinase family, and the activated CDCP1 promotes migration and invasion of malignant tumor cells. 26 , 27 , 28 For tumor cells, inhibition of CDCP1 activity or reduction of CDCP1 expression level can limit anchorage-independent survival which plays a vital role in tumor metastasis. 29 , 30 Based on the experiments above, we hypothesized that CDCP1 protein clusters may be functional domains that promote proliferation and metastasis of cancer cells.…”

Preclinical Evaluation of a Fluorescent Probe Targeting Receptor CDCP1 for Identification of Ovarian Cancer

“…CDCP1 is a 135 kDa cell surface protein that can undergo proteolytic cleavage in vitro and in vivo by serine proteases including matriptase and proteases of the plasminogen activation system. , After proteolysis, CDCP1 is present as a 70 kDa membrane spanning the carboxyl-terminal fragment (CTF) and a 65 kDa amino-terminal fragment (ATF) that binds to the plasma membrane via CDCP1-CTF or full-length CDCP1 or is shed from the cell surface and can be detected in conditioned cell culture media and colorectal cancer patient blood. , Because 10D7 binds to CDCP1-ATF, shedding of this fragment from the cell surface would prevent the antibody binding to CDCP1 to deliver a fluorescent payload such as ICG.…”

CUB Domain-Containing Protein 1 (CDCP1) is a rational target for the development of imaging tracers and antibody-drug conjugates for cancer detection and therapy

“…In addition, our flow cytometry results suggest that ex vivo evaluation of levels of cell surface CDCP1 in tumor biopsies or circulating malignant cells could also serve to stratify patients for CDCP1-targeted therapies. In addition, because we and others have shown that a region of CDCP1 can be shed from the cell surface and detected in colon cancer patient serum and function as a predictive biomarker of lung cancer onset [11,34,35], the detection of cell-shed CDCP1 may be suitable for selection of cancer patients suitable for CDCP1-targeted therapies.…”

“…These signalling events via CDCP1 are generally accompanied by its phosphorylation at residue tyrosine 734 (Y734) 10 . CDCP1-ATF is detectable in the serum of colorectal cancer patients 11 but appears to remain predominantly on the plasma membrane tethered to CDCP1-FL or CDCP1-CTF 12 , 13 .…”