Preclinical evaluation of M30 and M65 ELISAs as biomarkers of drug induced tumor cell death and antitumor activity

Abstract

M30 and M65 are ELISAs that detect different circulating forms of cytokeratin 18. Using the aurora kinase inhibitor AZD1152 and the SW620 human colon cancer xenograft, experiments were conducted to qualify preclinically both assays as serologic biomarkers of cell death. Using two different apoptotic markers, the kinetics of cell death induced by AZD1152 was first characterized in vitro in three different cell lines and shown to peak 5 to 7 days after drug addition. Treatment of non-tumor-bearing rats with AZD1152 (25 mg/kg) produced no alterations in circulating baseline values of M30 and M65 antigens. In treated, tumor-bearing animals, M30 detected a 2-to 3-fold (P < 0.05) increase in plasma antigen levels by day 5 compared with controls. This correlated to a 3-fold increase in the number of apoptotic cells detected on day 5 in SW620 xenografts using immunohistochemistry. By contrast, M65 did not detect a drug-induced increase in circulating antigen levels at day 5. However, M65 plasma levels correlated to changes in tumor growth in control animals (r 2 = 0.93; P < 0.01) and also followed the magnitude of the temporal effect of AZD1152 on tumor growth. An intermediate but active dose of AZD1152 (12.5 mg/kg) produced a less significant increase in M30 plasma levels at day 5. It was also confirmed that the plasma profiles of M30 and M65 mirrored closely those measured in whole tumor lysates. We conclude that M30 is a pharmacodynamic biomarker of AZD1152-induced apoptosis in the SW620 xenograft model, whereas M65 is a biomarker of therapeutic response.

Evaluation of Circulating Tumor Cells and Serological Cell Death Biomarkers in Small Cell Lung Cancer Patients Undergoing Chemotherapy

“…Consistent with our results, other studies have observed peak M30 and nDNA levels at 24 to 48 hours after chemotherapy. 27,31,32 However, this may not hold true for novel agents 36 where it would be prudent to perform pilot studies to select optimal time points.…”

Biological Functions of Cytokeratin 18 in Cancer

“…Nagashio and colleagues (53) reported that CK18 was upregulated in large cell neuroendocrine carcinoma compared with small cell lung carcinoma. Another study also showed substantially higher levels of positive CK18 immunostaining in adenocarcinoma and large cell neuroendocrine carcinoma than in squamous cell carcinoma and small cell lung carcinoma (59). In breast cancer, reduced CK8/18 expression in conjunction with the basal-like phenotype and family history may improve investigators' ability to identify which tumors are likely to be associated with a BRCA1 germline mutation, and thus may help streamline genetic testing (60).…”

Specific mitochondrial calcium overload induces mitochondrial fission in prostate cancer cells

“…Apoptosense is highly sensitive and is specific to apoptosis. Apoptosense kit has been utilized successfully in our (14,21,22), and other laboratories (23)(24)(25). Protein extract was added to 96-well plates pre-coated with mouse monoclonal M30 antibody, horseradish peroxidase tracer solution was added to the wells and incubated for 4 h. Color was developed by adding tetramethyl benzidine solution and the optical density was measured at 450 nm on a Spectra MAX340 microplate reader (Molecular Devices Corp., Sunnyvale, CA).…”