title: A caspase-3 'death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers.
abstract: Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical 'death-switch' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [(18)F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The 'death-switch' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of 'death-switched' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [(18)F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [(18)F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.
authors: Paterson Institute for Cancer Research, University of Manchester and Manchester Cancer Research Centre, Withington, Manchester, UK.
authors: K L
authors: KL
authors: Simpson
authors: C
authors: C
authors: Cawthorne
authors: C
authors: C
authors: Zhou
authors: C L
authors: CL
authors: Hodgkinson
authors: M J
authors: MJ
authors: Walker
authors: F
authors: F
authors: Trapani
authors: M
authors: M
authors: Kadirvel
authors: G
authors: G
authors: Brown
authors: M J
authors: MJ
authors: Dawson
authors: M
authors: M
authors: MacFarlane
authors: K J
authors: KJ
authors: Williams
authors: A D
authors: AD
authors: Whetton
authors: C
authors: C
authors: Dive
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D015415:Biomarkers / Q000097:blood; D053148:Caspase 3 / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000000981:diagnostic imaging / Q000188:drug therapy / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use; D018260:Gelsolin / Q000378:metabolism; D024243:HMGB1 Protein / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D053538:Keratin-18 / Q000097:blood; D051379:Mice; D016513:Mice, SCID; D000078792:Midkine; D049268:Positron-Emission Tomography; D040901:Proteomics; D011859:Radiography; D019275:Radiopharmaceuticals; D014183:Transplantation, Heterologous
chemical_list: D000970:Antineoplastic Agents; D015415:Biomarkers; D016207:Cytokines; D018260:Gelsolin; D024243:HMGB1 Protein; D053538:Keratin-18; D019275:Radiopharmaceuticals; D000078792:Midkine; D004317:Doxorubicin; D053148:Caspase 3
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