Much contemporary cancer research focuses on drug discovery such as rapamycin mimetics that induce autophagy in cancer cells and have been tested in clinical trials (1). Here we describe new simplified technology to detect autophagy, which will provide opportunities for the discovery of new or repurposed drugs for cancer treatments.
Activation of the CT Receptor is associated with pre-apoptotic cell stress (2). Further, expression of Calcitonin (CT) Receptor coincides with autophagy and quiescence in skeletal muscle satellite stem cells. Expression is lost with cell division and differentiation in response to injury. The question remains how general is the association of CT Receptor expression with autophagy?
CalRexinTM (www.apopbiosciences.com Apop Biosciences) is a monoclonal antibody that binds the N-terminal, extracellular domain of CT Receptor and is internalised into stressed, living cells. It was conjugated to the pH-sensitive fluorescent dye pHrodo Red and deployed to image autophagy on the high throughput Operetta platform (3). Autophagic HeLa and MG63 cells were induced with either rapamycin or serum deprivation. Data presented here demonstrate the intracellular accumulation of CalRexinTM:pHrodo Red into autolysosomes (pH 4-5, coincident with LysoTracker) during autophagy compared to the isotype control. In HeLa cells in which either ATG5 or FIP200 had been knocked out inhibiting elongation, the central process of formation of autophagosomes in autophagy, there was minimal accumulation of CalRexinTM:pHrodo Red. Futher, treatment with inhibitors of autophagy, chloroquine that inhibits fusion of autophagosomes with lysosomes or 3-methyladenine which inhibits formation of the essential factor IP3 (via IP3 kinase) in elongation, inhibited the increased fluorescence intensity associated with autophagy.
These data provide further expanded evidence (3) that CalRexinTM:pHrodo Red can be deployed to image autophagy in a simple assay and has potential for high content/throughput screening to identify candidate cancer drugs that promote autophagy.
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Wookey PJ, et al. Methods to measure calcitonin receptor activity, up-regulated in cell stress, apoptosis and autophagy. F1000Research 10:1019, 2021. DOI:10.12688/f1000research.72845.1