Synthesis and Evaluation of Cytosolic Phospholipase A₂ Activatable Fluorophores for Cancer Imaging

Activatable fluorophores selective to cytosolic phospholipase A₂ (cPLA₂) were synthesized and evaluated for their ability to image triple negative breast cancer cells. The activatable constructs were synthesized by esterification of a small molecule fluorophore with a fatty acid resulting in ablated fluorescence. Selectivity for cPLA₂ was generated through the choice of fluorophore and fatty acid. Esterification with arachidonic acid was sufficient to impart specificity to cPLA₂ when compared to esterification with palmitic acid. <i>In vitro</i> analysis of probes incorporated into phosphatidylcholine liposomes demonstrated that a nonselective phospholipase (sPLA₂ group IB) was able to hydrolyze both arachidonate and palmitate coupled fluorophores resulting in the generation of fluorescence. Of the four fluorophores tested, DDAO (7-hydroxy-9<i>H</i>-(1,3-dichloro-9,9-dimethylacridin-2-one)) was observed to perform optimally <i>in vitro</i> and was analyzed further in 4175-Luc+ cells, a metastatic triple negative human breast cancer cell line expressing high levels of cPLA₂. In contrast to the <i>in vitro</i> analysis, DDAO arachidonate was shown to activate selectively in 4175-Luc+ cells compared to the control DDAO palmitate as measured by fluorescence microscopy and quantitated with fluorescence spectroscopy. The addition of two agents known to activate cPLA₂ enhanced DDAO arachidonate fluorescence without inducing any change to DDAO palmitate. Inhibition of cPLA₂ resulted in reduced fluorescence of DDAO arachidonate but not DDAO palmitate. Together, we report the synthesis of a cPLA₂ selective activatable fluorophore capable of detecting cPLA₂ in triple negative breast cancer cells