2025 Theses Doctoral

Development of an optical pooled screen to investigate regulators of ferroptosis sensitivity

Decker, Aubrianna M.

Ferroptosis is an iron-dependent regulated form of cell death characterized by the lethal accumulation of lipid peroxidation and it is of particular interest as a way to target therapy-resistant mesenchymal cancer cells. Ferroptosis regulation is complex and sensitivity to ferroptotic induction can be influenced by numerous different pathways controlling metabolic and oxidative states of the cell. This complexity motivated the investigation of many potential regulators in a high-throughput knockout screen method. It has been demonstrated that ferroptotic induction is associated with the upregulation and translocation to the plasma membrane of Transferrin Receptor 1 (TfR1/TFRC). This translocation is a subcellular spatial phenotype captured via imaging that is intractable to typical screening readout methods that rely on dissociation and lysis of the cells.

In order to set up a system with which to explore regulators of this spatial phenotype of ferroptosis, as well as other optical features of ferroptosis in the future, we developed and implemented an optical pooled screen customized for the TfR1 phenotype. Targeted pooled screening allows smaller (academic) laboratories to investigate pathways of interest consisting of hundreds of genes in a single study.

Our method is also compatible with multiplexed readout and thus allows for analysis of perturbation associated effects on other pathways and organelles of interest in a single screen. Here we implement a CRISPRmap optical pooled screen method customized for investigating ferroptosis regulation, which identified LPL and MT1A as potential regulators of ferroptosis. This screening method has revealed that targeting lipases and metallothionines may be possible strategies for leveraging ferroptosis in cancer therapeutics.