An integrated method for single cell proteomics with simultaneous measurements of intracellular drug concentration implicates new mechanisms for adaptation to KRAS(G12D) inhibitors

It is well established that a population of single human cells will often respond to the same drug treatment in a heterogeneous manner. In the context of chemotherapeutics, these diverse responses may lead to individual adaptation mechanisms and ultimately multiple distinct methods of resistance. The obvious question from a pharmacology perspective is how intracellular concentrations of active drug varies between individual cells, and what role does that variation play in drug response heterogeneity? To date, no integrated methods for rapidly measuring intracellular drug levels while simultaneously measuring drug responses have been described. This study describes a method for single cell preparation that allows proteins to be extracted and digested from single cells while maintaining conditions for small molecules to be simultaneously measured. The method as described allows up to 40 cells to be analyzed per instrument per day. When applied to a KRAS(G12D) small molecule inhibitor I observe a wide degree of intracellular levels of the drug, and that proteomic responses largely stratify based on the concentration of drug within each single cell. Further work is in progress to develop and standardize this method and – more importantly – to normalize drug measurements against direct measurements of cell volume. However, these preliminary results appear promising for the identification of single cells with unique drug response mechanisms. All data described in this study has been made publicly available through the ProteomeXchange consortium under accession PXD046002.