Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment

Oncogenic KRAS mutations are frequent in colorectal cancer, presenting substantial challenges due to constitutive activation and resistance to molecular-targeted therapies driven by mutation-specific biochemical properties. In this study, using single-molecule imaging, we quantitatively analyzed diffusional behaviors of oncogenic KRAS mutants and associated signaling molecules to elucidate their signaling mechanisms and therapeutic implications. Upon EGF stimulation, KRAS molecules exhibited transient trapping and reduced diffusion due to interactions with SOS1 and BRAF, leading to the temporary formation of signaling complexes. Our results demonstrate that analysis of the temporal fraction and frequency of transient trapping events offers a more sensitive and precise evaluation of KRAS activation levels than western blotting. Furthermore, the study of dynamics of individual oncogenic KRAS molecules provides a more accurate assessment of the therapeutic efficacy of various molecular-targeted drugs. Consequently, we propose a highly sensitive strategy to enhance the therapeutic targeting of oncogenic KRAS in living colorectal cancer cells.