Targeted cancer therapies block cancer growth and spread using small molecules. Many molecular targets for an epidermal growth factor receptor (EGFR) selectively compete with the adenosine triphosphate-binding site of its tyrosine kinase domain. Detection of molecular targeted agents and their metabolites in cells/tissues by label-free imaging is attractive because dyes or fluorescent labels may be toxic or invasive. Here, label-free Raman microscopy is applied to show the spatial distribution of the molecular targeted drug erlotinib within the cell. The Raman images show that the drug is clustered at the EGFR protein at the membrane and induces receptor internalization. The changes within the Raman spectrum of erlotinib measured in cells as compared to the free-erlotinib spectrum indicate that erlotinib is metabolized within cells to its demethylated derivative. This study provides detailed insights into the drug targeting mechanism at the atomic level in cells. It demonstrates that Raman microscopy will open avenues as a non-invasive and label-free technique to investigate pharmacokinetics at the highest possible resolution in living cells.

Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label‐free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC‐MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.

Mutational acquired resistance is a major challenge in cancer therapy. Somatic tumours harbouring some oncogenic mutations are characterised by a high mortality rate. Surprisingly, preclinical evaluation methods do not show clearly resistance of mutated cancers to some drugs. Here, we implemented Raman spectral imaging to investigate the oncogenic mutation resistance to epidermal growth factor receptor targeting therapy. Colon cancer cells with and without oncogenic mutations such as KRAS and BRAF mutations were treated with erlotinib, an inhibitor of epidermal growth factor receptor, in order to detect the impact of these mutations on Raman spectra of the cells. Clinical studies suggested that oncogenic KRAS and BRAF mutations inhibit the response to erlotinib therapy in patients, but this effect is not observed in vitro. The Raman results indicate that erlotinib induces large spectral changes in SW-48 cells that harbour wild-type KRAS and BRAF. These spectral changes can be used as a marker of response to therapy. HT-29 cells (BRAF mutated) and SW-480 cells (KRAS mutated) display a smaller and no significant response, respectively. However, the erlotinib effect on these cells is not observed when phosphorylation of extracellular-signal-regulated kinase and AKT is monitored by Western blot, where this phosphorylation is the conventional in vitro test. Lipid droplets show a large response to erlotinib only in the case of cells harbouring wild-type KRAS and BRAF, as indicated by Raman difference spectra. This study shows the great potential of Raman spectral imaging as an in vitro tool for detecting mutational drug resistance.