Use of sensitive, non-destructive and straightforward 3D SERS for investigating the cellular uptake processes of functionalised nanotags in entire cell volume.

Breast cancer is one of the leading causes of cancer death in women. Novel in vitro tools that integrate three-dimensional (3D) tumor models with highly sensitive chemical reporters can provide useful information to aid biological characterization of cancer phenotype and understanding of drug activity. The combination of surface-enhanced Raman scattering (SERS) techniques with microfluidic technologies offers new opportunities for highly selective, specific, and multiplexed nanoparticle-based assays. Here, we explored the use of functionalized nanoparticles for the detection of estrogen receptor alpha (ERα) expression in a 3D tumor model, using the ERα-positive human breast cancer cell line MCF-7. This approach was used to compare targeted versus nontargeted nanoparticle interactions with the tumor model to better understand whether targeted nanotags are required to efficiently target ERα. Mixtures of targeted anti-ERα antibody-functionalized nanotags (ERα-AuNPs) and nontargeted (against ERα) anti-human epidermal growth factor receptor 2 (HER2) antibody-functionalized nanotags (HER2-AuNPs), with different Raman reporters with a similar SERS signal intensity, were incubated with MCF-7 spheroids in microfluidic devices and spectroscopically analyzed using SERS. MCF-7 cells express high levels of ERα and no detectable levels of HER2. 2D and 3D SERS measurements confirmed the strong targeting effect of ERα-AuNP nanotags to the MCF-7 spheroids in contrast to HER2-AuNPs (63% signal reduction). Moreover, 3D SERS measurements confirmed the differentiation between the targeted and the nontargeted nanotags. Finally, we demonstrated how nanotag uptake by MCF-7 spheroids was affected by the drug fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). These results illustrate the potential of using SERS and microfluidics as a powerful in vitro platform for the characterization of 3D tumor models and the investigation of SERD activity.