Illuminating the lateral organization of cell-surface CD24 and CD44 through plasmon coupling between Au nanoparticle immunolabels.

CD44 and CD24 are important cell surface glycoproteins whose relative expression levels are used to identify so-called cancer stem cells (CSCs). While current diagnostic applications of CD44 and CD24 focus primarily on their expression levels, we demonstrate here that noble metal nanoparticle (NP) immunolabeling in combination with plasmon coupling microscopy (PCM) can reveal more subtle differences, such as the spatial organization of these surface species on subdiffraction limit length scales. We quantified both expression and spatial clustering of CD44 and CD24 on MCF7 and SKBR3 breast cancer cells through analysis of the labeling intensity and the electromagnetic coupling of the NP labels, respectively. The labeling intensity was well correlated with the receptor expression, but the inspection of the labeled cell surface in the optical microscope revealed that the NP immunolabels were not homogeneously distributed. Consistent with a heterogeneous spatial distribution of the targeted CD24 and CD44 in the plasma membrane, a significant fraction of the NPs were organized into clusters, which were easily detectable in the optical microscope as discrete spots with colors ranging from green to orange. To further quantify the spatial organization of the targeted proteins, we characterized individual NP clusters through spatially resolved elastic scattering spectroscopy. The statistical analysis of the single cluster spectra revealed a higher clustering affinity for CD24 than for CD44 in the investigated cancer models. This preferential clustering was removed upon lipid raft disruption through cholesterol sequestration. Overall, these observations confirm a preferential enrichment of CD24 in lipid rafts and a more random distribution of CD44 in the plasma membrane as cause for the observed differences in protein clustering.