Tumor-associated macrophages (TAMs) are immune cells that perform essential roles within the tumor microenvironment. These macrophages can change their functional characteristics in response to local environmental cues, switching between an M1-like phenotype (anti-tumor) or an M2-like phenotype (tumor-promoting). Unfortunately, TAMs are largely polarized into the M2-like phenotype, contributing to tumor growth and immunosuppression. Understanding and modulating TAM polarization is essential in cancer research and therapy. Therefore, strategies to modulate and reprogram M2 macrophages towards an M1-like phenotype have been explored as potential approaches to boost anti-cancer immune responses and enhance the effectiveness of cancer therapies.

One of the significant challenges in targeting M2 macrophages is the need for specific and high-affinity targeting ligands that can distinguish them from the anti-tumor M1-like TAMs. Recently, Pun et al. reported that the polypeptide M2pep (YEQDPWGVKWWY) preferentially binds to M2 macrophages; however, further studies are needed. In order to investigate the selectivity of M2pep towards M2-like TAMs using fluorescence-based techniques, we report here the synthesis and characterization of the M2pep substrate labelled with a fluorophore (Rhodamine B). This peptide comprising a 12-amino acid sequence with three additional amino acids (GGG) was synthesized and coupled to Rhodamine B at its N-termini by microwave-assisted solid phase synthesis, using the Fmoc strategy. The structure of the labelled peptide was confirmed by NMR, UV-Vis and fluorescence spectroscopy and mass spectrometry.