The most abundant immune cell type in the tumor microenvironment are tumor associated macrophages (TAMs), affecting tumor progression, metastasis and therapeutic response. When exploring TAMs as targets in novel cancer immunotherapy approaches, their repolarization from the M2 (anti-inflammatory/pro-tumorigenic) into the M1 (pro-inflammatory/anti-tumorigenic) phenotype is an intriguing strategy.

Major bottlenecks of RNAi-based knockdown of M2 promoting genes include cellular siRNA delivery and correct intracellular processing in macrophages as a cell type notoriously hard-to-transfect. The cationic polymer polyethylenimine (PEI) is widely explored for delivering nucleic acids. Further advanced nanocarriers are tyrosine-modified polymers based on PEI or polypropylenimine dendrimers (PPI) for highly efficient siRNA delivery in vitro and in vivo.

Here, we explored a panel of PEI- or PPI-based nanoparticles for siRNA-mediated gene knockdown in macrophages and subsequent TAM repolarization. The tyrosine-modified linear 10 kDa PEI (LP10Y) or branched 5 kDa PEI (P5Y) as well as a tyrosine-modified PPI (PPI-Y) were most efficient for gene knockdown in macrophage cell lines or primary macrophages, independent of their polarization. Knockdown of STAT6 or STAT3 led to repolarization of M2 macrophages, as indicated by alterations in various M2 and M1 marker levels. This highly specific approach also demonstrated non-redundant functions of STAT3 and STAT6. Macrophage re-polarization from M2 to M1 upon PPI‑Y/siRNA-mediated STAT6 knockdown increased tumor cell phagocytosis in a co-culture model.

In conclusion, we identify certain tyrosine-modified PEI- or PPI-based nanoparticles as efficient for macrophage transfection, and the specific, siRNA-mediated STAT6 knockdown as promising approach for macrophage repolarization and enhancement of their tumor suppressive role.