Combined siRNA Targeting IκBα and Stat6 Promotes Macrophage Reprogramming toward Cancer Immunotherapy

Tumor-associated macrophages (TAMs) are central regulators of the tumor microenvironment (TME). Their polarization state can critically influence tumor development and treatment resistance. In the TME, the pro-tumor M2-like phenotype is the most prevalent among TAMs and contributes to tumor progression by promoting immunosuppression, angiogenesis, and metastasis. Thus, reprogramming TAMs from an M2 to an antitumor M1-like phenotype is a promising strategy to enhance cancer immunotherapy by inhibiting these processes, stimulating inflammatory responses, and activating cytotoxic immune cells. In this study, an array of small interfering RNA (siRNA) targeting candidate genes to repolarize macrophages, i.e., the inhibitor of nuclear factor kappa B alpha (IκBα), the colony-stimulating factor 1 receptor (Csf-1r), and the signal transducer and activator of transcription 6 (Stat6), was delivered by lipid nanoparticles (LNPs). While knockdown of all targeted genes was successful, silencing IκBα or Stat6 altered M1/M2 marker expression toward an M1-like polarization. Furthermore, combining IκBα and Stat6 produced the most effective macrophage reprogramming, achieving simultaneous downregulation of M2 markers and upregulation of M1 markers. Our data demonstrates that this dual-targeting strategy strongly induced potent reprogramming of M2-polarized macrophages, establishing it as a superior immunotherapeutic framework.