Immunomodulation of Tumor Associated Macrophages by Targeted, siRNA-Delivering Nanoparticles

Abstract

Tumor associated macrophages (TAMs) play an important role in establishing a pro-tumor environment in many tumor types. Produce low levels of inflammatory cytokines which creates pro-tumorigenic smoldering inflammation and also TAMs break down the surrounding extracellular matrix, secrete growth factors, and inhibit the adaptive immune response. TAMs are an attractive therapeutic target in cancer treatment, and strategically ablating these cells has been shown to remove their trophic effects. A more elegant solution would be to target these pro-tumor macrophages with a therapeutic agent that can alter their behavior to a strongly immunogenic phenotype capable of stimulating tumor immunity. The NF-κB pathways control macrophage phenotype and the inflammatory response. By selectively manipulating NF-κB in TAMs with siRNA, it should be possible to eradicate the TAM phenotype and recapitulate the normal immune response. I have developed a strategy to activate a classical immunogenic phenotype in TAMs. This strategy would stimulate tumor immunity by specifically activating the classical NF-κB pathway by knocking down the inhibitor of the pathway (IκBα).

Using mannosylated endosomal escape nanoparticles MnNP, I have transfected macrophages with siRNA targeting key NF-κB proteins. In vitro experiments show that MnNP rapidly and strongly transfect a small population of cells, as opposed to hydroxyl capped nanoparticles (OHNP) and commercial agents, which transfect many cells to a lesser degree and with less specificity. Analysis of IκBα knockdown macrophages shows a decrease in immunosuppressive cytokines, and an increase in a potent CD8+ T-cell recruiting cytokine, indicating a shift from a TAM-like phenotype, to an immunocompetent phenotype. In in vivo mouse models of human cancer, MnNP delivered nucleotides are taken up by mammary tumor TAMs to a greater extent than phagocytized, free siRNA and are protected within the core of the particle. Similarly, OHNP are not taken up by TAMs in solid ovarian tumors, while MnNP are able to evade non-specific uptake by cells in the ascites fluid and are taken up by tumor TAMs. In an intratracheal delivery model, MnNP have enhanced delivery to lung metastasis TAMs while OHNP are taken up nonspecifically by phagocytic granulocytes and monocytes.