Exosomes specifically regulate macrophages to enhance anti-tumor effects

Abstract

Primary liver cancer, the sixth most prevalent malignant tumor and the third highest cause of cancer-related deaths globally, is usually diagnosed at the advanced stages when treatment options are scarce. This makes the development of innovative treatments crucial. Tumor-associated macrophages (TAMs), which are abundant within the hepatocellular carcinoma (HCC) tumor microenvironment, play a pivotal role in tumor progression. Reprogramming the tumor-promoting M2-like TAMs into the M1 phenotype may restore anti-tumor immunity and initiate therapeutic effects.

Exosomes are naturally occurring nanovesicles with diverse physiological functions, they hold potentials as drug delivery platforms due to their ability to transport molecules and be taken up by distant cells. Modifying these exosomes to target TAMs may offer a novel approach to liver cancer treatment. In this research, a parental cell-based exosome engineering method was utilized to design targeted peptides, leveraging unique proteins or receptors on human and mouse M2 macrophages. These peptides were fused to the N-terminus of exosomal outer membrane protein Lamp2b, enabling the display of the targeting moiety on the exosome surface. A dual luciferase reporter system validated the targeting ability of engineered exosomes towards different cells.

Galectin 9, a galectin that binds β-galactoside, and CD36, a transmembrane glycoprotein receptor, were both found to have significant effects on macrophage polarization and HCC tumor growth. After confirming the clinical relevance of Galectin 9 or CD36-positive M2 macrophages in HCC, two pairs of gRNAs for the human and mouse LGALS9 or CD36 genes were designed to establish a CRISPR-Cas9 gene knockout system. Further investigations by CRISPR-Cas9 knockout systems demonstrated that the absence of Galectin 9 or CD36 in M2-like macrophages led to their reprogramming towards the M1 polarized state, resulting in altered tumor-promoting functions. Moreover, exosomes engineered with targeting peptides exhibited enhanced uptake by M2-like macrophages compared to non-targeted exosomes.

Our research validated the targeting capability of engineered exosomes towards human or murine M2 macrophages, both in vitro and in vivo. In the orthotopic liver cancer mouse model, targeted exosomes, equipped with CRISPR-Cas9 knockout systems, significantly diminished M2 macrophage-induced tumor growth. This innovative approach utilized exosomes as a targeted drug delivery system to modulate macrophages within the HCC tumor microenvironment (TME). The effective targeting of M2-like macrophages by the engineered exosomes, which achieved through the incorporation of CRISPR-Cas9 knockout systems, facilitated the reprogramming of macrophages towards the anti-tumor M1 phenotype. It’s strongly suggested that the engineered exosomes have the potential to enhance anti-tumor activity. These findings highlighted the promise of exosome-mediated therapy as a strategy to effectively inhibit tumor progression and metastasis in HCC.