Supporting data for "Exosomes specifically regulate immune cells to enhance anti-tumor effects"

Primary liver cancer—the sixth most prevalent malignant tumor and the third highest cause of cancer-related deaths globally—is typically diagnosed at an advanced stage 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 could restore anti-tumor immunity and initiate therapeutic effects.

Exosomes are naturally occurring nanovesicles with diverse physiological functions, and they hold potential as drug delivery platforms. Modifying these exosomes to target TAMs offers 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 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.

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. The gRNAs were used to establish a CRISPR-Cas9 gene knockout system, which was then integrated into targeted exosomes for therapeutic exploration. 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.

Our research validated that these engineered exosomes could target human or murine M2 macrophages both in vitro and in vivo. Using the orthotopic liver implant mouse model, it was discovered that engineered exosomes significantly diminished M2 macrophage-induced tumor growth. Thus, our study suggests that engineered exosomes can enhance anti-tumor activity by reprogramming TAMs, offering fresh insights into the development of HCC therapeutic strategies.