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.

原发性肝癌——全球第六大常见恶性肿瘤，同时也是癌症相关死亡的主要原因之一——通常在晚期被诊断出，此时治疗选择极为有限。这使得开发创新治疗方法变得至关重要。肿瘤相关巨噬细胞（TAMs），在肝细胞癌（HCC）的肿瘤微环境中含量丰富，在肿瘤进展中扮演着核心角色。将促进肿瘤的M2样TAMs重编程为M1表型，能够恢复抗肿瘤免疫并启动治疗效应。外泌体是自然界中存在的具有多种生理功能的纳米囊泡，并且作为药物递送平台具有潜在价值。针对TAMs修改这些外泌体为肝癌治疗提供了一种新的方法。在本研究中，采用基于亲本细胞的工程化外泌体设计方法，利用人类和鼠M2巨噬细胞上独特的蛋白或受体设计靶向肽。这些肽被融合到外泌体膜蛋白Lamp2b的N端，从而使得靶向基团在外泌体表面展示。双荧光素酶报告系统验证了工程化外泌体对不同细胞的靶向能力。在确认Galectin 9或CD36阳性M2巨噬细胞在HCC中的临床相关性后，设计了针对人类和鼠LGALS9或CD36基因的成对gRNAs。这些gRNAs被用于建立CRISPR-Cas9基因敲除系统，并将其整合到靶向外泌体中进行治疗探索。Galectin 9，一种与β-半乳糖苷结合的半乳糖蛋白，以及CD36，一种跨膜糖蛋白受体，均被证实对巨噬细胞极化和HCC肿瘤生长具有显著影响。我们的研究证实，这些工程化外泌体能够在体外和体内靶向人类或鼠M2巨噬细胞。使用原位肝植入小鼠模型，发现工程化外泌体显著减少了M2巨噬细胞诱导的肿瘤生长。因此，我们的研究表明，工程化外泌体可以通过重编程TAMs来增强抗肿瘤活性，为HCC治疗策略的开发提供了新的见解。