Laboratory of Cellular Immunology, IRCCS Humanitas Research Hospital, Rozzano-Milan, Italy.
Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
Front Immunol. 2024 Jan 8;14:1334800. doi: 10.3389/fimmu.2023.1334800. eCollection 2023.
In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors.
NCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry and multispectral immunophenotyping.
We have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice.
Mannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs , and reduce tumor progression and metastasis spread in mouse tumors.
在肿瘤微环境（TME）中，肿瘤相关巨噬细胞（TAMs）发挥关键的免疫抑制作用，限制了免疫系统对抗癌症的能力。Toll 样受体（TLR）配体，如聚（I：C）或瑞喹莫德（R848），能够将 TAMs 重新编程为 M1 样抗肿瘤效应细胞。我们的工作目标是开发和评估负载聚（I：C）+R848 的聚合物纳米胶囊（NCs），以提高药物稳定性和全身毒性，并评估它们在实体瘤 TME 中对 TAMs 的靶向和治疗活性。
通过溶剂置换和层层自组装方法制备 NCs，并通过动态光散射和纳米颗粒跟踪分析进行表征。透明质酸（HA）通过化学方法与甘露糖结合，用于 NCs 的涂层以靶向 TAMs。通过 Alamar Blue、ELISA 和流式细胞术评估负载 TLR 配体的 NCs 的毒性和免疫刺激活性，使用原代人单核细胞衍生的巨噬细胞。对于实验，使用 CMT167 肺癌模型和转移到肺部的 MN/MCA1 纤维肉瘤模型；通过流式细胞术和多光谱免疫表型分析评估肿瘤浸润白细胞。
我们开发了负载聚（I：C）+R848 的聚合物 NCs。在一系列 5 个先导原型中，基于其物理化学性质（大小、电荷、稳定性）和表征，选择了鱼精蛋白-NCs，它们表现出良好的原代巨噬细胞生物相容性，并能够刺激其产生 T 细胞吸引趋化因子（CXCL10、CCL5），并诱导 M1 样巨噬细胞对肿瘤细胞的细胞毒性。在小鼠肿瘤模型中，肿瘤内注射聚（I：C）+R848-鱼精蛋白-NCs 可显著抑制肿瘤生长和肺转移。在原位小鼠肺癌模型中，静脉注射负载额外一层透明质酸-甘露糖以提高 TAM 靶向性的聚（I：C）+R848-鱼精蛋白-NCs，具有良好的抗肿瘤疗效，且无明显全身毒性。虽然 T 细胞数量（CD8、CD4 或 Treg）没有明显变化，但在治疗小鼠中 TAM 重编程得到证实，治疗小鼠中间质肺泡巨噬细胞相对减少，具有更高的 CD86 表达，但相同细胞中的 CD206 和 Arg1 表达降低。
负载聚（I：C）+R848 的甘露糖-HA-鱼精蛋白-NCs 成功地重新编程 TAMs，减少了小鼠肿瘤的进展和转移扩散。
