An Engineered Extracellular Vesicle With Enhanced Tumor and Lymph Nodes Targeting as siRNA Delivery System for Robust Tumor Immunotherapy

Abstract

Tumor-derived extracellular vesicles (EVs) are a class of natural nanocarriers with phospholipid bilayers that show great promise as personalized cancer vaccine platforms due to their ability to carry tumor-specific antigens. However, their immunotherapeutic potential is hindered by limited tissue-specific targeting. In this study, we engineered tumor cell-derived EVs using an immunomodulatory peptide, DP7-C, to generate DP7-C engineered EVs (DP-EVs). These DP-EVs exhibited significantly enhanced accumulation in both lymph nodes and tumor tissues. Additionally, they demonstrated improved cellular uptake and facilitated more efficient endosomal escape. To further enhance the therapeutic efficacy, programmed cell death 1 ligand 1 targeting small interfering RNA (siPD-L1) was loaded into the DP-EVs, resulting in DP-EVs/siPD-L1. This formulation enabled concurrent suppression of PD-L1 expression in both dendritic cells (DCs) and tumor cells. In vivo experiments showed that DP-EVs/siPD-L1 significantly inhibited tumor growth and prolonged survival in tumor-bearing mice. The observed antitumor effect was attributed to the immune activation in the lymph nodes and the remodeling of the immunosuppressive tumor microenvironment (TME). Collectively, our findings demonstrate that DP-EVs/siPD-L1 functions as an effective therapeutic vaccine, which synergistically activates antitumor immunity and reverses immunosuppression through targeted PD-L1 blockade. This engineered EV platform represents a promising and translatable strategy for cancer immunotherapy.

Keywords

extracellular vesicles / immune response activation / siRNA delivery / tumor immunotherapy / tumor microenvironment reprogramming

References

[1] Y. Wang, C. Liu, C. Fang, et al., “Engineered Cancer Nanovaccines: A New Frontier in Cancer Therapy,” Nanomicro Letter 17, no. 1 (2024): 30.

[2] D. R. Wang, X. L. Wu, and Y. L. Sun, “Therapeutic Targets and Biomarkers of Tumor Immunotherapy: Response versus Non-response,” Signal transduction and targeted therapy 7, no. 1 (2022): 331.

[3] T. Fan, M. Zhang, J. Yang, Z. Zhu, W. Cao, and C. Dong, “Therapeutic Cancer Vaccines: Advancements, Challenges, and Prospects,” Signal transduction and targeted therapy 8, no. 1 (2023): 450.

[4] J. Liu, S. S. Liew, J. Wang, and K. Pu, “Bioinspired and Biomimetic Delivery Platforms for Cancer Vaccines,” Advanced Materials 34, no. 1 (2022): e2103790.

[5] L. Lin, Y. Pei, Z. Li, and D. Luo, “Progress and Challenges of mRNA Vaccines,” Interdisciplinary Medicine 1, no. 1 (2023): e20220008.

[6] R. Kalluri, “The Biology and Function of Extracellular Vesicles in Immune Response and Immunity,” Immunity 57, no. 8 (2024): 1752–1768.

[7] R. Kalluri, “The Biology and Function of Exosomes in Cancer,” Journal of Clinical Investigation 126, no. 4 (2016): 1208–1215.

[8] M. Mathieu, L. Martin-Jaular, G. Lavieu, and C. Théry, “Specificities of Secretion and Uptake of Exosomes and Other Extracellular Vesicles for Cell-to-cell Communication,” Nature Cell Biology 21, no. 1 (2019): 9–17.

[9] G. van Niel, G. D'Angelo, and G. Raposo, “Shedding Light on the Cell Biology of Extracellular Vesicles,” Nature Reviews Molecular Cell Biology 19, no. 4 (2018): 213–228.

[10] R. Kalluri and V. S. LeBleu, “The Biology, Function, and Biomedical Applications of Exosomes,” Science 367, no. 6478 (2020): eaau6977.

[11] X. Wang, Y. Zhang, Y. Chung, et al., “Tumor Vaccine Based on Extracellular Vesicles Derived From γδ-T Cells Exerts Dual Antitumor Activities,” Journal of Extracellular Vesicles 12, no. 9 (2023): e12360.

[12] S. Liu, X. Wu, S. Chandra, et al., “Extracellular Vesicles: Emerging Tools as Therapeutic Agent Carriers,” Acta pharmaceutica Sinica B 12, no. 10 (2022): 3822–3842.

[13] X. Huang, T. He, X. Liang, et al., “Advances and applications of nanoparticles in cancer therapy,” MedComm—Oncology 3, no. 1 (2024): e67.

[14] I. Kaymak, K. S. Williams, J. R. Cantor, and R. G. Jones, “Immunometabolic Interplay in the Tumor Microenvironment,” Cancer Cell 39, no. 1 (2021): 28–37.

[15] S. Zhu, T. Zhang, L. Zheng, et al., “Combination Strategies to Maximize the Benefits of Cancer Immunotherapy,” Journal of hematology & oncology 14, no. 1 (2021): 156.

[16] D. J. Irvine and E. L. Dane, “Enhancing Cancer Immunotherapy With Nanomedicine,” Nature Reviews Immunology 20, no. 5 (2020): 321–334.

[17] J. Y. Sun, D. Zhang, S. Wu, et al., “Resistance to PD-1/PD-L1 Blockade Cancer Immunotherapy: Mechanisms, Predictive Factors, and Future Perspectives,” Biomarker Research 8 (2020): 35.

[18] Y. Xia, S. Fu, Q. Ma, Y. Liu, and N. Zhang, “Application of Nano-Delivery Systems in Lymph Nodes for Tumor Immunotherapy,” Nanomicro Letter 15, no. 1 (2023): 145.

[19] S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl, “Duplexes of 21-nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells,” Nature 411, no. 6836 (2001): 494–498.

[20] R. Kanasty, J. R. Dorkin, A. Vegas, and D. Anderson, “Delivery Materials for siRNA Therapeutics,” Nature Materials 12, no. 11 (2013): 967–977.

[21] G. Kara, G. A. Calin, and B. Ozpolat, “RNAi-based Therapeutics and Tumor Targeted Delivery in Cancer,” Advanced Drug Delivery Reviews 182 (2022): 114113.

[22] M. Cully, “Exosome-based Candidates Move Into the Clinic,” Nature reviews Drug discovery 20, no. 1 (2021): 6–7.

[23] T. Smyth, M. Kullberg, N. Malik, P. Smith-Jones, M. W. Graner, and T. J. Anchordoquy, “Biodistribution and Delivery Efficiency of Unmodified Tumor-derived Exosomes,” official journal of the Controlled Release Society 199 (2015): 145–155.

[24] E. J. Park, O. Prajuabjinda, Z. Y. Soe, et al., “Exosomal Regulation of Lymphocyte Homing to the Gut,” Blood advances 3, no. 1 (2019): 1–11.

[25] T. Tian, H. X. Zhang, C. P. He, et al., “Surface Functionalized Exosomes as Targeted Drug Delivery Vehicles for Cerebral Ischemia Therapy,” Biomaterials 150 (2018): 137–149.

[26] X. Wu, Z. Wang, X. Li, et al., “In Vitro and in Vivo Activities of Antimicrobial Peptides Developed Using an Amino Acid-based Activity Prediction Method,” Antimicrobial Agents and Chemotherapy 58, no. 9 (2014): 5342–5349.

[27] X. Wu, Z. Li, X. Li, et al., “Synergistic Effects of Antimicrobial Peptide DP7 Combined With Antibiotics Against Multidrug-resistant Bacteria,” Drug design, development and therapy 11 (2017): 939–946.

[28] R. Zhang, L. Tang, Y. Tian, et al., “Cholesterol-modified DP7 Enhances the Effect of Individualized Cancer Immunotherapy Based on Neoantigens,” Biomaterials 241 (2020): 119852.

[29] Y. Tian, Q. Hu, R. Zhang, et al., “Rational Design of Innate Defense Regulator Peptides as Tumor Vaccine Adjuvants,” NPJ Vaccines 6, no. 1 (2021): 75.

[30] K. Men, M. Liu, X. Zhang, et al., “Identification of Potent siRNA Delivery Peptides Using Computer Modeling,” Advanced science (Weinheim, Baden-Wurttemberg, Germany) 11, no. 14 (2024): e2308345.

[31] H. Zhang, L. Liu, J. Liu, et al., “Roles of Tumor-associated Macrophages in Anti-PD-1/PD-L1 Immunotherapy for Solid Cancers,” Molecular cancer 22, no. 1 (2023): 58.

[32] U. Švajger, N. Tešić, and P. Rožman, “Programmed Death Ligand 1 (PD-L1) Plays a Vital Part in DC Tolerogenicity Induced by IFN-γ,” International Immunopharmacology 99 (2021): 107978.

[33] H. B. Gupta, C. A. Clark, B. Yuan, et al., “Tumor Cell-intrinsic PD-L1 Promotes Tumor-initiating Cell Generation and Functions in Melanoma and Ovarian Cancer,” Signal transduction and targeted therapy 1 (2016): 16030.

[34] C. A. Clark, H. B. Gupta, and T. J. Curiel, “Tumor Cell-intrinsic CD274/PD-L1: A Novel Metabolic Balancing Act With Clinical Potential,” Autophagy 13, no. 5 (2017): 987–988.

[35] R. Chen, T. Feng, S. Cheng, et al., “Evaluating the Defect Targeting Effects and Osteogenesis Promoting Capacity of Exosomes From 2D- and 3D-cultured human Adipose-derived Stem Cells,” Nano Today 49 (2023): 101789.

[36] J. Liu, X. Zhang, Y. Cheng, and X. Cao, “Dendritic Cell Migration in Inflammation and Immunity,” Cell Mol Immunol 18, no. 11 (2021): 2461–2471.

[37] M. Liu, D. Xie, D. Hu, et al., “In Situ Cocktail Nanovaccine for Cancer Immunotherapy,” Advanced science (Weinheim, Baden-Wurttemberg, Germany) 10, no. 31 (2023): e2207697.

[38] K. Xu, B. Han, Y. Bai, et al., “MiR-451a Suppressing BAP31 Can Inhibit Proliferation and Increase Apoptosis Through Inducing ER Stress in Colorectal Cancer,” Cell Death & Disease 10, no. 3 (2019): 152.

[39] L. Tao, W. Shu-Ling, H. Jing-Bo, et al., “MiR-451a Attenuates Doxorubicin Resistance in Lung Cancer via Suppressing Epithelialmesenchymal Transition (EMT) Through Targeting c-Myc,” Biomedicine & Pharmacotherapy 125 (2020): 109962.

[40] Y. Zhong, F. Yu, L. Yang, et al., “HOXD9/miR-451a/PSMB8 Axis Is Implicated in the Regulation of Cell Proliferation and Metastasis via PI3K/AKT Signaling Pathway in human Anaplastic Thyroid Carcinoma,” Journal of Translational Medicine 21, no. 1 (2023): 817.

[41] Z. Hu, Y. Zhao, J. Jiang, et al., “Exosome-derived miR-142-5p From Liver Stem Cells Improves the Progression of Liver Fibrosis by Regulating Macrophage Polarization Through CTSB,” Environmental Toxicology 38, no. 8 (2023): 1860–1873.

[42] W. Zhou, X. Chen, Y. Zhou, et al., “Exosomes Derived From Immunogenically Dying Tumor Cells as a Versatile Tool for Vaccination Against Pancreatic Cancer,” Biomaterials 280 (2022): 121306.

[43] H. C. Verdera, J. J. Gitz-Francois, R. M. Schiffelers, and P. Vader, “Cellular Uptake of Extracellular Vesicles Is Mediated by Clathrin-independent Endocytosis and Macropinocytosis,” Official journal of the Controlled Release Society 266 (2017): 100–108.

[44] M. Zhang, W. Shao, T. Yang, et al., “Conscription of Immune Cells by Light-Activatable Silencing NK-Derived Exosome (LASNEO) for Synergetic Tumor Eradication,” Advanced science (Weinheim, Baden-Wurttemberg, Germany) 9, no. 22 (2022): e2201135.

[45] Y. Meng, Z. Yao, X. Ke, et al., “Extracellular Vesicles-based Vaccines: Emerging Immunotherapies Against Cancer,” Journal of Controlled Release 378 (2025): 438–459.

[46] Y. Li, A. Sharma, and I. G. H. Schmidt-Wolf, “Evolving Insights Into the Improvement of Adoptive T-cell Immunotherapy Through PD-1/PD-L1 Blockade in the Clinical Spectrum of Lung Cancer,” Molecular cancer 23, no. 1 (2024): 80.

[47] L. Shu, X. Li, Z. Liu, et al., “Bile Exosomal miR-182/183-5p Increases Cholangiocarcinoma Stemness and Progression by Targeting HPGD and Increasing PGE2 Generation,” Hepatology 79, no. 2 (2024): 307–322.

[48] M. Q. Cao, A. B. You, X. D. Zhu, et al., “miR-182-5p Promotes Hepatocellular Carcinoma Progression by Repressing FOXO3a,” Journal of hematology & oncology 11, no. 1 (2018): 12.

[49] J. Xue, A. Zhou, Y. Wu, et al., “miR-182-5p Induced by STAT3 Activation Promotes Glioma Tumorigenesis,” Cancer research 76, no. 14 (2016): 4293–4304.

[50] H. Wang, Z. Ma, X. Liu, et al., “MiR-183-5p Is Required for Non-small Cell Lung Cancer Progression by Repressing PTEN,” Biomedicine & Pharmacotherapy 111 (2019): 1103–1111.

[51] Z. Wang, F. Xia, T. Feng, B. Jiang, W. Wang, and X. Li, “OTUD6B-AS1 Inhibits Viability, Migration, and Invasion of Thyroid Carcinoma by Targeting miR-183-5p and miR-21,” Front Endocrinol (Lausanne) 11 (2020): 136.

[52] J. O. Yoo, S. Y. Kwak, H. J. An, I. H. Bae, M. J. Park, and Y. H. Han, “miR-181b-3p Promotes Epithelial-mesenchymal Transition in Breast Cancer Cells Through Snail Stabilization by Directly Targeting YWHAG,” Biochimica Et Biophysica Acta 1863, no. 7 Pt A (2016): 1601–1611.

[53] Y. Jiang, Q. Qiu, X. Jing, et al., “Cancer-associated Fibroblast-derived Exosome miR-181b-3p Promotes the Occurrence and Development of Colorectal Cancer by Regulating SNX2 Expression,” Biochemical and Biophysical Research Communications 641 (2023): 177–185.

[54] C. E. Green, J. Clarke, R. Bicknell, and A. M. Turner, “Pulmonary MicroRNA Changes Alter Angiogenesis in Chronic Obstructive Pulmonary Disease and Lung Cancer,” Biomedicines 9, no. 7 (2021): 830.

[55] R. L. Causin, L. S. da Silva, A. F. Evangelista, et al., “MicroRNA Biomarkers of High-Grade Cervical Intraepithelial Neoplasia in Liquid Biopsy,” BioMed research international 2021 (2021): 6650966.

[56] X. Qin, J. Zhang, Y. Lin, X. M. Sun, J. N. Zhang, and Z. Q. Cheng, “Identification of MiR-211-5p as a Tumor Suppressor by Targeting ACSL4 in Hepatocellular Carcinoma,” Journal of translational medicine 18, no. 1 (2020): 326.

[57] G. Jiang, L. Wen, W. Deng, Z. Jian, and H. Zheng, “Regulatory Role of miR-211-5p in Hepatocellular Carcinoma Metastasis by Targeting ZEB2,” Biomedicine & Pharmacotherapy 90 (2017): 806–812.

[58] K. Wang, W. Jin, P. Jin, X. Fei, X. Wang, and X. Chen, “miR-211-5p Suppresses Metastatic Behavior by Targeting SNAI1 in Renal Cancer,” Molecular Cancer Research 15, no. 4 (2017): 448–456.

[59] J. Shi, G. An, Y. Guan, et al., “miR-328-3p Mediates the Anti-tumor Effect in Osteosarcoma via Directly Targeting MMP-16,” Cancer cell international 19 (2019): 104.

[60] Z. Liu, T. Wang, Y. She, et al., “N(6)-methyladenosine-Modified circIGF2BP3 Inhibits CD8(+) T-Cell Responses to Facilitate Tumor Immune Evasion by Promoting the Deubiquitination of PD-L1 in Non-Small Cell Lung Cancer,” Molecular cancer 20, no. 1 (2021): 105.

[61] Q. Wang, J. Shang, Y. Zhang, Y. Zhou, and L. Tang, “MiR-451a Restrains the Growth and Metastatic Phenotypes of Papillary Thyroid Carcinoma Cells via Inhibiting ZEB1,” Biomedicine & Pharmacotherapy 127 (2020): 109901.

[62] J. Cui, J. Wang, Y. Shen, and D. Lin, “Suppression of HELLS by miR-451a Represses mTOR Pathway to Hinder Aggressiveness of SCLC,” Genes Genomics 43, no. 2 (2021): 105–114.

[63] E. Minna, P. Romeo, M. Dugo, et al., “miR-451a Is Underexpressed and Targets AKT/mTOR Pathway in Papillary Thyroid Carcinoma,” Oncotarget 7, no. 11 (2016): 12731–12747.

[64] Y. Liu, H. Z. Yang, Y. J. Jiang, and L. Q. Xu, “miR-451a Is Downregulated and Targets PSMB8 in Prostate Cancer,” The Kaohsiung Journal of Medical Sciences 36, no. 7 (2020): 494–500.

[65] G. Y. Wei, M. Hu, L. Zhao, and W. S. Guo, “MiR-451a Suppresses Cell Proliferation, Metastasis and EMT via Targeting YWHAZ in Hepatocellular Carcinoma,” European Review for Medical and Pharmacological Sciences 23, no. 12 (2019): 5158–5167.

[66] S. A. Melo, L. B. Luecke, C. Kahlert, et al., “Glypican-1 Identifies Cancer Exosomes and Detects Early Pancreatic Cancer,” Nature 523, no. 7559 (2015): 177–182.

RIGHTS & PERMISSIONS

2026 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.