Nidogen 1-enriched extracellular vesicles drive HCC progression by inducing mregDC differentiation and Treg infiltration

Abstract

Aim: Nidogen 1 (NID1) is a secreted pro-tumorigenic protein. While extracellular vesicle (EV)-NID1 is known to accelerate hepatocellular carcinoma (HCC) progression, its role in reshaping the HCC immune microenvironment remains undefined. This study investigated how EV-NID1 modulates dendritic cells (DCs) to establish an immunosuppressive niche that drives liver cancer growth.

Methods: The TIMER3.0 database was used to assess the correlations of NID1 with DCs and regulatory T cells (Tregs). A Transwell co-culture system using bone marrow-derived DCs was employed to evaluate the paracrine effects of HCC cells. EVs were isolated by differential ultracentrifugation, and their identity and purity were verified. Orthotopic and subcutaneous HCC mouse models were established to assess immune cell infiltration in vivo. DC adoptive transfer and Treg depletion assays were conducted to elucidate the underlying mechanism.

Results: High NID1 expression in HCC was associated with increased infiltration of myeloid DCs and Tregs, and their co-occurrence predicted poor patient survival. In vitro, EV-NID1 induced DC maturation and promoted their polarization toward mature DCs enriched in immunoregulatory molecules (mregDC), accompanied by elevated secretion of C-C chemokine ligand 17 (CCL17). These polarized DCs promoted CD4⁺ T cell proliferation and FOXP3⁺ Treg formation. In vivo, NID1-high tumors exhibited accelerated growth and increased infiltration of mregDCs and Tregs in tumors, lymph nodes, and spleens. NID1 knockdown reduced tumor growth, whereas adoptive transfer of EV-NID1-pulsed DCs partially rescued tumor progression. Furthermore, Treg depletion markedly suppressed the growth of NID1-high tumors.

Conclusions: This study suggests that NID1-enriched EVs derived from HCC promote tumor progression by inducing a regulatory phenotype in DCs and the expansion of Tregs.

Keywords

NID1 / extracellular vesicles / dendritic cells / Tregs / hepatocellular carcinoma

References

[1] Oh JH,Jun DW. The latest global burden of liver cancer: A past and present threat Clin Mol Hepatol 2023 29 355 7 PMC10121295

[2] Bray F,Laversanne M,Sung H.et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J Clin 2024 74 229 63

[3] Vo Quang E,Shimakawa Y,Nahon P. Epidemiological projections of viral-induced hepatocellular carcinoma in the perspective of WHO global hepatitis elimination Liver Int 2021 41 915 27

[4] Luo X,Lu Y,Zhang Y.et al. USP32 facilitates tumor development and is correlated with poor clinical outcomes in hepatocellular carcinoma patients and is modulated by the E2F7/miR-218-5p axis LabMed Discovery 2025 2 100102

[5] Kumar MA,Baba SK,Sadida HQ.et al. Extracellular vesicles as tools and targets in therapy for diseases Signal Transduct Target Ther 2024 9 27 PMC10838959

[6] Marar C,Starich B,Wirtz D. Extracellular vesicles in immunomodulation and tumor progression Nat Immunol 2021 22 560 70 PMC9389600

[7] Paskeh MDA,Entezari M,Mirzaei S.et al. Emerging role of exosomes in cancer progression and tumor microenvironment remodeling J Hematol Oncol 2022 15 83 PMC9238168

[8] Olejarz W,Dominiak A,Żołnierzak A,Kubiak-Tomaszewska G,Lorenc T. Tumor-Derived Exosomes in Immunosuppression and Immunotherapy J Immunol Res 2020 2020 6272498 PMC7261328

[9] Morrissey SM,Zhang F,Ding C.et al. Tumor-derived exosomes drive immunosuppressive macrophages in a pre-metastatic niche through glycolytic dominant metabolic reprogramming Cell Metab 2021 33 2040 58.e10

[10] Guan L,Wu B,Li T.et al. HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8(+) T-cell infiltration into tumors Nat Commun 2022 13 4078

[11] Gao J,Li S,Xu Q.et al. Exosomes Promote Pre-Metastatic Niche Formation in Gastric Cancer Front Oncol 2021 11 652378 PMC8180914

[12] Komarova EY,Suezov RV,Nikotina AD.et al. Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma Sci Rep 2021 11 21314 PMC8556270

[13] Hopf M,Göhring W,Kohfeldt E,Yamada Y,Timpl R. Recombinant domain IV of perlecan binds to nidogens, laminin-nidogen complex, fibronectin, fibulin-2 and heparin Eur J Biochem 1999 259 917 25

[14] Urooj T,Wasim B,Mushtaq S.et al. Increased NID1 expression among breast cancer lung metastatic women; a comparative analysis between naive and treated cases Recent Pat Anticancer Drug Discov 2020 15 59 69

[15] Zhang B,Xu C,Liu J,Yang J,Gao Q,Ye F. Nidogen-1 expression is associated with overall survival and temozolomide sensitivity in low-grade glioma patients Aging 2021 13 9085 107 PMC8034893

[16] Xue T,Yeung CLS,Mao X.et al. Development of a broadly potent neutralizing antibody targeting Nidogen 1 effectively inhibits cancer growth and metastasis in preclinical tumor models J Transl Int Med 2025 13 78 92 PMC11921815

[17] Alečković M,Wei Y,LeRoy G.et al. Identification of Nidogen 1 as a lung metastasis protein through secretome analysis Genes Dev 2017 31 1439 55 PMC5588926

[18] Rokavec M,Jaeckel S,Hermeking H. Nidogen-1/NID1 function and regulation during progression and metastasis of colorectal cancer Cancers 2023 15 5316 PMC10670298

[19] Mao X,Tey SK,Yeung CLS.et al. Nidogen 1-enriched extracellular vesicles facilitate extrahepatic metastasis of liver cancer by activating pulmonary fibroblasts to secrete tumor necrosis factor receptor 1 Adv Sci 2020 7 2002157 PMC7640351

[20] Xue W,Kitzing T,Roessler S.et al. A cluster of cooperating tumor-suppressor gene candidates in chromosomal deletions Proc Natl Acad Sci U S A 2012 109 8212 7 PMC3361457

[21] Moon CY,Belabed M,Park MD,Mattiuz R,Puleston D,Merad M. Dendritic cell maturation in cancer Nat Rev Cancer 2025 25 225 48 PMC11954679

[22] Del Prete A,Salvi V,Soriani A.et al. Dendritic cell subsets in cancer immunity and tumor antigen sensing Cell Mol Immunol 2023 20 432 47 PMC10203372

[23] Upadhaya P,Lamenza FF,Shrestha S.et al. Berry extracts and their bioactive compounds mitigate LPS and DNFB-mediated dendritic cell activation and induction of antigen specific T-Cell effector responses Antioxidants 2023 12 1667 PMC10525528

[24] Plebanek MP,Xue Y,Nguyen YV.et al. A lactate-SREBP2 signaling axis drives tolerogenic dendritic cell maturation and promotes cancer progression Sci Immunol 2024 9 eadi4191 PMC11926670

[25] Maier B,Leader AM,Chen ST.et al. A conserved dendritic-cell regulatory program limits antitumour immunity Nature 2020 580 257 62 PMC7787191

[26] Zheng M,Yu J,Tian Z. Characterization of the liver-draining lymph nodes in mice and their role in mounting regional immunity to HBV Cell Mol Immunol 2013 10 143 50 PMC4003044

[27] Li H,Chiang CL,Kwak KJ.et al. Extracellular vesicular analysis of glypican 1 mRNA and protein for pancreatic cancer diagnosis and prognosis Adv Sci 2024 11 e2306373 PMC10953589

[28] Heidegger S,Stritzke F,Dahl S.et al. Targeting nucleic acid sensors in tumor cells to reprogram biogenesis and RNA cargo of extracellular vesicles for T cell-mediated cancer immunotherapy Cell Rep Med 2023 4 101171 PMC10518594

[29] Chen G,Huang AC,Zhang W.et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response Nature 2018 560 382 6 PMC6095740

[30] Ricklefs FL,Alayo Q,Krenzlin H.et al. Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles Sci Adv 2018 4 eaar2766 PMC5842038

[31] Wang X,Yang X,Huang C.et al. Tumor-derived extracellular vesicle PD-1 promotes tumor immune evasion via disruption of peripheral T cell homeostasis Cancer Lett 2025 612 217486

[32] Théry C,Duban L,Segura E,Véron P,Lantz O.et al. Indirect activation of naïve CD4+ T cells by dendritic cell-derived exosomes Nat Immunol 2002 3 1156 62

[33] Salimu J,Webber J,Gurney M,Al-Taei S,Clayton A,Tabi Z. Dominant immunosuppression of dendritic cell function by prostate-cancer-derived exosomes J Extracell Vesicles 2017 6 1368823 PMC5613918

[34] Chung AE,Dong LJ,Wu C,Durkin ME. Biological functions of entactin Kidney Int 1993 43 13 9

[35] Jagroop R,Martin CJ,Moorehead RA. Nidogen 1 regulates proliferation and migration/invasion in murine claudin-low mammary tumor cells Oncol Lett 2021 21 52 PMC7709544

[36] Alshoubaki YK,Nayer B,Lu YZ.et al. Tregs delivered post-myocardial infarction adopt an injury-specific phenotype promoting cardiac repair via macrophages in mice Nat Commun 2024 15 6480 PMC11294480

[37] Zbinden A,Layland SL,Urbanczyk M.et al. Nidogen-1 mitigates ischemia and promotes tissue survival and regeneration Adv Sci 2021 8 2002500 PMC7887573

[38] Caracuel-Peramos R,Rodríguez-Baena FJ,Redondo-García S.et al. Loss of the extracellular protease ADAMTS1 reveals an antitumorigenic program involving the action of NIDOGEN-1 on macrophage polarization Oncoimmunology 2025 14 2508057 PMC12101600

[39] Sage J,Leblanc-Noblesse E,Nizard C.et al. Cleavage of nidogen-1 by cathepsin S impairs its binding to basement membrane partners PLoS One 2012 7 e43494 PMC3429489

[40] Mayer U,Mann K,Timpl R,Murphy G. Sites of nidogen cleavage by proteases involved in tissue homeostasis and remodelling Eur J Biochem 1993 217 877 84

[41] Dionisi M,De Archangelis C,Battisti F.et al. Tumor-derived microvesicles enhance cross-processing ability of clinical grade dendritic cells Front Immunol 2018 9 2481 PMC6230586

[42] Rughetti A,Rahimi H,Belleudi F.et al. Microvesicle cargo of tumor-associated MUC1 to dendritic cells allows cross-presentation and specific carbohydrate processing Cancer Immunol Res 2014 2 177 86

[43] Zhang H,Tang K,Zhang Y.et al. Cell-free tumor microparticle vaccines stimulate dendritic cells via cGAS/STING signaling Cancer Immunol Res 2015 3 196 205

[44] Kvedaraite E,Ginhoux F. Human dendritic cells in cancer Sci Immunol 2022 7 eabm9409

[45] Fan Z,Wu C,Chen M.et al. The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation Acta Pharm Sin B 2022 12 1041 53 PMC9069407

[46] Thumkeo D,Punyawatthananukool S,Prasongtanakij S.et al. PGE₂-EP2/EP4 signaling elicits immunosuppression by driving the mregDC-Treg axis in inflammatory tumor microenvironment Cell Rep 2022 39 110914

[47] Yeung CLS,Cui L,Wang J.et al. Small extracellular vesicle-derived nidogen 1 promotes HCC carcinogenesis via transglutaminase 3-mediated YAP/TAZ signaling J Extracell Vesicles 2026 15 e70359 PMC13479469

[48] Chen S,Huang X,Xue Y.et al. Nanotechnology-based mRNA vaccines Nat Rev Methods Primers 2023 3 63 PMC12312698

[49] Guo Y,Wang H,Liu S.et al. Engineered extracellular vesicles with DR5 agonistic scFvs simultaneously target tumor and immunosuppressive stromal cells Sci Adv 2025 11 eadp9009 PMC11734719