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. 2020 Aug 19;7(21):2002157.
doi: 10.1002/advs.202002157. eCollection 2020 Nov.

Nidogen 1-Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1

Xiaowen Mao  1 , Sze Keong Tey  1 , Cherlie Lot Sum Yeung  1 , Ernest Man Lok Kwong  1 , Yi Man Eva Fung  2 , Clive Yik Sham Chung  3 , Lung-Yi Mak  4   5 , Danny Ka Ho Wong  4   5 , Man-Fung Yuen  4   5 , James Chung Man Ho  4 , Herbert Pang  6 , Maria Pik Wong  1 , Carmen Oi-Ning Leung  7 , Terence Kin Wah Lee  7 , Victor Ma  8 , William Chi-Shing Cho  8 , Peihua Cao  9   10 , Xiaoping Xu  9 , Yi Gao  9   11 , Judy Wai Ping Yam  1   5
Affiliations

Nidogen 1-Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1

Xiaowen Mao et al. Adv Sci (Weinh). .

Abstract

In hepatocellular carcinoma (HCC) patients with extrahepatic metastasis, the lung is the most frequent site of metastasis. However, how the lung microenvironment favors disseminated cells remains unclear. Here, it is found that nidogen 1 (NID1) in metastatic HCC cell-derived extracellular vesicles (EVs) promotes pre-metastatic niche formation in the lung by enhancing angiogenesis and pulmonary endothelial permeability to facilitate colonization of tumor cells and extrahepatic metastasis. EV-NID1 also activates fibroblasts, which secrete tumor necrosis factor receptor 1 (TNFR1), facilitate lung colonization of tumor cells, and augment HCC cell growth and motility. Administration of anti-TNFR1 antibody effectively diminishes lung metastasis induced by the metastatic HCC cell-derived EVs in mice. In the clinical perspective, analysis of serum EV-NID1 and TNFR1 in HCC patients reveals their positive correlation and association with tumor stages suggesting the potential of these molecules as noninvasive biomarkers for the early detection of HCC. In conclusion, these results demonstrate the interplay of HCC EVs and activated fibroblasts in pre-metastatic niche formation and how blockage of their functions inhibits distant metastasis to the lungs. This study offers promise for the new direction of HCC treatment by targeting oncogenic EV components and their mediated pathways.

Keywords: extracellular vesicles; hepatocellular carcinoma; nidogen 1; pre‐metastatic niche; tumor necrosis factor receptor 1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
EVs from metastatic MHCC97L cells promote HCC tumorigenesis and metastasis. A) Migration and invasion assays of LO2 and PLC/PRF/5 cells pretreated with EVs derived from MIHA, MHCC97L, or MHCCLM3 cells. Cells treated with PBS were included as controls. Representative images of fixed and crystal violet‐stained migrated and invaded cells at the end of the experiment are shown. B) Schematic diagram of the EV education mouse model. Nude mice were injected with EVs derived from MIHA or MHCC97L cells via tail vein once a week for 3 weeks (15 µg per week) prior to orthotopic liver implantation of tumor seeds derived from naïve luciferase‐labeled MHCC97L cells (n = 5). Analysis of liver tumors formed and distant lung metastases was performed 5 weeks after liver implantation. C) Bioluminescence imaging of animals at the end of the experiment. D) Image of excised livers. Measurement of liver tumor size is plotted. E) Bioluminescence imaging of dissected lung tissues. Quantification of the luciferase signal is shown. Three independent experiments were performed in triplicate for assays shown in (A) and (B). Data are represented as the mean ± SEM; :p < 0.05; ::p < 0.01; :::p < 0.001; NS, not significant from Student's t‐test.
Figure 2
Figure 2
EVs from metastatic MHCC97L cells enhance endothelial leakiness and hepatoblasts colonization in the lungs. A) Tissue distribution of EVs in tissues of mice. 24 h after mice were intravenously injected with EVs derived from MHCC97L CD63‐GFP cells, the mice were subjected to euthanasia, perfused, and fixed for 24 h before tissue dissection. Tissue sections were examined under confocal microscopy. Black and white images reveal the detection of EVs in different tissues (upper panel). EVs are indicated by the arrowhead. Images of GFP+ EVs and DAPI‐stained nuclei are shown (lower panel). Quantification of the signal in five random fields of three tissue sections per organ is shown. Scale bar: 10 µm. B) Tube formation assay of HUVECs pretreated with the indicated EVs. Quantification of capillary‐like tubular structures formed is shown. C) Analysis of lung vessel leakiness after tail vein injection of MHCC97L‐EVs, Texas Red‐Dextran, and FITC‐Lectin. The arrowhead indicates the area of endothelial leakiness. Scale bar: 20 µm. D) Analysis of lung colonization of murine p53−/−; Myc hepatoblasts (1 × 105) 2 weeks after coinjection with the indicated EVs (10 µg) via tail vein (n = 5). E) Image of bioluminescence signals of mice at the end of the experiment. Quantification of the luciferase signal is shown. F) Bioluminescence imaging of dissected lung tissues and quantification of the luciferase signal. G) Representative image of a dissected lung after fixation. H) Representative images of hematoxylin and eosin (H&E) staining of lung tissues. Examples of metastatic lesions are indicated by arrowheads. Insets show the enlarged area of the metastatic lesions. Magnification, 5 ×; Scale bar, 200 µm. Three independent experiments were performed in triplicate for assays shown in (A)–(C). Data are represented as the means ± SEM; :p < 0.05; ::p < 0.01; :::p < 0.001; ::::p < 0.0001; NS, not significant from Student's t‐test.
Figure 3
Figure 3
NID1 level in EVs correlates with the metastatic potential of cells and tumor burden in mice. A) Protein was extracted from EVs derived from MIHA, MHCCLM3, and MHCC97L cells and was subjected to mass spectrometry analysis (technical triplicate/sample). Venn diagram illustrating the number of proteins that were commonly and uniquely expressed in EVs of the indicated cell lines. B) Analysis of the distribution of cellular components of proteins commonly identified in MHCCLM3‐ and MHCC97L‐EVs using FunRich3.1.3. C) Volcano plots of proteins that were significantly modulated by at least fourfold in MHCCLM3‐EVs (left) and MHCC97L‐EVs (right) when compared to proteins of MIHA‐EVs with p‐value < 0.05. D) Top ten upregulated proteins identified in MHCCLM3‐EVs ranked by p‐value. Significance of their upregulation in MHCC97L‐EVs is listed accordingly. E) Immunoblots showing NID1 expression in the total cell lysate (TCL) and EVs of MIHA, MHCCLM3, and MHCC97L cells. F) Analysis of NID1 expression in EVs derived from MIHA cells and different HCC cell lines was performed in duplicate by ELISA. G) Collection of blood from mice before and after orthotopic liver implantation of luciferase‐labeled MHCC97L tumor seed (n = 5). EVs were isolated from the serum and subjected to protein extraction. Serum EV‐NID1 level was analyzed in duplicate using ELISA. Data are represented as the mean ± SEM; :p < 0.05; ::p < 0.01; :::p < 0.001 from Student's t‐test.
Figure 4
Figure 4
EV‐NID1 is a functional component that drives HCC motility, tumorigenesis, and metastasis. A) ELISA analysis of NID1 levels in EVs derived from MHCC97L (97L) control (CTL‐KD) and NID1 knockdown cells (NID1‐KD1 and NID1‐KD2) and control (XPack) and NID1 overexpressing cells (XP‐NID1) established in HLE and Hep3B cells. The analysis was performed in triplicate. B) Examination of the migratory potential and invasiveness of MIHA and PLC/PRF/5 cells pretreated with MHCC97L CTL‐KD‐ and NID1‐KD‐EVs. C) Examination of the migratory potential and invasiveness of MIHA and PLC/PRF/5 cells pretreated with HLE XPack‐ and XP‐NID1‐EVs. D) Examination of the migratory potential and invasiveness of MIHA and PLC/PRF/5 cells pretreated with Hep3B XPack‐ and XP‐NID1‐EVs. E) EV mouse model comparing the effects of EVs from MHCC97L CTL‐KD and NID1‐KD cells on HCC tumorigenesis and metastasis (n = 5). Image showing the luciferase signal of the animals at the end of the experiment. Quantification of the luciferase signal is shown. F) Bioluminescence imaging of dissected liver tissues. Quantification of the luciferase signal is shown. G) Bioluminescence imaging of dissected lung tissues. Quantification of the luciferase signal is shown. Three independent experiments were performed in triplicate for assays shown in (C) and (D). Data are represented as the mean ± SEM; :p < 0.05; ::p < 0.01; :::p < 0.001; NS, not significant from Student's t‐test.
Figure 5
Figure 5
EVs with reduced NID1 levels show a diminished ability to increase vascular permeability, enhance angiogenesis, and facilitate colonization of hepatoblasts in the lung. A) Analysis of lung vessel leakiness after tail vein injection of PBS, MHCC97L CTL‐KD‐EVs, or NID1‐KD‐EVs; Texas Red‐Dextran and FITC‐Lectin. The arrowhead indicates the area of endothelial leakiness. Scale bar: 20 µm. B) Tube formation of HUVECs pretreated with EVs. Quantification of the capillary‐like tubular structures formed is shown. Three independent experiments were performed in triplicate. C) In vivo angiogenesis plug formation assay performed by subcutaneous coinjection of PLC/PRF/5 cells with PBS, MHCC97L CTL‐KD‐EVs, or NID1‐KD‐EVs. Representative images showing H&E staining and immunohistochemistry of dissected tumors using anti‐CD31 antibody are shown. The inset shows the enlarged area of the tumors. Scale bar: 100 µm. The number of microvessels is counted. D) Analysis of lung colonization of murine p53−/−; Myc hepatoblasts (1 × 105) after coinjection with EVs (10 µg) via tail vein (n = 4). Bioluminescence imaging of mice at the end of the experiment. Quantification of the luciferase signal is shown. E) Bioluminescence imaging of dissected lung tissues. Quantification of the luciferase signal is shown. F) Representative image of dissected lung after fixation. G) Representative images of H&E staining of lung tissues. Examples of metastatic lesions are indicated by arrowheads. Insets show the enlarged area of the metastatic lesions. Magnification, 2.5 ×; Scale bar, 500 µm. Data are represented as the mean ± SEM; :p < 0.05; ::p < 0.01; :::p < 0.001; NS, not significant from Student's t‐test.
Figure 6
Figure 6
TNFR1 secreted by EV‐NID1‐activated pulmonary fibroblasts promotes HCC cell motility and colonization in the lungs. A) Immunohistochemistry of metastatic lesions in lungs tissues obtained from mice injected with PBS, MHCC97L CTL‐KD‐EVs of NID1‐KD‐EVs using anti‐α‐SMA antibody. Magnification, 20 ×; Scale bar, 25 µm. B) Immunoblotting of S100A4 expression in MRC‐5 cells treated with EVs for 24 h. C) Immunofluorescence in MRC‐5 cells after a 24 h incubation with PKH67‐labeled MHCC97L‐EVs. Scale bar: 20 µm. D) Analysis of TNFR1 copy number and concentration of soluble TNFR1 in MRC‐5 cells pretreated with the indicated EVs using qPCR and ELISA, respectively. E) Diagram illustrating the collection of conditioned medium from MRC‐5 cells pretreated with EVs for functional assays. F) Colony formation assay performed with Hep3B incubated with the conditioned medium from MRC‐5 cells incubation with EVs from CTL‐KD or NID1‐KD cells for 72 h. Anti‐TNFR1 neutralizing antibody was added to neutralize the activity of soluble TNFR1 (Ab) (0.4 µg mL−1) in the conditioned medium. Representative image shows the fixed and crystal violet‐stained colonies. G) Migration and invasion assays performed using PLC/PRF/5 cells pretreated as described in (F). Representative image shows the fixed and crystal violet‐stained migratory and invasive cells. H) Bioluminescence imaging of mice (n = 6) subjected to intravenous coinjection of murine p53−/−; Myc hepatoblasts (1 × 105) with PBS, IgG (10 µg), or anti‐TNFR1 antibody (TNFR1 Ab) (10 µg). Quantification of the luciferase signal is shown. I) Ex vivo bioluminescence imaging of lung tissues. Quantification of the luciferase signal is shown. J) Representative images of H&E staining of lung tissues. Examples of metastatic lesions are indicated by arrowheads. Insets show the enlarged area of the metastatic lesions. Magnification, 2.5 ×; Scale bar, 500 µm. Three independent experiments were performed in triplicate for assays shown in (D)–(G). Data are represented as the mean ± SEM; :p < 0.05; ::p < 0.01; :::p < 0.001; NS, not significant from Student's t‐test.
Figure 7
Figure 7
EV‐NID1 and serum TNFR1 levels correlate with the tumor stage of HCC. A) ELISA analysis of NID1 expression in circulating EVs obtained from sera collected from individuals without liver disease (Control) (n = 12), patients with early (n = 43) and late stage (n = 22) HCC (left). ELISA analysis of serum TNFR1 in the same subjects (right). ELISA was performed in duplicate. B) Correlation between EV‐NID1 and serum TNFR1 levels determined in (A) using Pearson correlation test. C) ROC curves of EV‐NID1, serum TNFR1, and combined EV‐NID1 and serum TNFR1 for discriminating control subjects and patients with early stage HCC. D) ROC curves of AFP, AFP in combination with EV‐NID1, or serum TNFR1 for discriminating control subjects and patients with early stage HCC. E) Proposed signaling mediated by EV‐NID1. The EV‐NID1 level increases with HCC development. EV‐NID1 derived from metastatic HCC cells promotes liver tumor development and distant metastasis to the lungs. EV‐NID1 increases pulmonary vessel leakiness, angiogenesis, and colonization of cancer cells to the lungs and activates pulmonary fibroblasts to secrete TNFR1, which in turn promotes HCC cell growth and motility. ROC, receiver operating characteristic. Data are represented as the mean ± SEM; :p < 0.05 and ::p < 0.01 from Student's t‐test.
Figure 8
Figure 8
Treatment with TNFR1 neutralizing antibody effectively inhibits tumor growth and metastasis in mice implanted with metastatic tumor seed. A) Schematic diagram of the treatment regimen applied to mice implanted with luciferase‐labeled MHCC97L cells in the liver. Mice were administered PBS, IgG, or anti‐TNFR1 antibody (200 µg) via peritoneal injection every 4 days for 28 days (n = 5). B) Bioluminescence imaging of animals at the end of the experiment. Quantification of the luciferase signal is shown. The size of the liver tumors was measured and plotted. C) Ex vivo bioluminescence imaging of livers. Quantification of the luciferase signal is shown. D) Representative image of H&E staining of liver tissues showing the boundary of tumors obtained from (C). Dotted line indicates the bulging growth fronts of liver tumor. Arrows indicate the cluster of tumors nearby the liver‐tumor boundary. Magnification, 20 ×; Scale bar, 100 nm. E) Ex vivo bioluminescence imaging of lungs. Quantification of the luciferase signal is shown. F) Body weight of the mice was measured twice a week and plotted against time. Data are represented as the mean ± SEM; :p < 0.05, ::p < 0.01, :::p < 0.001 and NS, not significant from Student's t‐test.
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