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Immune modulation of liver sinusoidal endothelial cells by melittin nanoparticles suppresses liver metastasis.

ABSTRACT: Liver sinusoidal endothelial cells (LSECs) are responsible for the immunologic tolerance of liver which is a common site for visceral metastases, suggesting its potential role as an target for cancer immunotherapy. However, targeted modulation of LSECs is still not achieved thus far. Here, we report LSECs are specifically targeted and modulated by melittin nanoparticles (α-melittin-NPs). Intravital imaging shows that LSECs fluoresce within 20 s after intravenous injection of α-melittin-NPs. α-melittin-NPs trigger the activation of LSECs and lead to dramatic changes of cytokine/chemokine milieu in the liver, which switches the hepatic immunologic environment to the activated state. As a result, α-melittin-NPs resist the formation of metastatic lesions with high efficiency. More strikingly, the survival rate reaches 80% in the spontaneous liver metastatic tumor model. Our research provides support for the use of α-melittin-NPs to break LSEC-mediated immunologic tolerance, which opens an avenue to control liver metastasis through the immunomodulation of LSECs.

SUBMITTER: Yu X

PROVIDER: S-EPMC6361944 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Immune modulation of liver sinusoidal endothelial cells by melittin nanoparticles suppresses liver metastasis.

Yu Xiang X Chen Lu L Liu Jianqiao J Dai Bolei B Xu Guoqiang G Shen Guanxin G Luo Qingming Q Zhang Zhihong Z

Nature communications 20190204 1

Liver sinusoidal endothelial cells (LSECs) are responsible for the immunologic tolerance of liver which is a common site for visceral metastases, suggesting its potential role as an target for cancer immunotherapy. However, targeted modulation of LSECs is still not achieved thus far. Here, we report LSECs are specifically targeted and modulated by melittin nanoparticles (α-melittin-NPs). Intravital imaging shows that LSECs fluoresce within 20 s after intravenous injection of α-melittin-NPs. α-me ...[more]

PMID: 30718511

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Project description:Phenotypic transformation of liver sinusoidal endothelial cells is one of the most important stages of liver metastasis progression. The miRNA effects on liver sinusoidal endothelial cells during liver metastasis have not yet been analysed. Herein, analysis of miRNA expression in these cells during colorectal liver metastasis revealed repressed expression of microRNA-20a. Importantly, downregulation of miR-20a occurs in parallel with upregulation of its known protein targets. To restore normal miR-20a levels in liver sinusoidal endothelial cells, we developed chondroitin sulfate-sorbitan ester nanoparticles conjugated with miR-20a in a delivery system that specifically targets liver sinusoidal endothelial cells. The restoration of normal mir-20a levels in these cells induced downregulation of the expression of its protein targets, and this also resulted in a reduction of in vitro LSEC migration and a reduction of in vivo activation and tumor-infiltrating capacity and ability of the tumor decreased by approximately 80% in a murine liver metastasis model. Animals: Balb/c mice (6- to 8-week-old males) were obtained from Charles River Laboratories Spain SA (Barcelona, Spain). All the procedures were approved by the Ethical Committee for Animal Experimentation (CEEA) of the University of the Basque Country (EHU/UPV) in accordance with institutional, national and international guidelines regarding the protection and care of animal use for scientific purposes. Mice were kept in the animal facility of EHU/UPV and had access to standard chow and water ad libitum. Colorectal cancer cells: Murine colorectal cancer C26 cells (ATCC, Manassas, VA) syngeneic with Balb/c mice were used. The cells were grown under standard conditions in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 microg/ml) and amphotericin B (0.25 microg/ml), all purchased from Life technologies, Carlsbad, CA. Control and tumor-activated LSECs culture: Control and tumour activated primary cultures of LSECs were isolated form livers with colorectal cancer metastasis or from healthy ones. To do so, Balb/c mice were anesthetized with isofluorano and small cut done in their left broadside. Then, 50 microl for each mice of C26 colon carcinoma cells were inoculated in the spleen at 2 x106 cells/ml concentration and the peritoneum and skin were sutured. The control mice were inoculated with PBS 1x buffer. Fourteen days later, all mice were sacrificed and the tumour activated LSECs and control LSECs were isolated by differential centrifugation. To do so, mice were perfused with collagenase P from Clostridium histolyticum (Sigma-Aldrich, St.Louis, MO, USA) through cava vein. The obtained cell suspension was twice centrifuged resulting in a parenchymal (PC)-enriched pellet and a non PC-enriched supernatant. Then, the non PC-enriched supernatant was layered on Percoll gradients (25% on top of 50%) to obtain LSEC. After a centrifugation the interphase between the two density cushions was recollected with purified non-PC enriched LSECs and Kupffer cells (KC). Then, the KCs were separated by adherence and isolated LSECs were washed and cultured with RPMI 1640 medium (Sigma-Aldrich, St.Louis, MO, USA) supplemented with 10% FBS and used in different experiments 24 hours after the isolation. (Life technologies, Carlsbad, CA). miRNA microarray analysis : The purified total RNA was analysed using Agilent mouse miRNA microarrays. Release 18.0, 8x60K (v18) microarray slides (Agilent Technologies, USA), with 1,200 mouse miRNAs represented, were used. Briefly, 100 ng of total RNA was labelled and hybridized following the standard Agilent Protocol for the miRNA Microarray System with a miRNA Complete Labelling and Hybridization Kit, including Agilent miRNA Spike-ins, and the results were scanned using an Agilent Microarray Scanner G2565CA (Agilent Technologies, USA). The scanned TIFF image files were processed using Agilent Feature Extraction Software vs 10.7.3.1 to extract raw data and obtain QC reports. The microarray raw data were normalized using the quantile method. To find the statistically significant differentially expressed miRNAs (DEMs) between two groups of samples, we calculated the mean of each probe across all the samples of the group. Next, we filtered out all the probes whose absolute value of the difference of mean values between the two groups was less than a selection threshold thetaDEM = 4 (that corresponds to a fold change of 2 in log2 scale), and we applied a Student's t-test with a significance threshold alphaDEM = 0.05. The PCA and the hierarchical clustering of genes and samples were performed with one minus the correlation metric and the unweighted average distance (UPGMA) linkage method. All the analysis algorithms and graphics were implemented functions in Matlab.

Project description:BackgroundLiver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs; liver resident macrophages) form the body's most effective scavenger cell system for the removal of harmful blood-borne substances, ranging from modified self-proteins to pathogens and xenobiotics. Controversies in the literature regarding the LSEC phenotype pose a challenge when determining distinct functionalities of KCs and LSECs. This may be due to overlapping functions of the two cells, insufficient purification and/or identification of the cells, rapid dedifferentiation of LSECs in vitro, or species differences. We therefore characterized and quantitatively compared expressed gene products of freshly isolated, highly pure LSECs (fenestrated SE-1/FcγRIIb2+) and KCs (CD11b/c+) from Sprague Dawley, Crl:CD (SD), male rats using high throughput mRNA-sequencing and label-free proteomics.ResultsWe observed a robust correlation between the proteomes and transcriptomes of the two cell types. Integrative analysis of the global molecular profile demonstrated the immunological aspects of LSECs. The constitutive expression of several immune genes and corresponding proteins of LSECs bore some resemblance with the expression in macrophages. LSECs and KCs both expressed high levels of scavenger receptors (SR) and C-type lectins. Equivalent expression of SR-A1 (Msr1), mannose receptor (Mrc1), SR-B1 (Scarb1), and SR-B3 (Scarb2) suggested functional similarity between the two cell types, while functional distinction between the cells was evidenced by LSEC-specific expression of the SRs stabilin-1 (Stab1) and stabilin-2 (Stab2), and the C-type lectins LSECtin (Clec4g) and DC-SIGNR (Clec4m). Many immune regulatory factors were differentially expressed in LSECs and KCs, with one cell predominantly expressing a specific cytokine/chemokine and the other cell the cognate receptor, illustrating the complex cytokine milieu of the sinusoids. Both cells expressed genes and proteins involved in antigen processing and presentation, and lymphocyte co-stimulation.ConclusionsOur findings support complementary and partly overlapping scavenging and immune functions of LSECs and KCs. This highlights the importance of including LSECs in studies of liver immunity, and liver clearance and toxicity of large molecule drugs and nano-formulations.

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Immune modulation of liver sinusoidal endothelial cells by melittin nanoparticles suppresses liver metastasis.

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Immune modulation of liver sinusoidal endothelial cells by melittin nanoparticles suppresses liver metastasis.

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