Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice

Guangfu Li, Dai Liu, Eric T. Kimchi, Jussuf T. Kaifi, Xiaoqiang Qi, Yariswamy Manjunath, Xinjian Liu, Tye Deering, Diego M. Avella, Todd Fox, Don C. Rockey, Todd D. Schell, Mark Kester, Kevin F. Staveley-O'Carroll

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Abstract

Background & Aims: Ceramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors. Methods: Immune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachloride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8+ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8+ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay. Results: Injection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8+ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8+ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF. Conclusions: In mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8+ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

Original languageEnglish (US)
Pages (from-to)1024-1036.e9
JournalGastroenterology
Volume154
Issue number4
DOIs
StatePublished - Mar 2018

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Liver
Growth
Neoplasms
Macrophages
Neoplasm Antigens
T-Lymphocytes
Injections
N-caproylsphingosine
Immunization
Macrophage Colony-Stimulating Factor
Apoptosis
Intraperitoneal Injections
Tail
Hepatocellular Carcinoma
Veins
Reactive Oxygen Species
Luminescent Measurements
Phenotype
Sphingolipids
Carbon Tetrachloride

All Science Journal Classification (ASJC) codes

  • Hepatology
  • Gastroenterology

Cite this

Li, G., Liu, D., Kimchi, E. T., Kaifi, J. T., Qi, X., Manjunath, Y., ... Staveley-O'Carroll, K. F. (2018). Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice. Gastroenterology, 154(4), 1024-1036.e9. https://doi.org/10.1053/j.gastro.2017.10.050
Li, Guangfu ; Liu, Dai ; Kimchi, Eric T. ; Kaifi, Jussuf T. ; Qi, Xiaoqiang ; Manjunath, Yariswamy ; Liu, Xinjian ; Deering, Tye ; Avella, Diego M. ; Fox, Todd ; Rockey, Don C. ; Schell, Todd D. ; Kester, Mark ; Staveley-O'Carroll, Kevin F. / Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice. In: Gastroenterology. 2018 ; Vol. 154, No. 4. pp. 1024-1036.e9.
@article{b127e798e97e434bad16a79295d5e014,
title = "Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice",
abstract = "Background & Aims: Ceramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors. Methods: Immune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachloride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8+ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8+ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay. Results: Injection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8+ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8+ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF. Conclusions: In mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8+ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.",
author = "Guangfu Li and Dai Liu and Kimchi, {Eric T.} and Kaifi, {Jussuf T.} and Xiaoqiang Qi and Yariswamy Manjunath and Xinjian Liu and Tye Deering and Avella, {Diego M.} and Todd Fox and Rockey, {Don C.} and Schell, {Todd D.} and Mark Kester and Staveley-O'Carroll, {Kevin F.}",
year = "2018",
month = "3",
doi = "10.1053/j.gastro.2017.10.050",
language = "English (US)",
volume = "154",
pages = "1024--1036.e9",
journal = "Gastroenterology",
issn = "0016-5085",
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Li, G, Liu, D, Kimchi, ET, Kaifi, JT, Qi, X, Manjunath, Y, Liu, X, Deering, T, Avella, DM, Fox, T, Rockey, DC, Schell, TD, Kester, M & Staveley-O'Carroll, KF 2018, 'Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice', Gastroenterology, vol. 154, no. 4, pp. 1024-1036.e9. https://doi.org/10.1053/j.gastro.2017.10.050

Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice. / Li, Guangfu; Liu, Dai; Kimchi, Eric T.; Kaifi, Jussuf T.; Qi, Xiaoqiang; Manjunath, Yariswamy; Liu, Xinjian; Deering, Tye; Avella, Diego M.; Fox, Todd; Rockey, Don C.; Schell, Todd D.; Kester, Mark; Staveley-O'Carroll, Kevin F.

In: Gastroenterology, Vol. 154, No. 4, 03.2018, p. 1024-1036.e9.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice

AU - Li, Guangfu

AU - Liu, Dai

AU - Kimchi, Eric T.

AU - Kaifi, Jussuf T.

AU - Qi, Xiaoqiang

AU - Manjunath, Yariswamy

AU - Liu, Xinjian

AU - Deering, Tye

AU - Avella, Diego M.

AU - Fox, Todd

AU - Rockey, Don C.

AU - Schell, Todd D.

AU - Kester, Mark

AU - Staveley-O'Carroll, Kevin F.

PY - 2018/3

Y1 - 2018/3

N2 - Background & Aims: Ceramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors. Methods: Immune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachloride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8+ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8+ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay. Results: Injection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8+ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8+ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF. Conclusions: In mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8+ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

AB - Background & Aims: Ceramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors. Methods: Immune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachloride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8+ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8+ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay. Results: Injection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8+ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8+ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF. Conclusions: In mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8+ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

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