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

Abstract

BACKGROUND & AIMSCeramide, 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.METHODSImmune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachlo-ride 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.RESULTSInjection 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.CONCLUSIONSIn 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.

Ceramide and palmitic acid inhibit macrophage-mediated epithelial–mesenchymal transition in colorectal cancer

“…Inhibition of IL-10 secretion by M2-TAMs resulted in impairment of EMT, migratory, and proliferative capacities of colorectal cancer cells. PA and Cer are non-toxic lipids and natural components of the plasma membrane, and delivered in vivo at significantly high concentrations, as recently demonstrated for Cer [36], and could be powerful anti-tumor agents.…”

“…Cer, a sphingolipid metabolite, plays an important role in many pathobiological processes, such as growth arrest, apoptosis, cellular signaling adhesion, and trafficking of immune cells [41,46]. Cer is also a powerful tumor suppressor, and has recently been shown to induce anti-tumor immunity by targeting M2-TAMs through inhibition of ROS signaling [36]. Albeit being different lipid species, PA and Cer block M2-TAM polarization.…”

“…Increases in intracellular Cer were reported in many cell types, in response to a variety of stimuli, including inflammatory cytokines TNF-α, IL-1, and IFN-γ [32], as well as apoptosis-inducing stimuli [33][34][35]. More recently, C-6 Cer delivered in nanoliposomes in vivo was shown to directly reduce the number of TAMs and M2-like markers in hepatocellular cancer [36].…”

Novel Sphingolipid-Based Cancer Therapeutics in the Personalized Medicine Era

“…It has been hypothesized that vinblastine and other microtubule destabilizers such as chloroquine and vincristine synergize with C 6 ceramide by reducing autophagosome formation and switching ceramide-induced autophagy into ceramide-induced cell death (Adiseshaiah et al, 2013; Lima, Milstien, & Spiegel, 2017; Young et al, 2016). A recent study showed that the use of CNL combined with adoptive T cell transfer immunotherapy in mice bearing liver tumors was able to reduce the number of tumor-associated macrophages and decreased the ability of these immune cells to suppress the anti-tumor properties (Li et al, 2018). …”

Nanomaterial Manipulation of Immune Microenvironment in the Diseased Liver

“…Li et al developed a C6‐ceremide loaded nanoliposomes (LipC6) for systemic application as an ROS scavenger, which can modulate the function of tumor‐associated macrophages through stimulating pro‐M1 differentiation, thereby break tumor‐induced immunotolerance in HCC. After the combination with the adoptive T cell transfer and immunization approach, LipC6 blocks the onset of tolerance of tumor antigen specific CD8 + T cells and facilitates increased proliferation and IFN‐γ production in response to tumor antigen stimulation, leading to the eradication of established HCC tumors . Similarly, i.v.…”