Enhanced Drug Delivery with Oil-in-Water Nanoemulsions: Stability and Sustained Release of Doxorubicin.

In this study, oil-in-water nanoemulsions are prepared, an isotropic mixture of oil, surfactant, and cosurfactants. The nanoemulsions exhibit stable structures and are capable of efficiently encapsulating hydrophobic drugs such as doxorubicin (Dox). Compared to polymeric micelles, nanoemulsions demonstrate enhanced stability and loading capacity for Dox. Furthermore, nanoemulsions release Dox steadily over 14 days, with 51.6% released within the initial 24 h and up to 80% over the subsequent period. These properties suggest that nanoemulsions can mitigate the side effects related to the burst release of Dox, thereby improving therapeutic efficacy and safety. Additionally, nanoemulsion-treated cardiomyocytes show increased viability compared to those treated with free Dox, indicating the potential of nanoemulsions to alleviate Dox-induced cardiotoxicity. Overall, nanoemulsions hold promise as versatile and efficient drug carriers for improving cancer treatment outcomes.

Immunosuppressive nanoparticles containing recombinant PD-L1 and methotrexate alleviate multi-organ inflammation.

Multi-organ inflammatory diseases are one of the most serious autoimmune diseases worldwide. The regulation of immune responses by immune checkpoint proteins influences the development and treatment of cancer and autoimmune diseases. In this study, recombinant murine PD-L1 (rmPD-L1) was used for controlling T cell immunity to treat multi-organ inflammation. To enhance the immunosuppressive effect, we incorporated methotrexate, an anti-inflammatory drug, into hybrid nanoparticles (HNPs) and decorated the surface of HNPs with rmPD-L1 to produce immunosuppressive HNPs (IsHNPs). IsHNP treatment effectively targeted PD-1-expressing CD4 and CD8 T cells in the splenocytes; additionally, it promoted the production of Foxp3-expressing regulatory T cells, which suppressed the differentiation of helper T cells. IsHNP treatment also inhibited anti-CD3 antibody-mediated activation of CD4 and CD8 T cells in mice in vivo. This treatment protected mice from multi-organ inflammation induced by the adoptive transfer of naïve T cells to recombination-activating gene 1 knockout mice. The results of this study imply the therapeutic potential of IsHNPs in the treatment of multi-organ inflammation and other inflammatory diseases.

Dual-functional alginate and collagen-based injectable hydrogel for the treatment of cancer and its metastasis.

BACKGROUND: Immunotherapies have been gaining attention for the prevention of cancer recurrence and metastasis. Cancer immunotherapy can induce memory cells to target cancer-specific antigens and, thus, selectively kill cancer cells. However, there are difficulties in inducing cancer antigen-specific immunity due to limited knowledge regarding cancer antigens. In this study, we synthesized a dual-functional hydrogel to induce antigen generation and immune activation. RESULTS: To elicit a cancer self-antigen-specific immune response, we synthesized an alginate-collagen-based injectable hydrogel, called thermally responsive hydrogel (pTRG), which was incorporated with indocyanine green and the immune stimulator polyinosinic:polycytidylic acid (poly I:C). pTRG was evaluated for its anticancer and anti-metastatic effects against CT-26 carcinoma and 4T1 breast tumor in mice by combining photothermal therapy (PTT) and immunotherapy. Near-infrared (NIR) irradiation promoted temperature elevation in pTRG, consequently exerting a therapeutic effect on mouse tumors. Lung metastasis was prevented in cured CT-26 tumor-injected mice following pTRG treatment via cancer antigen-specific T cell immunity. Moreover, pTRG successfully eliminated the original tumor in 4T1 tumor-bearing mice via PTT and protected them from lung metastasis. To further evaluate the carrier function of TRGs, different types of immunotherapeutic molecules were incorporated into TRGs, which led to the effective elimination of the first CT-26 tumor and the prevention of lung metastasis. CONCLUSIONS: Our data demonstrate that TRG is a efficient material not only for treating primary tumors but also for preventing metastasis and recurrence.

Recombinant programmed cell death protein 1 functions as an immune check point blockade and enhances anti-cancer immunity.

Effective cancer therapy aims to treat not only primary tumors but also metastatic and recurrent cancer. Immune check point blockade-mediated immunotherapy showed promising effect against tumors; however, it still has a limited effect in metastatic or recurrent cancer. Here, we extracted recombinant murine programmed death-1 (rmPD-1) proteins. The extracted rmPD-1 effectively bound to CT-26 and 4T1 cells expressing PD-L1 and PD-L2. The rmPD-1 did not alter the activation of dendritic cells (DCs); however, rmPD-1 promoted T cell-mediated anti-cancer immunity against CT-26 tumors in mice. Moreover, rmPD-1 decorated thermal responsive hybrid nanoparticles (piHNPs) promoted apoptotic and necrotic cell death of CT-26 cells in response to laser irradiation at 808 nm consequently, it promoted anti-tumor effects against the 1st challenged CT-26 tumors in mice. In addition, piHNP-mediated cured mice from 1st challenged CT-26 was also prevented the 2nd challenged lung metastatic tumor growth, which was dependent of cancer antigen-specific memory T cell immunity. It was also confirmed that the lung metastatic growth of 2nd challenged 4T1 breast cancer was also prevented in cured mice from 1st challenged 4T1 by piHNP. Thus, these data demonstrate that rmPD-1 functions as an immune checkpoint blockade for the treatment of tumors, and piHNPs could be a novel therapeutic agent for preventing cancer metastasis and recurrence.

Escherichia coli Mimetic Gold Nanorod-Mediated Photo- and Immunotherapy for Treating Cancer and Its Metastasis.

Most cancer-related deaths are due to metastasis or recurrence. Therefore, the ultimate goal of cancer therapy will be to treat metastatic and recurrent cancers. Combination therapy for cancer will be one of trial for effective treating metastasis and recurrence. In this study, Escherichia coli-mimetic nanomaterials are synthesized using Escherichia coli membrane proteins, adhesion proteins, and gold nanorods, which are named E. coli mimetic AuNRs (ECA), for combination therapy against cancer and its recurrence. ECA treatment with 808 nm laser irradiation eliminates CT-26 or 4T1 tumors via a photothermal effect. ECA with laser irradiation induces activation of immune cells in the tumor-draining lymph nodes. The mice cured from CT-26 or 4T1 tumor by ECA are rechallenged with those cancer in the lung metastatic form, and the results showed that ECA treatment for the first CT-26 or 4T1 tumor challenge prevents cancer infiltration to the lung in the second challenge. This preventive effect of ECA against tumor growth in the second challenge is aided by cancer antigen-specific T cell immunity. Overall, these findings show that ECA is a nanomaterial with dual functions as a photothermal therapy for treating primary cancers and as immunotherapy for preventing recurrence and metastasis.

Comparison of the immune activation capacities of fucoidan and laminarin extracted from Laminaria japonica.

Laminaria japonica is a brown alga and is composed primarily of polysaccharides. Fucoidan and laminarin are the major polysaccharides of L. japonica and exhibit biological activities, including immune modulation and anti-coagulant and antioxidant effects in animals and humans. In this study, we evaluated the ability of fucoidan and laminarin from L. japonica to induce immune cell activation and anti-cancer immunity, which has not yet been studied. The injection of fucoidan to mice promoted the upregulation of major histocompatibility complex and surface activation molecules in splenic dendritic cell subsets, whereas laminarin showed a weaker immune activation ability. In addition, fucoidan treatment elicited inflammatory cytokine production; however, laminarin did not induce the production of these cytokines. Regarding cytotoxic cell activities, fucoidan induced the activation of lymphocytes, including natural killer and T cells, whereas laminarin did not induce cell activation. Finally, fucoidan enhanced the anticancer efficacy of anti-programmed Death-Ligand 1 (PD-L1) antibody against Lewis lung carcinoma, whereas laminarin did not promote the cancer inhibition effect of anti-PD-L1 antibody. Thus, these data suggest that fucoidan from L. japonica can be used as an immune stimulatory molecule to enhance the anticancer activities of immune checkpoint inhibitors.

Immunomodulatory effects of polysaccharides from marine algae for treating cancer, infectious disease, and inflammation.

A significant rise in the occurrence and severity of adverse reactions to several synthetic drugs has fueled considerable interest in natural product-based therapeutics. In humans and animals, polysaccharides from marine microalgae and seaweeds have immunomodulatory effects. In addition, these polysaccharides may possess antiviral, anticancer, hypoglycemic, anticoagulant, and antioxidant properties. During inflammatory diseases, such as autoimmune diseases and sepsis, immunosuppressive molecules can serve as therapeutic agents. Similarly, molecules that participate in immune activation can induce immune responses against cancer and infectious diseases. We aim to discuss the chemical composition of the algal polysaccharides, namely alginate, fucoidan, ascophyllan, and porphyran. We also summarize their applications in the treatment of cancer, infectious disease, and inflammation. Recent applications of nanoparticles that are based on algal polysaccharides for the treatment of cancer and inflammatory diseases have also been addressed. In conclusion, these applications of marine algal polysaccharides could provide novel therapeutic alternatives for several diseases.

Mice Plasmacytoid Dendritic Cells Were Activated by Lipopolysaccharides Through Toll-Like Receptor 4/Myeloid Differentiation Factor 2.

Plasmacytoid dendritic cells (pDCs) are known to respond to viral infections. However, the activation of pDCs by bacterial components such as lipopolysaccharides (LPS) has not been well studied. Here, we found that pDCs, conventional dendritic cells (cDCs), and B cells express high levels of toll-like receptor 4 (TLR4), a receptor for LPS. Moreover, LPS could effectively bind to not only cDCs but also pDCs and B cells. Intraperitoneal administration of LPS promoted activation of splenic pDCs and cDCs. LPS treatment led to upregulation of interferon regulatory factor 7 (IRF7) and induced production of interferon-alpha (IFN-α) in splenic pDCs. Furthermore, LPS-dependent upregulation of co-stimulatory molecules in pDCs did not require the assistance of other immune cells, such as cDCs. However, the production levels of IFN-α were decreased in cDC-depleted splenocytes, indicating that cDCs may contribute to the enhancement of IFN-α production in pDCs. Finally, we showed that activation of pDCs by LPS requires the TLR4 and myeloid differentiation factor 2 (MD2) signaling pathways. Thus, these results demonstrate that the gram-negative component LPS can directly stimulate pDCs via TLR4/MD2 stimulation in mice.

Enhancement of Immune Checkpoint Inhibitor-Mediated Anti-Cancer Immunity by Intranasal Treatment of Ecklonia cava Fucoidan against Metastatic Lung Cancer.

Although fucoidan, a well-studied seaweed-extracted polysaccharide, has shown immune stimulatory effects that elicit anticancer immunity, mucosal adjuvant effects via intranasal administration have not been studied. In this study, the effect of Ecklonia cava-extracted fucoidan (ECF) on the induction of anti-cancer immunity in the lung was examined by intranasal administration. In C57BL/6 and BALB/c mice, intranasal administration of ECF promoted the activation of dendritic cells (DCs), natural killer (NK) cells, and T cells in the mediastinal lymph node (mLN). The ECF-induced NK and T cell activation was mediated by DCs. In addition, intranasal injection with ECF enhanced the anti-PD-L1 antibody-mediated anti-cancer activities against B16 melanoma and CT-26 carcinoma tumor growth in the lungs, which were required cytotoxic T lymphocytes and NK cells. Thus, these data demonstrated that ECF functioned as a mucosal adjuvant that enhanced the immunotherapeutic effect of immune checkpoint inhibitors against metastatic lung cancer.

Carrier-free micellar CpG interacting with cell membrane for enhanced immunological treatment of HIV-1.

Unmethylated CpG motifs activate toll-like receptor 9 (TLR9), leading to sequence- and species-specific immune stimulation. Here, we engineered a CpG oligodeoxyribonucleotide (ODN) with multiple hydrophobic moieties, so-called lipid-modified uracil, which resulted in a facile micelle formation of the stimulant. The self-assembled CpG nanostructure (U4CpG) containing the ODN 2216 sequence was characterized by various spectroscopic and microscopic methods together with molecular dynamics simulations. Next, we evaluated the nano-immunostimulant for enhancement of anti-HIV immunity. U4CpG treatment induced activation of plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells in healthy human peripheral blood, which produced type I interferons (IFNs) and IFN-γ in human peripheral blood mononuclear cells (PBMCs). Moreover, we validated the activation and promotion efficacy of U4CpG in patient-derived blood cells, and HIV-1 spread was significantly suppressed by a low dosage of the immunostimulant. Furthermore, U4CpG-treated PBMC cultured medium elicited transcription of latent HIV-1 in U1 cells indicating that U4CpG reversed HIV-1 latency. Thus, the functions of U4CpG in eradicating HIV-1 by enhancing immunity and reversing latency make the material a potential candidate for clinical studies dealing with viral infection.

Intramitochondrial Disulfide Polymerization Controls Cancer Cell Fate.

Recent advances in supramolecular chemistry research have led to the development of artificial chemical systems that can form self-assembled structures that imitate proteins involved in the regulation of cellular function. However, intracellular polymerization systems that operate inside living cells have been seldom reported. In this study, we developed an intramitochondrial polymerization-induced self-assembly system for regulating the cellular fate of cancer cells. It showed that polymeric disulfide formation inside cells occurred due to the high reactive oxygen species (ROS) concentration of cancer mitochondria. This polymerization barely occurs elsewhere in the cell owing to the reductive intracellular environment. The polymerization of the thiol-containing monomers further increases the ROS level inside the mitochondria, thereby autocatalyzing the polymerization process and creating fibrous polymeric structures. This process induces dysfunction of the mitochondria, which in turn activates cell necroptosis. Thus, this in situ polymerization system shows great potential for cancer treatment, including that of drug-resistant cancers.

Escherichia coli adhesin protein-conjugated thermal responsive hybrid nanoparticles for photothermal and immunotherapy against cancer and its metastasis.

BACKGROUND: Advanced cancer therapy is targeted at primary tumors and also recurrent or metastatic cancers. Combinational cancer treatment has recently shown high efficiency against recurrent and metastatic cancers. In this study, we synthesized a thermal responsive hybrid nanoparticle (TRH) containing FimH, an immune stimulatory recombinant protein, for the induction of a combination of photothermal therapy (PTT) and immunotherapy against cancer and its metastasis. METHODS: The hybrid nanoparticle was incorporated with a near-infrared (NIR) absorbent, indocyanine green, and decorated with FimH on its surface to form F-TRH. F-TRH was evaluated for its anticancer and antimetastatic effects against CT-26 carcinoma in mice by combining PTT and immunotherapy. RESULTS: NIR laser irradiation elicited an elevation of temperature in F-TRH, which induced apoptosis in CT-26 carcinoma cells in vitro. In addition, F-TRH and NIR laser irradiation promoted photothermal-mediated therapeutic effects against CT-26 and 4T1 tumors in mice. The release of FimH from F-TRH in response to elevated temperature and apoptotic bodies of cancer cells via PTT elicited dendritic cell-mediated cancer antigen-specific T-cell responses, which subsequently inhibited the second challenge of CT-26 and 4T1 cell growth in the lung. CONCLUSIONS: These data demonstrate the potential use of F-TRH for immuno-photothermal therapy against cancer and its recurrence and metastasis.

Ecklonia cava fucoidan has potential to stimulate natural killer cells in vivo.

Fucoidan is a sulfated polysaccharide, derived from various marine brown seaweeds, that has immunomodulatory effects. In this study, we analyzed the effects of five different fucoidans, which were extracted from Ascophyllum nodosum, Undaria pinnatifida, Macrocystis pyrifera, Fucus vesiculosus, and Ecklonia cava, on natural killer (NK) cell activation in mice. Among these, E. cava fucoidan (ECF) promoted an increase in the number of NK cells in the spleen and had the strongest effect on the activation of NK cells. Additionally, we observed that DC stimulation was required for NK cell activation and that ECF had the most potent effect on splenic dendritic cells (DC). Finally, ECF treatment effectively prevented infiltration of CT-26 carcinoma cells in the lungs of BALB/c mice in an NK cell dependent manner. Collectively, these results suggest that ECF could be a suitable candidate for enhancing NK cell-mediated anti-cancer immunity.

Astragalus membranaceus polysaccharides potentiate the growth-inhibitory activity of immune checkpoint inhibitors against pulmonary metastatic melanoma in mice.

Astragalus membranaceus (A. membranaceus) is commonly used in various herbal formulations to treat several human and animal diseases. Polysaccharides, which are the major bioactive components in the A. membranaceus, exhibit various bioactive properties. However, the ability of A. membranaceus polysaccharides (APS) to activate the mucosal immune response has not been examined. We examined the effect of intranasal administration of APS on mucosal immune cell activation and the growth-inhibitory activity against pulmonary metastatic melanoma in mice by combination treatment with immune checkpoint blockade. The intranasal treatment of APS increased the number of lineage-CD11c+ dendritic cell (DCs) in the mesenteric lymph nodes (mLN) through the upregulation of CC-chemokine receptor 7 expression. Moreover, intranasal treatment of APS activated DCs, which further stimulated natural killer (NK) and T cells in the mLN. The APS/anti-PD-L1 antibody combination inhibited the pulmonary infiltration of B16 melanoma cells. The depletion of NK cells and CD8 T cells in mice mitigated the anti-cancer effect of this combination, thereby highlighting the critical role of NK cells and CD8 T cells in mediating anti-cancer immunity. These findings demonstrated that APS could be used as a topical mucosal adjuvant to enhance the immune check point inhibitor anti-cancer effect.

Water Dynamics in Unilateral Biportal Endoscopic Spine Surgery and Its Related Factors: An In Vivo Proportional Regression and Proficiency-Matched Study.

BACKGROUND: Stable water dynamics during endoscopic spine surgery improves the surgeon's comfort and patient's outcomes. We aimed to measure the water dynamics during spinal surgery and identify the factors that facilitate stable water dynamics. METHODS: This open-label, prospective, proficiency-matched, in vivo study included patients with single-level degenerative spinal disease. After assessing their heights and balancing the matched instrument, we measured the irrigation fluid pressure in various situations. We performed multiple regression analysis based on odds ratio (OR), confidence interval (CI), and relationships (proficiency-matched) with possible instrumental and physical characteristics. The instrumental factors were the presence and length of a rigid cannulation, and the physical characteristics were body mass index (body mass index [BMI]), skin-to-dura depth, height (interaction with BMI), and body weight (interaction with BMI). RESULTS: Of the 36 patients, 29 were included. The mean pressure of the operation cavity was 16.66 ± 9.12 cm H2O (12.25 ± 6.71 mm Hg). Water pressure with the rigid cannulation (9.41 ± 2.94 cm H2O [6.92 ± 2.16 mm Hg]) was significantly lower than that without cannulation (23.43 ± 7.57 cm H2O [17.26 ± 5.57 mm Hg], P < 0.01). Water pressure correlated with cannular length (OR = -1.08, CI = -1.79, -0.37, P < 0.01) and BMI (OR = 0.56, CI = 0.12, 0.99, P < 0.01). BMI showed a proportional relationship (r = 0.84, P < 0.01). CONCLUSIONS: During biportal endoscopy, we suggest maintaining water pressure between 4.41 cm H2O (2.41 mm Hg) and 31.00 cm H2O (22.83 mm Hg). Compared to physical characteristics, placement of the cannula and appropriate cannula length are important factors that affect water dynamics.

Valorization of waste-cooking oil into sophorolipids and application of their methyl hydroxyl branched fatty acid derivatives to produce engineering bioplastics.

Waste-cooking oil (WCO) is defined as vegetable oil that has been used to fry food at high temperatures. The annual global generation of WCO is 41-67 million tons. Without proper treatment, most WCO is abandoned in sinks and the solid residue of WCO is disposed of in landfills, resulting in serious environmental problems. Recycling and valorizing WCO have received considerable attention to reduce its negative impact on ecosystems. To convert WCO into a high value-added compound, we aimed to produce sophorolipids (SLs) that are industrially important biosurfactants, using WCO as a hydrophobic substrate by the fed-batch fermentation of Starmerella bombicola. The SLs concentration was increased ~3.7-fold compared with flask culture (315.6 vs. 84.8 g/L), which is the highest value ever generated from WCO. To expand the applications of SLs, we prepared methyl hydroxy branched fatty acids (MHBFAs) from SLs, which are important chemicals for various industries yet difficult to produce by chemical methods, using a bio-chemical hybrid approach. We synthesized bio-based plastics using MHBFAs as co-monomers. Compared with the control polymer without MHBFAs, even the incorporation of 1 mol% into polymer chains improved mechanical properties (such as ultimate tensile strength, 1.1-fold increase; toughness, 1.3-fold increase). To the best of our knowledge, this is the first attempt to apply MHBFAs from SLs derived from WCO to building blocks of plastics. Thus, we extended the valorization areas of WCO to one of the world's largest industries.

Comparison of human peripheral blood dendritic cell activation by four fucoidans.

Brown seaweed is an important source of fucoidan, which displays immunomodulatory effects by activating various immune cells. However, these effects of fucoidans from various sources of brown seaweed have not yet been explored in human blood dendritic cells. We studied fucoidans extracted from Ecklonia cava, Macrocystis pyrifera, Undaria pinnatifida, and Fucus vesiculosus for their effects on human monocyte-derived dendritic cells (MODC) and human peripheral blood DC (PBDC) activation. Ecklonia cava fucoidan (ECF) strongly upregulated co-stimulatory molecules, major histocompatibility complex class I and II, and the production of proinflammatory cytokines in MODCs and PBDCs compared to those by the other three fucoidans. Moreover, ECF elicited the strongest effect in the induction of syngeneic T cell proliferation and IFN-γ production compared to those of other fucoidans. These results suggest that ECF could be a suitable candidate molecule for enhancing immune activation in humans compared to that with the other three fucoidans.

Monophosphoryl lipid A-induced activation of plasmacytoid dendritic cells enhances the anti-cancer effects of anti-PD-L1 antibodies.

Monophosphoryl lipid A (MPLA) is a toll-like receptor 4 ligand that promotes immune activation in mice and humans, without undesired inflammation. Immunotherapy by the combining immune checkpoint blockade and MPLA has shown promising anti-cancer effects in both mice and humans. In this study, we explored how MPLA enhanced the anti-cancer effects of anti-PD-L1 antibodies (Abs). Anti-cancer immunity induced by the combination of anti-PD-L1 Abs and MPLA failed in CD4 and CD8 cell-depleted mice. Moreover, the combination treatment of anti-PD-L1 Abs and MPLA synergistically enhanced the activation of plasmacytoid dendritic cells (pDCs) in the mouse in vivo, while conventional DCs were not. In addition, mice treated with anti-PD-L1 Abs and MPLA were not protected from B16 melanoma by blockade of interferon-alpha receptor (IFNAR). The combination of anti-PD-L1 Abs and MPLA also promoted human peripheral blood pDC activation and induced IFN-α-dependent T cell activation. Therefore, these results demonstrate that MPLA enhances anti-PD-L1 Ab-mediated anti-cancer immunity through the activation and IFN-α production of pDCs.

Ubiquitin Activating Enzyme UBA6 Regulates Th1 and Tc1 Cell Differentiation.

Ubiquitination is a crucial mechanism in regulating the immune response, setting the balance between immunity and tolerance. Here, we investigated the function of a poorly understood alternative branch of the ubiquitin-activating E1 enzyme UBA6 in activating immune cells. UBA6 expression levels were elevated in T cells by toll-like receptor agonists and anti-CD3/28 antibody stimulation, but not in dendritic cells, macrophages, B cells, and natural killer cells. Additionally, we generated T cell-specific UBA6-deficient mice and found that UBA6-deficient CD4 and CD8 T cells elevated the production of interferon-gamma (IFN-γ). Moreover, the transfer of UBA6-deficient CD4 and CD8 T cells in RAG1-knockout mice exacerbated the development of multi-organ inflammation compared with control CD4 and CD8 T cell transfer. In human peripheral blood CD4 and CD8 T cells, basal levels of UBA6 in lupus patients presented much lower than those in healthy controls. Moreover, the IFN-γ production efficiency of CD4 and CD8 T cells was negatively correlated to UBA6 levels in patients with lupus. Finally, we found that the function of UBA6 was mediated by destabilization of IκBα degradation, thereby increasing NF-κB p65 activation in the T cells. Our study identifies UBA6 as a critical regulator of IFN-γ production in T cells by modulating the NF-κB p65 activation pathway.

Polysaccharide from Codium fragile Induces Anti-Cancer Immunity by Activating Natural Killer Cells.

Natural polysaccharides exhibit beneficial immune modulatory effects, including immune stimulatory and anti-cancer activities. In this study, we examined the effect of Codium fragile polysaccharide (CFP) on natural killer (NK) cell activation, and its effect on tumor-bearing mice. Intravenous CFP treatment of C57BL/6 mice resulted in the upregulation of CD69, which is a marker associated with NK cell activation. In addition, intracellular levels of interferon (IFN)-γ and the cytotoxic mediators perforin and granzyme B were markedly increased in response to the CFP treatment of splenic NK cells. IFN-γ production by NK cells was directly induced by CFP, whereas the upregulation of CD69 and cytotoxic mediators required IL-12. Finally, intraperitoneal treatment with CFP prevented CT-26 (murine carcinoma) tumor cell infiltration in the lungs, without significantly reducing the body weight. In addition, treatment with CFP prevented B16 melanoma cell infiltration in the lung of C57BL/6 mice. Moreover, the anti-tumor effect was diminished by the depletion of NK cells. Therefore, these data suggest that CFP may be used as an NK cell stimulator to produce a phenomenon that contributes to anti-cancer immunity.