Dendritic cell-induced activation of adaptive and innate antitumor immunity

While studying Ag-pulsed syngeneic dendritic cell (DC) immunization, we discovered that surprisingly, unpulsed DCs induced protection against tumor lung metastases resulting from i.v. injection of a syngeneic BALB/c colon carcinoma CT26 or a syngeneic C57BL/6 lung carcinoma LL/2. Splenocytes or immature splenic DCs did not protect. The protection was mediated by NK cells, in that it was abrogated by treatment with anti-asialo-GM1 but not anti-CD8, and was induced by CD1(-/-) DCs unable to stimulate NKT cells, but did not occur in beige mice lacking NK cells. Protection correlated with increased NK activity, and increased infiltration of NK but not CD8(+) cells in lungs of tumor-bearing mice. Protection depended on the presence of costimulatory molecules CD80, CD86, and CD40 on the DCs, but surprisingly did not require DCs that could make IL-12 or IL-15. Unexpectedly, protection sensitive to anti-asialo-GM1 and increased NK activity were still present 14 mo after DC injection. As NK cells lack memory, we found by depletion that CD4(+) not CD8(+) T cells were required for induction of the NK antitumor response. The role of DCs and CD4(+) T cells provides a novel mechanism for NK cell induction and innate immunity against cancer that may have potential in preventing clinical metastases.     

Role of organ-associated NK cells in decreased formation of experimental metastases in lung and liver

Mice treated with anti-asialo GM1 (asGM1) serum exhibited increased formation of experimental metastases in lung and liver after i.v. challenge with B16 melanoma or Lewis lung carcinoma. This increased metastasis formation coincided with decreased splenic NK activity and increased survival of i.v. injected radiolabeled tumor cells. In contrast, the injection of mice with the pyran copolymer maleic anhydride divinyl ether (MVE-2) augmented NK activity in the spleen and significantly depressed the formation of experimental metastases in the lungs and liver. However, a single or double administration of anti-asGM1 antiserum to MVE-2-pretreated mice failed to inhibit the immunoprophylaxis associated with MVE-2 administration, although it did decrease splenic NK activity and also increased the survival of i.v.-injected radiolabeled tumor cells. To address the mechanism for this dichotomy, we examined NK activity not only in the spleen but also in the blood, lungs, and livers of MVE-2-treated mice. Levels of NK activity in the lungs and liver were several-fold higher than those observed in spleen and blood. However, MVE-2-augmented NK activity in lung and liver was more resistant to depletion by the standard regimen of anti-asGM1 treatment than was NK activity in blood and spleen, and required two high-dose administrations of a higher titered antiserum for depletion of the augmented response. This high-dose regimen removed all detectable NK activity from the lung and liver, and concomitantly eliminated the metastasis-inhibiting effect of MVE-2. These data are consistent with a role for organ-associated NK cells in inhibiting metastasis formation during the extravasation and/or early postextravasation phases of the metastatic process. The results also suggest that biologic effects of NK activity in spleen and blood can be dissociated from those mediated by NK activity in other organs by use of different treatment regimens with anti-asGM1 serum. Finally, because NK activity in target organs can be augmented to an even greater extent than in the blood and spleen by at least some biologic response modifiers (BRMs), organ-associated NK activity should be considered as a possible mechanism for the therapeutic effects of BRM treatment.     

The Association of the Nm23-M1 Protein and β-Tubulin Correlates with Cell Differentiation

The Nm23 protein is a nucleoside diphosphate kinase (NDPK) and is thought to play a critical role in metastatic behavior. It has been reported that a NDPK activity is present in microtubules assembled in vitro. Since microtubule assembly is determinant in cell growth and differentiation, we investigated whether Nm23-M1 forms molecular complexes with β-tubulin in murine cells either actively proliferating or differentiating. For this purpose a polyclonal antibody against the GST-Nm23-M1 fusion protein was generated and employed to detect Nm23-M1/β-tubulin complexes in murine tumor cells derived from the Lewis lung carcinoma (3LL) and in undifferentiated and differentiated myogenic cells (C2C12). Immunoblotting and immunoprecipitation experiments performed using the anti-fusion protein antibody demonstrated that the Nm23-M1 protein is detectable in in vitro tumor cell lines and in in vivo primary tumors but not in spontaneous lung metastases. These data are in good agreement with data previously reported. Immunoprecipitation experiments demonstrated that the Nm23-M1 protein forms complexes with β-tubulin in in vitro tumor cell lines, but not in primary tumors. Furthermore, the Nm23-M1 protein forms complexes with β-tubulin in myogenic cells prior to and after differentiation. Interestingly, however, the level of the Nm23-M1/β-tubulin complexes is remarkably increased in differentiated myotubes. In conclusion, the results indicate that the Nm23-M1 protein forms molecular complexes with β-tubulin and that the number of complexes increases during the differentiation process of murine cells.     

Tumor-inhibitory and liver-protective effects of <Emphasis Type="Italic">Phellinus igniarius</Emphasis> extracellular polysaccharides

The tumor-inhibitory and liver-protective effects of crude extracellular polysaccharides (EPS) extracted from the liquid mycelial culture of the mushroom Phellinus igniarius were studied in mice. The mice were injected with murine sarcoma S180 and murine hepatoma H22. Crude EPS at 100, 200, 400 mg kg⁻¹ body weight was administered to EPS groups each day in the twelve consecutive days. The result showed that EPS 200 mg kg⁻¹ body weight significantly inhibited S180 and H22 at 65.0 and 46.3%, respectively. Moreover, EPS could not only keep the numbers of WBC, RBC, PLT and the concentration of HGB in a normal range, but also normalize the activities of AST, ALT and ALP. For example, in EPS-treated mice, AST significantly reduced with the percentage of A/G reverse in S180 (P < 0.05) and H22 (P < 0.01) when the mice took EPS 200 mg kg⁻¹ body weight. In conclusion, it was remarkable that P. igniarius EPS exhibited antitumor activity related to dosage and protected liver function by sustaining the blood routine as well as keeping the blood biochemical indexes normal.     

Production and characterization of the exopolysaccharide produced by <Emphasis Type="Italic">Paenibacillus jamilae</Emphasis> grown on olive mill-waste waters

Paenibacillus jamilae, a strain isolated from compost prepared with olive-mill wastewaters, produced an extracellular polysaccharide (EPS) when it was grown in a culture containing olive-mill waste waters (OMWW) as sole carbon and energy sources. Maximal EPS production in 100 mL batch-culture experiments (5.1 g L⁻¹) was reached with a concentration of 80% of OMWW as fermentation substrate (v/v). Although an inhibitory effect was observed on growth and EPS production when OMWW concentration was increased, an appreciable amount of EPS (2.7 g L⁻¹) was produced with undiluted OMWW. Sepharose CL-2B chromatography showed that the EPS presented two fractions, EPS I (>2000 kDa) and EPS II (500 kDa). Both fractions were characterized by GC-MS as two different acidic heteropolysaccharides containing glucose, galactose and mannose as the major components. The performed study made evident the possibility of using OMWW as substrate for the production of EPS by P. jamilae with a satisfactory yield.     

Exopolysaccharide production of <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> BCRC 14759 and <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> BCRC 14615

In the present study, the production of exopolysaccharides (EPS) by 13 strains of Lactobacillus and 6 strains of Bifidobacterium in a chemical defined medium (CDM) supplemented with 30 g lactose/l was first compared. The highest EPS production of the Lactobacillus strains was found in L. salivarius BCRC 14759 while among the Bifidobacterium strains examined, B. bifidum BCRC 14615 showed the highest EPS production. Analyzes of the effect of lactose concentration and cultivation temperature on EPS production revealed that L. salivarius produced the highest amount of EPS (45.3 mg/l) in CDM supplemented with 5 g lactose/l at 40°C while B. bifidum produced the highest EPS (17.0 mg/l) in CDM supplemented with 40 g lactose/l at 35°C. α-Phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose-4-epimerase exhibited a markedly notable activity compared with other enzymes examined in the cell extract of both test organisms. This indicates their possible involvement in the biosynthesis of EPS.     

Expression of theJE/MCP-1 gene suppresses metastatic potential in murine colon carcinoma cells

The purpose of this study was to determine whether the expression of theJE/MCP-1 gene encoding for the monocyte chemottractant protein, MCP-1 (also known as monocyte chemotactic and activating factor MCAF, TDCF, and SMC-CF) can influence the metastatic properties of tumor cells. The highly metastatic murine colon carcinoma CT-26 cells, syngeneic to BALB/c mice that do not produce endogenous JE/MCP-1 protein, were transfected with a BCMGS-Neo expression vector (control) or a vector containing full-lengthJE cDNA. CT-26 parental cells, CT-26 Neo, and CT-26 JE/MCP-1-positive cells were injected into syngeneic or nude mice. The CT-26 JE/MCP-1-positive cells produced significantly fewer lung metastases. The decrease in incidence of metastasis was not due to the inability of the transfected cells to arrest in the lung vasculature or to differences in cell cycle time. CT-26 cells producing JE/MCP-1 were highly susceptible to lysis by syngeneic macrophages treated with subthreshold concentrations of lipopolysaccharide. In addition, culture supernatants of JE/MCP-1-expressing cells plus lipopolysaccharide synergistically activated tumoricidal properties in syngeneic macrophages. This activity was blocked by anti-JE/MCP-1 antibodies, indicating the involvement of the JE/MCP-1 molecule in this process. Moreover, purified JE/MCP-1 added to lipopolysaccharide-containing medium resulted in significant activation of macrophages against parental CT-26 cells. These data suggest that, in addition to its chemotactic properties, JE/MCP-1 can synergize with bacterial endotoxins to activate macrophages to become tumoricidal and, hence, could suppress metastasis.     

Successful adoptive immunotherapy with OK432-inducible activated natural killer cells in tumor-bearing mice

We had demonstrated that the NK cell mediated cytotoxicity of murine spleen cells could be augmented byin vivo priming and subsequentin vitro challenge with a streptococcal preparation OK432, and the cell surface phenotype of induced killer cells was Thy-1⁺, asialo GM1⁺, suggesting that the activated cells were of NK lineage (OK-NK cell). We had also clarified that IL-2 played a major role in inducing the OK-NK cells via the production of IFN-. In this study, we examined the effect of adoptive transfer of OK-NK cells on syngeneic tumors in mice. Mice were implanted with SP2 myeloma cells intraperitoneally (i.p.), or C26 colon adenocarcinoma cells subcutaneously to make the models of peritonitis carcinomatosa or solid tumor, and the OK-NK cells were transferred i.p. or intratumorally, adoptively. By the adoptive transfer of OK-NK cells, 92% of mice bearing SP2-tumor had be cured. The tumor growth of C26-solid tumor was inhibited, and the survival rate of mice bearing C26-tumor was significantly increased. The intratumoral remnants of¹²⁵I-labelled OK-NK cells were 61, 27 and 8% at 4, 12 and 36h after intratumoral transfer, respectively. By multiple transfer of OK-NK cells, the antitumor effect was more effectively augmented than that of a single transfer. Results in this study suggested that OK-NK cells could be useful for the therapy of cancer patients.     

Antimetastatic effect of Lactobacillus casei YIT9018 (LC 9018) on a highly metastatic variant of B16 melanoma in C57BL/6J mice

Summary The effect of Lactobacillus casei YIT9018 (LC 9018) on a highly metastatic variant of B16 melanoma, B16-BL6, was determined in C57BL/6 mice. Intralesional (i.l.) injection of LC 9018 inhibited tumor growth and prolonged the survival after s.c. inoculation of B16-BL6 into C57BL/6 mice. Injection of LC 9018 i.v. protected the mice against pulmonary metastasis after i.v. inoculation of B16-BL6. Injection of LC 9018 i.l. before surgical excision of the primary tumor inhibited axillary lymph node metastasis and i.v. injection of LC 9018 after surgical excision of the primary tumor inhibited both axillary lymph node and lung metastases. On the other hand, the combination of i.l. and i.v. injections of LC 9018 markedly inhibited both lymph node and lung metastases. Natural killer cell activity of axillary lymph node cells was augmented by the injection of LC 9018 into a front footpad, while the cytolytic activity of axillary lymph node cells was significantly enhanced. However, the cytolytic activity was diminished by depleting whole lymph node cells of the plastic adherent cells. Furthermore, alveolar macrophage-mediated cytotoxic activity was augmented by the i.v. injection of LC 9018.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript> production within tumor microenvironment inversely correlated with infiltration of CD56<Superscript>dim</Superscript> NK cells in gastric and esophageal cancer: possible mechanisms of NK cell dysfunction

Human NK cells can be divided into two subsets, CD56^dimCD16(+)NK and CD56^brightCD16(−)NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed the relationship between CD56^dim/CD56^bright NK cells and H₂O₂ in tumor-infiltrating NK cells in patients with gastric (n = 50) and esophageal (n = 35) cancer. The ratio of CD56^dim NK cells infiltrating tumors gradually decreased according to disease progression. H₂O₂ was abundantly produced within tumor microenvironments, and there was an inverse correlation between CD56^dim NK cell infiltration and H₂O₂ production. CD56^dim NK cells are more sensitive to apoptosis induced by physiological levels of H₂O₂ than CD56^bright NK cells. Furthermore, the exposure of NK cells to H₂O₂ resulted in the impairment of ADCC activity. In conclusion, H₂O₂ produced within tumor microenvironments inversely correlated with the infiltration of CD56^dim NK cells, possibly due to their preferentially induced cell death. These observations may explain one of the mechanisms behind NK cell dysfunction frequently observed in tumor microenvironments.     

Role of histamine in natural killer cell-mediated resistance against tumor cells

The formation of lung metastases by i.v.-injected B16 melanoma (F1 and F10 strain) cells in Swiss albino, C57BL/6, and BALB/c mice was reduced by a single dose of histamine given 24 h before tumor cell inoculation. The antimetastatic effect of histamine was specifically mediated by histamine H2-receptors (H2R): it was blocked by the H2R antagonist ranitidine and mimicked by dimaprit, a specific H2R agonist but not by an H2R-inactive structural analog of this compound, nor-dimaprit, or the H1R agonist 2-thiazolyl-ethylamide. A single dose of any of the H2R antagonists ranitidine, tiotidine, famotidine, or cimetidine drastically augmented metastasis. Effects of H2R-interactive compounds on B16 metastasis required intact NK cells, as judged by the inability of histamine or ranitidine to affect B16 metastasis after NK cell depletion in vivo using antibodies to asialo-GM1. NK-cell-mediated lysis of YAC-1 lymphoma cells in vivo was enhanced by histamine and reduced by ranitidine within 4 h after inoculation of tumor cells. The antimetastatic effect of IL-2 was potentiated by histamine; in some experiments, combined treatment with a low dose of IL-2 (6000 U/kg) and histamine completely eliminated metastasis, whereas concomitant treatment with ranitidine abrogated antimetastatic effects of IL-2; animals treated with ranitidine and IL-2 displayed the same level of enhanced metastasis as those treated with ranitidine alone. The presented data are suggestive of an earlier unrecognized role for histamine in NK cell-mediated resistance against metastatic tumor cells.     

CD26 expression determines lung metastasis in mutant F344 rats: involvement of NK cell function and soluble CD26

The association between CD26 expression, tumor cell adhesion, metastasis, and natural killer (NK) cell function was investigated in a CD26 mutant Fischer 344 (F344/DuCrj) substrain from Japanese breeders (F344JAP) in comparison with wild-type F344 substrains from US (F344/Crl) and Hannover (HAN; F344/Ztm) breeders. F344JAP rats lack the dipeptidyl peptidase IV activity of CD26 and show a reduced cell surface expression of the mutated CD26 glycoprotein. In vivo adhesion of vital dye-labeled MADB106 tumor cells, tumor colonization, CD26 enzymatic activity, and CD26 immunoreactivity in lungs and soluble CD26-like protein expression in serum were markedly reduced in F344JAP rats. These findings demonstrate that CD26 protein expression exerts a key role in lung metastasis. In addition, NK cell cytotoxicity against MADB106 cells was diminished in the mutant F344 substrain, suggesting that CD26 enzymatic activity sustains NK cytotoxicity. Interestingly, tumor cells lacked CD26 immunoreactivity in vitro, but displayed CD26 immunoreactivity in situ after in vivo inoculation as well as after incubation with rat serum, indicating that soluble CD26-like protein assembles in tumor cells during in vivo passage, which may interact with the process of tumor adhesion and metastasis. Overall, these findings indicate that altered expression and function of a single enzyme-the CD26 protein--can drastically change the outcome of metastatic disease.     

Effects of paclitaxel-producing fungal endophytes on growth and paclitaxel formation of <Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells

Effects of a fungal endophyte, Fusarium mairei, on growth and paclitaxel formation of Taxus cuspidata cells were investigated by adding fungal endophyte culture supernatant (FECS) to suspension cultures of T. cuspidata cells. The main effective chemical responsible for paclitaxel formation in FECS was an exopolysaccharide (EPS) of molecular weight ~2 kDa. FECS fractions except EPS stimulated growth of Taxus cells but had no effects on paclitaxel accumulation. Additionally, elicitation efficiency of FECS based on different culture conditions was studied. EPS content in FECS was related to FECS culture conditions. FECS with long cultivation and high-aeration cultivation contained higher EPS content and resulted in higher paclitaxel yield than that with short cultivation and low-aeration cultivation. The maximum yield of paclitaxel from Taxus cultures, elicited by FECS with 9-day cultivation, was 4.7-fold that of the control cultures.     

Chemoattractant Signaling between Tumor Cells and Macrophages Regulates Cancer Cell Migration,Metastasis and Neovascularization

Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1α and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.     

Using size‐controlled multicellular spheroids of murine adenocarcinoma cells to efficiently establish pulmonary tumors in mice

Previous studies demonstrated that multicellular spheroids developed using polydimethylsiloxane‐based microwells exhibited superior functions, such as insulin secretion from pancreatic cells, over suspended cells. To successfully apply these spheroids, the effect of spheroid size on cellular functions must be determined. In this study, using murine adenocarcinoma colon26 cells, the authors examined whether such spheroids were useful for developing tumor‐bearing animal models, which requires the efficient and stable engraftment of cancer cells at implanted sites and/or metastatic sites. The authors prepared microwells with widths of 360, 450, 560, and 770 μm through a micromolding technique, and obtained colon26 spheroids with average diameters of 169, 240, 272, and 341 μm, respectively. Small and medium spheroids were subsequently used. mRNA levels of integrin β1, CD44, and fibronectin, molecules involved in cell adhesion, increased with increasing colon26 spheroid size. Approximately 1.5 × 10⁴ colon26 cells in suspension or in spheroids were intravenously inoculated into BALB/c mice. At 21 days after inoculation, the lung weight of both colon26 spheroid groups, especially the group injected with small spheroids, was significantly higher than that of mice in the suspended colon26 cell group. These results indicate that controlling cancer cell spheroid size is crucial for tumor development in tumor‐bearing mouse models.     

Nutrient regulation of bacterial growth and production of anti-tumor metabolites in <Emphasis Type="Italic">Stigmatella</Emphasis> WXNXJ-B fermentation

The metabolites produced by Stigmatella WXNXJ-B inhibited the growth of tumor cells. The aims of this research were to evaluate the inhibition potency to different tumor cell lines and to study the effects of ammonium, phosphate and iron salts on bacterial growth and production of bioactive metabolites in Stigmatella WXNXJ-B fermentation. The results showed that the chloroform extract (CE-ME) showed the strongest growth inhibition bioactivity on mouse melanoma cell line (B16), murine colon carcinoma cell line (CT-26), human liver carcinoma cell line (HepG2) and human breast cancer cell line (MDA-MB231) in vitro and the IC₅₀ values were 9.94, 7.33, 11.34 and 11.66 μg ml⁻¹ respectively. The IC₅₀ value was above 700 μg ml⁻¹ on normal mouse spleen cells. Morphology happened changes in B16 cells treated with CE-ME. The anti-tumor metabolites were mainly produced during the stationary phase of the bacterial growth. Cell growth was stimulated at the phosphate concentration below 5 mM, but it was inhibited partly with 10 mM phosphate. The production of bioactive substances was inhibited by the phosphate. Ammonium increased the cell growth by 250% at 5 mM addition. The inhibition rate to B16 cells was increased to 89% at the concentration of 40 mM ammonium. The bacteria showed the best growth with 4 mM iron. Iron had little effect on the production at 2 mM, but bigger inhibition effect at higher iron concentration.     

Inhibition of colon cancer growth and metastasis by NK4 gene repetitive delivery in mice

NK4, originally prepared as a competitive antagonist for hepatocyte growth factor (HGF), is a bifunctional molecule that acts as an HGF-antagonist and angiogenesis inhibitor. When the expression plasmid for NK4 gene was administered into mice by hydrodynamics-based delivery, the repetitive increase in the plasma NK4 protein level was achieved by repetitive administration of NK4 gene. Mice were subcutaneously implanted with colon cancer cells and weekly given with the NK4 plasmid. The repetitive delivery and expression of NK4 gene inhibited angiogenesis and invasiveness of colon cancer cells in subcutaneous tumor tissue and this was associated with suppression of primary tumor growth. By fifty days after tumor implantation, cancer cells naturally metastasized to the liver, whereas NK4 gene expression potently inhibited liver metastasis. Inhibition of the HGF-Met receptor pathway and tumor angiogenesis by NK4 gene expression has potential therapeutic value toward inhibition of invasion, growth, and metastasis of colon cancer.     

Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

Yes‐associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030‐BrM3(K‐ras^G12C mutation) and PC9‐BrM3 (EGFR^Δexon19 mutation) had a significantly decreased p‐YAP(S127)/YAP ratio compared to parental H2030 (K‐ras^G12C mutation) and PC9 (EGFR^Δexon19 mutation) cells (P < .05). H2030‐BrM3 cells had significantly increased YAP mRNA and expression of Hippo downstream genes CTGF and CYR61 compared to parental H2030 cells (P < .05). Inhibition of YAP by short hairpin RNA (shRNA) and small interfering RNA (siRNA) significantly decreased mRNA expression in downstream genes CTGF and CYR61 in H2030‐BrM3 cells (P < .05). In addition, inhibiting YAP by YAP shRNA significantly decreased migration and invasion abilities of H2030‐BrM3 cells (P < .05). We are first to show that mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells had significantly decreased metastatic tumour burden and survived longer than control mice (P < .05). Collectively, our results suggest that YAP plays an important role in promoting lung adenocarcinoma brain metastasis and that direct inhibition of YAP by shRNA suppresses H2030‐BrM3 cell brain metastasis in a murine model.