Comparison of genetic susceptibility to lung adenocarcinoma and squamous cell carcinoma in Japanese patients using a novel panel for cancer-related drug-metabolizing enzyme genes.

The differences in genetic susceptibility to lung adenocarcinoma and squamous cell carcinoma remain unclear. We developed a customized, targeted gene sequencing panel for efficient and sensitive identification of germline variants, including whole-gene deletion types for cancer-related drug-metabolizing enzyme genes in lung adenocarcinoma and squamous cell carcinoma. The minor allele frequencies of the variants, confirmed as clinically significant in the Japanese population, did not differ significantly from those of normal participants listed in the public database. Genotype analysis comparing lung adenocarcinoma (n = 559) and squamous cell carcinoma (n = 151) indicated that the variants of DPYD (rs190771411, Fisher's exact test, P = 0.045; rs200562975, P = 0.045) and ALDH2 (rs568781254, P = 0.032) were associated with an increased risk of squamous cell carcinoma compared to adenocarcinoma. Conversely, whole-gene deletion of CYP2A6 was associated with adenocarcinoma but not squamous cell carcinoma. Notably, whole-gene deletion of CYP2A6 was confirmed in 22 patients with lung adenocarcinoma but not in any patients with squamous cell carcinoma. Most patients with whole-gene deletion of CYP2A6 were female non-smokers. The discovery of a whole-gene deletion of CYP2A6 in patients with lung adenocarcinoma may have an important role in clinical practice and advance our understanding of CYP2A6 germline variants and their association with carcinogenesis or their susceptibility to lung adenocarcinoma.

Activation of NLRP3 Inflammasome Complexes by Beta-Tricalcium Phosphate Particles and Stimulation of Immune Cell Migration in vivo.

Beta-tricalcium phosphate (ß-TCP) serves as a bone substitute in clinical practice because it is resorbable, biocompatible, osteointegrative, and osteoconductive. Particles of ß-TCP are also inflammatory mediators although the mechanism of this function has not been fully elucidated. Regardless, the ability of ß-TCP to stimulate the immune system might be useful for immunomodulation. The present study aimed to determine the effects of ß-TCP particles on NLR family pyrin domain containing 3 (NLRP3) inflammasome complexes. We found that ß-TCP activates NLRP3 inflammasomes, and increases interleukin (IL)-1ß production in primary cultured mouse dendritic cells (DCs) and macrophages, and human THP-1 cells in caspase-1 dependent manner. In THP-1 cells, ß-TCP increased also IL-18 production, and NLRP3 inflammasome activation by ß-TCP depended on phagocytosis, potassium efflux, and reactive oxygen species (ROS) generation. We also investigated the effects of ß-TCP in wild-type and NLRP3-deficient mice in vivo. Immune cell migration around subcutaneously injected ß-TCP particles was reduced in NLRP3-deficient mice. These findings suggest that the effects of ß-TCP particles in vivo are at least partly mediated by NLRP3 inflammasome complexes.

BMP4 and PHLDA1 are plausible drug-targetable candidate genes for KRAS G12A-, G12D-, and G12V-driven colorectal cancer.

Despite the frequent detection of KRAS driver mutations in patients with colorectal cancer (CRC), no effective treatments that target mutant KRAS proteins have been introduced into clinical practice. In this study, we identified potential effector molecules, based on differences in gene expression between CRC patients carrying wild-type KRAS (n = 390) and those carrying KRAS mutations in codon 12 (n = 240). CRC patients with wild-type KRAS harboring mutations in HRAS, NRAS, PIK3CA, PIK3CD, PIK3CG, RALGDS, BRAF, or ARAF were excluded from the analysis. At least 11 promising candidate molecules showed greater than two-fold change between the KRAS G12 mutant and wild-type and had a Benjamini-Hochberg-adjusted P value of less than 1E-08, evidence of significantly differential expression between these two groups. Among these 11 genes examined in cell lines transfected with KRAS G12 mutants, BMP4, PHLDA1, and GJB5 showed significantly higher expression level in KRAS G12A, G12D, and G12V transfected cells than in the wild-type transfected cells. We expect that this study will lead to the development of novel treatments that target signaling molecules functioning with KRAS G12-driven CRC.

Metabolic Profiling of Human Gastric Cancer Cells Treated With Salazosulfapyridine.

PURPOSE: The adhesion molecule cluster of differentiation 44v9 interacts with and stabilizes the cystine/glutamate exchanger protein, which functions as a transporter of cystine. Stabilized cystine/glutamate exchanger increases extracellular cystine uptake and enhances glutathione production. Augmented levels of reduced glutathione mitigate reactive oxygen species and protect cancer cells from apoptosis. Salazosulfapyridine blocks cystine/glutamate exchanger activity and mitigates the supply of cystine to increase intracellular ROS production, thereby increasing cell susceptibility to apoptosis. This enhances the effect of anticancer drugs such as cisplatin. Currently, salazosulfapyridine is being developed as a promising anticancer agent. In the present study, we elucidated the molecular mechanism associated with salazosulfapyridine by investigating the salazosulfapyridine-induced changes in glutathione metabolism in cultured gastric cancer cell lines. METHODS: The effect of salazosulfapyridine treatment on glutathione metabolism was investigated in 4 gastric cancer (AGS, MKN1, MKN45, and MKN74) and 2 colorectal cancer (HCT15 and HCT116) cell lines using metabolomic analyses. In addition, the effect of inhibition of the reduced form of nicotinamide adenine dinucleotide phosphate by 2-deoxyglucose on glutathione metabolism was evaluated. RESULTS: Under hypoxia, enhanced glycolysis resulted in lactate accumulation with an associated reduction in nicotinamide adenine dinucleotide phosphate. Salazosulfapyridine treatment decreased the cysteine content and inhibited the formation of glutathione. Combined treatment with salazosulfapyridine and 2-deoxyglucose significantly inhibited cell proliferation. 2-Deoxyglucose, an inhibitor of glycolysis, depleted nicotinamide adenine dinucleotide phosphate required for the formation of glutathione. CONCLUSIONS: Our results indicate that in cancer cells having a predominant glycolytic pathway, metabolomic analyses under hypoxic conditions enable the profiling of global metabolism. In addition, inhibiting the supply of nicotinamide adenine dinucleotide phosphate by blocking glycolysis is a potential treatment strategy for cancer, in addition to cystine blockade by salazosulfapyridine.

A T-cell-engaging B7-H4/CD3-bispecific Fab-scFv Antibody Targets Human Breast Cancer.

PURPOSE: The B7 homolog 4 (B7-H4, VTCN1) is an immune checkpoint molecule that negatively regulates immune responses and is known to be overexpressed in many human cancers. Previously, we generated a mouse anti-human B7-H4 mAb that did not have a significant antitumor effect in vivo probably because of molecule instability. In this study, we designed a B7-H4/CD3-bispecific antibody (BsAb) and investigated its antitumor activity in vitro and in vivo using a humanized mouse model. EXPERIMENTAL DESIGN: cDNAs of the antibody-binding fragment (Fab)-single-chain variable fragment (scFv) and scFv-scFv of the anti-B7-H4/CD3 BsAb were synthesized, and the BsAb antibodies were produced in HEK293 cells. The antitumor activity against human breast cancer cells by human peripheral blood mononuclear cells (hPBMC) with BsAb was measured by lactate dehydrogenase release in vitro, and in vivo using hPBMC-transplanted MHC class I- and class II-deficient NOG mice. RESULTS: hPBMCs with anti-B7-H4/CD3 BsAbs successfully lysed the human breast cancer cell line MDA-MB-468 (EC50: 0.2 ng/mL) and other B7-H4+ cell lines in vitro. When BsAb was injected in a humanized mouse model, there was an immediate and strong antitumor activity against MDA-MB-468, HCC-1954, and HCC-1569 tumors and CD8+ and granzyme B+ CTL infiltration into the tumor, and there were no adverse effects after long-term observation. CD8+ T-cell depletion by an anti-CD8 antibody mostly reduced the antitumor effect of BsAb in vivo. CONCLUSIONS: An anti-B7-H4/CD3 BsAb may be a good therapeutic tool for patients with B7-H4+ breast cancers.

Antitumor Effect of Programmed Death-1 (PD-1) Blockade in Humanized the NOG-MHC Double Knockout Mouse.

PURPOSE: Humanized mouse models using NOD/Shi-scid-IL2rγnull (NOG) and NOD/LtSz-scid IL2rγnull (NSG) mouse are associated with several limitations, such as long incubation time for stem cell engraftment and the development of xenograft versus host disease in mice injected with peripheral blood mononuclear cells (PBMCs). To solve problems, we used humanized major histocompatibility class I- and class II-deficient NOG mice (referred to as NOG-dKO) to evaluate the antitumor effect of anti-programmed death-1 (PD-1) antibody. EXPERIMENTAL DESIGN: Humanized NOG-dKO mice, in which human PBMCs and human lymphoma cell line SCC-3, or glioblastoma cell line U87 were transplanted, were used as an immunotherapy model to investigate the effect of anti-PD-1 antibody. A biosimilar anti-PD-1 mAb generated in our laboratory was administered to humanized NOG-dKO mice transplanted with tumors. RESULTS: Within 4 weeks after transplantation, human CD45+ cells in antibody-treated mice constituted approximately 70% of spleen cells. The injection of anti-PD-1 antibody reduced by more 50% the size of SCC-3 and U87 tumors. In addition, induction of CTLs against SCC-3 cells and upregulation of natural killer cell activity was observed in the antibody-treated group. Tumor-infiltrating lymphocyte profiling showed that more exhausted marker (PD1+TIM3+LAG3+) positive T cells maintained in anti-PD-1 antibody-treated tumor. A greater number of CD8+ and granzyme-producing T cells infiltrated the tumor in mice treated with the anti-PD-1 antibody. CONCLUSIONS: These results suggest that NOG-dKO mice might serve as a good humanized immunotherapy model to evaluate the efficacy of anti-PD-1 antibody prior to the clinical treatment. Clin Cancer Res; 23(1); 149-58. ©2016 AACR.

Effects of beta-tricalcium phosphate particles on primary cultured murine dendritic cells and macrophages.

Beta-tricalcium phosphate (ß-TCP) is widely used for bone substitution in clinical practice. Particles of calcium phosphate ceramics including ß-TCP act as an inflammation mediators, which is an unfavorable characteristic for a bone substituent or a prosthetic coating material. It is thought that the stimulatory effect of ß-TCP on the immune system could be utilized as an immunomodulator. Here, in vitro effects of ß-TCP on primary cultured murine dendritic cells (DCs) and macrophages were investigated. ß-TCP particles enhanced expression of costimulatory surface molecules, including CD86, CD80, and CD40 in DCs, CD86 in macrophages, and MHC class II and class I molecules in DCs. DEC205 and CCR7 were up-regulated in ß-TCP-treated DCs. Production of cytokines and chemokines, including CCL2, CCL3, CXCL2, and M-CSF, significantly increased in DCs; CCL2, CCL3, CCL4, CCL5, CXCL2, and IL-11ra were up-regulated in macrophages. The results of the functional assays revealed that ß-TCP caused a prominent reduction in antigen uptake by DCs, and that conditioned medium from DCs treated with ß-TCP facilitated the migration of splenocytes in the transwell migration assay. Thus, ß-TCP induced phenotypical and functional maturation/activation of DCs and macrophages; these stimulating effects may contribute to the observed in vivo effect where ß-TCP induced extensive migration of immune cells. When compared to lipopolysaccharide (LPS), an authentic TLR ligand, the stimulatory effect of ß-TCP on the immune systems is mild to moderate; however, it may have some advantages as a novel immunomodulator. This is the first report on the direct in vitro effects of ß-TCP against bone marrow-derived DCs and macrophages.

Prevalence of low-penetrant germline TP53 D49H mutation in Japanese cancer patients.

Using whole exome sequencing data obtained from 1,685 Japanese cancer patients, we examined genetic variations of germline TP53 and found 10 types of non-synonymous single nucleotide variants. In the present study, we focused on 6 patients with germline D49H mutation located in the transactivation domain 2 of p53 protein, since the mutation seemed to be prevalent in cancer patients and to be pathogenic. According to the initial survey for family history of the proband with the germline TP53 D49H mutation, one osteosarcoma patient and his pedigree fulfill the criteria for Li-Fraumeni-like syndrome and the 2009 Chompret criteria for germline TP53 mutation screening. Since this patient possesses double germline mutations of TP53 D49H and A159D, further studies are required to evaluate contribution of the D49H mutation in this morbidity. The remaining 5 patients had family histories of cancer, but none fulfills the criteria either for the Li-Fraumeni/Li-Fraumeni-like syndromes or the 2009 Chompret criteria for germline TP53 mutation screening. It is possible to postulate that the germline TP53 D49H mutation is likely to be low-penetrant in some pedigrees. The present study also indicates that the survey for the germline TP53 mutation plays an important role in clinical practice as it will prevent mistaking cancer patients with unusual heredities for sporadic cases.

Characterization of beta-tricalcium phosphate as a novel immunomodulator.

Calcium phosphate (CaP) ceramics including hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP) have been widely used for bone substitution in orthopedic, maxillofacial and dental surgery, as well as in tumor resections. CaP particles are also known to cause inflammatory responses, which are thought to be an unfavorable characteristic of prosthetic coating materials. On the other hand, the immunostimulatory effect of ß-TCP induces an anti-tumor effect in xenograft tumor models in athymic mice. To date, in depth analysis of the biological effects of ß-TCP has not been studied in mice. In the present study, in vivo biological effects of ß-TCP were investigated by subcutaneously injecting ß-TCP particles into mice. This induced extensive migration of immune cells to the area surrounding the injection. In addition, we found that in vitro treatment with ß-TCP in murine monocyte/macrophage cells (J774A.1) induced up-regulation of surface expression of CD86, and increased production of TNF-α, MIP-1α, and sICAM-1. Furthermore, conditioned medium from J774A.1 cells treated with ß-TCP facilitated migration of murine splenocytes in a transwell migration assay. These findings clarify that ß-TCP induces an immunostimulatory effect in mice, and suggest a potential for ß-TCP as a novel adjuvant for cancer therapy.

Flt3 ligand enhances anti-tumor effects of antibody therapeutics.

Fms-like tyrosine kinase 3 ligand ([Flt3 ligand], FL) stimulates proliferation and development of a wide range of hematopoietic cells including hematopoietic stem cells and myeloid and lymphoid progenitor cells. FL also has been shown to have anti-tumor effects in a variety of in vivo tumor models. In this study, the effect of FL against tumor growth was investigated in the COLO-205 human colon tumor xenograft model. FL was delivered in vivo by the "hydrodynamics-based gene delivery of naked DNA" method. In this experimental setting, FL and/or the therapeutic antibody anti-carcinoembryonic antigen (CEA) monoclonal antibody was administered. FL alone or anti-CEA antibody alone induced significant growth inhibition; furthermore, FL plus antibody treatment produced synergistic anti-tumor effects. This study is the first demonstration of a synergistic anti-tumor effect between FL and antibody therapeutics.

The effect of 5-aminolevulinic acid on cytochrome c oxidase activity in mouse liver.

BACKGROUND: 5-Aminolevulinic acid (ALA) is a precursor of heme that is fundamentally important in aerobic energy metabolism. Among the enzymes involved in aerobic energy metabolism, cytochrome c oxidase (COX) is crucial. In this study, the effect of ALA on cytochrome c oxidase activity was measured. FINDINGS: c57BL/6N species of mice were administered ALA orally for 15 weeks. After ALA administration, mice were sacrificed and livers were obtained. COX activity in mitochondria from ALA-administered mouse livers was 1.5-fold higher than that in mitochondria from PBS-administered mouse livers (P < 0.05). Furthermore, ATP levels in ALA-administered mouse livers were much higher than those in PBS-administered mouse livers. These data suggest that oral administration of ALA promotes aerobic energy metabolism, especially COX activity. CONCLUSIONS: This is the first report of a drug that functions in aerobic energy metabolism directly. Since COX activity is decreased in various diseases and aging, the pharmacological effects of ALA will be expanding.

Innate immunity and cancer therapy.

Classical cancer immunotherapy utilizes the immune response against microbial components, and a sequence of immune responses produce antitumor effects. The identification of mammalian Toll-like receptors (TLRs), receptors for microbial components, has shed light on antigen recognition by the innate immune system and provided a molecular basis for our understanding of the relationship between innate immunity and antitumor activity. However, accumulating evidence has revealed another important role of TLRs in maintaining tissue homeostasis and has also shown that tumor cells utilize this function to create favorable conditions for growth and survival, suggesting that TLR signaling acts as a double-edged sword in cancer therapy. In this review, innate immunity-based cancer therapy will be discussed with special reference to TLR-targeting drugs.

Characterization of a MAGE-1-derived HLA-A24 epitope-specific CTL line from a Japanese metastatic melanoma patient.

A MAGE-1 HLA-A24 peptide-specific CTL line was characterized using a novel staining approach in the case of a metastatic melanoma patient who exhibited a remarkable clinical response in HLA-A24 peptide cocktail-pulsed dendritic cell (DCs) vaccine therapy. Briefly, pre- or post-vaccine peripheral blood mononuclear cells (PBMCs) from the vaccinated patient were stimulated several times with MAGE-1 A24 peptide-pulsed DCs and T2-A24 cells in vitro. Expanded MAGE-1 A24-specific CTL line was investigated in terms of immunological functions. The proportion of MAGE-1 A24 tetramer+ CTLs increased from 0.04% to 18.6%, and the absolute numbers of MAGE-1 tetramer+ CTLs increased up to 5,068-fold after stimulations. Expanded CTL line exhibited a strong cytotoxic activity against MAGE-1+ cancer cell line in the restriction of HLA. Finally, successful identification of MAGE-1 A24 peptide-specific T-cell receptor (TCR) cDNA from anti-TCR MoAbsorted CTL was obtained for the first time and the specific cytotoxicity in TCR gene-transduced naive T-cells was confirmed.

Ligation of tumour-produced mucins to CD22 dramatically impairs splenic marginal zone B-cells.

CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to alpha2,6- sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state.

Novel approach to the characterization of melanoma associated-peptide-specific CTL lines from Japanese metastatic melanoma patients.

Melanoma-associated antigens, MART-1, tyrosinase, gp100 and MAGEs, are typical melanoma-specific tumor antigens which can potently induce immune responses in metastatic melanoma patients treated with peptide vaccines. In the present study, we established a dendritic cell (DC)-based HLA-A2 melanoma-associated peptide (MART-1 or gp100)-specific CTL induction method and characterized the CTLs using HLA-A2 tetramer staining in 6 cases of HLA-A2+ melanoma treated with DC vaccines. Peripheral blood mononuclear cells (PBMC) from patients were stimulated twice with MART-1 A2 peptide-pulsed DCs in the presence of a low dose of IL-2. To boost CTL populations, CTL lines were further stimulated twice with MART-1 A2 peptide-pulsed T2 cells. The frequency of MART-1 A2 tetramer-positive CTLs increased from 0.16% (prior to stimulation) to 2.15% (after DC stimulation), and reached 46.5% on average (after additional T2 stimulation) in 4 cases which showed a successful expansion. The absolute numbers of MART-1 A2 tetramer-positive CTLs increased from 187- to 619-fold (average, 415-fold) compared to prior to DC stimulation. CTL assays using MART-1-specific CTL lines demonstrated potent killing activity against MART-1 peptide-pulsed T2 cells or HLA-A2+ melanoma cell lines in accordance with the frequency of tetramer-positive CTLs. Finally, we were successful in identifying melanoma peptide-specific T-cell receptor (TCR) cDNAs in 2 cases for MART-1 and 1 case for gp100 using the anti-TCR MoAb-based sorting as a novel approach instead of a conventional cell cloning, and confirmed peptide-specific IFN-gamma production in TCR cDNA-transduced naïve T cells. The results showed that cloned TCR cDNAs were efficient in reconstituting tumor-specific cytotoxicity and good candidates for novel immunotherapy.

Changes in the expression of CD106, osteogenic genes, and transcription factors involved in the osteogenic differentiation of human bone marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are well known to possess multipotential differentiation and are becoming a good tool for clinical research. However, specific markers for their purification and the mechanism of their osteogenic differentiation remain to be elucidated. In the present study, we compared the expression of CD106, and osteogenic differentiation-related proteins and genes in human bone marrow (BM)-derived MSCs, before and after differentiation by FACS, histochemical staining, immunohistochemical staining, RT-PCR, and real-time PCR. It was found that MSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105, and CD166, but negative for CD14, CD31, CD34, CD62E, CD45, and GlyA. Notably, CD106 was detected before osteogenic induction, but its expression was downregulated 10 fold after 2 weeks of osteogenic differentiation as determined by flow cytometry. The results of RT-PCR and real-time PCR revealed that the expression of CD106 mRNA in MSCs significantly decreased by 7.1-, 4.2-, and 5.1-fold, respectively after osteogenic, chondrogenic, and adipogenic differentiation. In contrast, other MSC-positive markers described above did not change significantly even after differentiation. Compared to levels in control cells, after 2 weeks of osteogenic differentiation, mRNA levels of alkaline phosphatase, bone sialoprotein, osteocalcin, and transcript factors RUNX2 and Osterix showed more than 2-fold, 5-fold, 1.5-fold, 2-fold, and 5-fold increase, respectively. Thus, we speculate that CD106 might be a useful surface marker for BMMSCs. Moreover, alkaline phosphatase, type I collagen, osteonectin, osteopontin, and biglycin were expressed in the early stages of osteogenic differentiation before bone sialoprotein and osteocalcin. The present study should help to provide a novel marker for isolating purified MSCs and characterizing osteogenic differentiation.

Proteomic analysis of a highly metastatic gastric cancer cell line using two-dimensional differential gel electrophoresis.

Stomach cancer is still a major cause of death in Asian people despite a complete cure after the resection of early cancers, mainly because peritoneal dissemination is difficult to treat. In the present study, we used two-dimensional differential gel electrophoresis (2-D DIGE) to identify specific proteins differentially expressed between a highly metastatic stomach cancer cell line MKN-45-P and its parental cell line MKN-45. We detected 27 protein spots in at least 2 of 3 experiments which showed statistically significant differences in abundance. All 27 protein spots were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and database-searching software. A proteomic analysis revealed 13 different proteins with some isoforms sharing different biochemical characteristics, and that 8 proteins were up-regulated, and 5 were down-regulated. The 13 proteins were mainly involved in protein synthesis (transfer RNA synthetase), metabolism (flavoprotein subunit, pyruvate kinase, adenylate kinase), receptor and signal transduction (annexins I and A2), the cytoskeleton (keratin 5, cytokeratin 8) and cell cycling (ts11). These results suggested that a proteomic approach including 2-D DIGE would be an efficient way to identify the proteins responsible for specific biological functions. Moreover, these observations might be novel findings leading to the prediction of postoperative peritoneal recurrence.

Clinical response in Japanese metastatic melanoma patients treated with peptide cocktail-pulsed dendritic cells.

BACKGROUND: Metastatic, chemotherapy-resistant melanoma is an intractable cancer with a very poor prognosis. As to immunotherapy targeting metastatic melanoma, HLA-A2+ patients were mainly enrolled in the study in Western countries. However, HLA-A24+ melanoma patients-oriented immunotherapy has not been fully investigated. In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on metastatic melanoma patients with HLA-A2 or A24 genotype. METHODS: Nine cases of metastatic melanoma were enrolled into a phase I study of monocyte-derived dendritic cell (DC)-based immunotherapy. HLA-genotype analysis revealed 4 cases of HLA-A*0201, 1 of A*0206 and 4 of A*2402. Enriched monocytes were obtained using OptiPreptrade mark from leukapheresis products, and then incubated with GM-CSF and IL-4 in a closed serum-free system. After pulsing with a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-2, MAGE-3 and MART-1 or MAGE-1) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Finally, thawed DCs were washed and injected subcutaneously (s.c.) into the inguinal region in a dose-escalation manner. RESULTS: The mean percentage of DCs rated as lin-HLA-DR+ in melanoma patients was 46.4 +/- 15.6 %. Most of DCs expressed high level of co-stimulatory molecules and type1 phenotype (CD11c+HLA-DR+), while a moderate number of mature DCs with CD83 and CCR7 positive were contained in DC products. DC injections were well tolerated except for transient liver dysfunction (elevation of transaminases, Grade I-II). All 6 evaluable cases except for early PD showed positive immunological responses to more than 2 melanoma peptides in an ELISPOT assay. Two representative responders demonstrated strong HLA-class I protein expression in the tumor and very high scores of ELISPOT that might correlate to the regression of metastatic tumors. Clinical response through DC injections was as follows : 1CR, 1 PR, 1SD and 6 PD. All 59 DC injections in the phase I study were tolerable in terms of safety, however, the maximal tolerable dose of DCs was not determined. CONCLUSIONS: These results suggested that peptide cocktail-treated DC-based immunotherapy had the potential for utilizing as one of therapeutic tools against metastatic melanoma in Japan.

Antitumor effect induced by dendritic cell (DC)-based immunotherapy against peritoneal dissemination of the hamster pancreatic cancer.

Establishing a method to control peritoneal dissemination is one of the most pressing issues in the postsurgical treatment of pancreatic cancer. In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on peritoneal disseminations of hamster pancreatic cancer cells, PGHAM-1. After the orthotopically inoculation of 2 x 10(6) PGHAM-1 cells, DC pulsed with PGHAM-1-derived tumor lysates, DC alone or PBS as a vehicle was injected intraperitoneally (i.p.) three times at weekly intervals. The group treated with DC or DC+lysate was found to have smaller disseminated tumors than the vehicle-treated. In addition, mean survival time in the DC+lysate groups was significantly longer than the PBS group. These findings suggested that DC-based immunotherapy might be efficient for the treatment of peritoneal disseminations of the pancreatic cancer.

NK cell activation by dendritic cells (DCs) requires the formation of a synapse leading to IL-12 polarization in DCs.

Mature dendritic cells (mDCs) can trigger the effector functions of natural killer (NK) cells. Knock-out, small-interfering RNA or neutralizing antibodies targeting interleukin 12 (IL-12) subunits revealed a critical role for IL-12 in NK cell interferon gamma (IFN-gamma) secretion promoted by mDCs. However, NK cell activation by DCs also required direct cell-to-cell contacts. DC-mediated NK cell activation involved the formation of stimulatory synapses between DCs and NK cells. The formation of DC/NK cell conjugates depended on cytoskeleton remodeling and lipid raft mobilization in DCs. Moreover, the disruption of the DC cytoskeleton using pharmacologic agents or the loss-of-function mutation of the Wiskott-Aldrich syndrome protein abolished the DC-mediated NK cell activation. Synapse formation promoted the polarized secretion of preassembled stores of IL-12 by DCs toward the NK cell. The synaptic delivery of IL-12 by DCs was required for IFN-gamma secretion by NK cells, as assessed using inhibitors of cytoskeleton rearrangements and transwell experiments. Therefore, the cross-talk between DCs and NK cells is dictated by functional synapses.