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Melanoma has been a prototype cancer to investigate tumor immunity and development of immunotherapy. The recent analyses on the patients treated with immune checkpoint inhibitors made a great progress of human tumor immunology. However, the response rate of PD-1/PD-L1 Ab therapy is about 30% so that the identification of biomarkers to select appropriate patients and immunotherapies as well as the improvement of efficacy possibly through combination treatment are required. Pretreatment immune status is different among patients, and correlated with prognosis and response to cancer therapies including immunotherapy. It may be defined by cancer cell’s genetic characteristics, patients’ immune reactivity, and environmental factors. In the T-cell-inflamed melanoma, adaptive immune resistance such as PD-1/PD-L1 interaction prevents melanoma attack by T cells specific for tumor antigens such as DNA mutation-derived neo-antigens. Blocking such resistant mechanisms may reinvigorate T cells to eradicate melanoma cells. Partially exhausted T cells expressing PD-1 and CTLA4 appear to be expanded following PD-1/PD-L1 blockade and may contribute to their antitumor effects. In T-cell non-inflamed melanoma, primary immune resistance including cancer cell-derived immunosuppressive factors, loss of immune-inducing factors from cancer cells, mesenchymal tumor microenvironment, and other genetic alternations prevents induction and tumor infiltration of antitumor T cells even in the presence of immunogenic tumor antigens. Depletion of such negative factors may convert nonresponders to responders, indicating that combination immunotherapy may augment the efficacy of current immunotherapies. Further understanding of immunobiology using multi-omics and systematic immune analyses is required to identify better biomarkers and therapeutic targets for the next-generation immunotherapy for melanoma.

We generated a novel mouse strain expressing transgenic human interleukin-15 (IL-15) using the severe immunodeficient NOD/Shi-scid-IL-2Rγ null (NOG) mouse genetic background (NOG-IL-15 Tg). Human natural killer (NK) cells, purified from the peripheral blood (hu-PB-NK) of normal healthy donors, proliferated when transferred into NOG-IL-15 Tg mice. In addition, the cell number increased, and the hu-PB-NK cells persisted for 3 months without signs of xenogeneic graft versus host diseases (xGVHD). These in vivo-expanded hu-PB-NK cells maintained the original expression patterns of various surface antigens, including NK receptors and killer cell immunoglobulin-like receptor (KIR) molecules. They also contained significant amounts of granzyme A and perforin. Inoculation of K562 leukemia cells into hu-PB-NK-transplanted NOG-IL-15 Tg mice resulted in significant suppression of tumor growth compared with non-transplanted mice. Furthermore, NOG-IL-15 Tg mice allowed for engraftment of in vitro-expanded NK cells prepared for clinical cell therapy. These cells exerted antibody-dependent cell-mediated cytotoxicity (ADCC) on Her2-positive gastric cancer cells in the presence of therapeutic anti-Her2 antibody, and subsequently suppressed tumor growth. Our results collectively suggest that the NOG-IL-15 Tg mice are a useful model for studying human NK biology and evaluating human NK cell-mediated in vivo cytotoxicity.

The AMPK-related kinase NUAK2 has been implicated in melanoma growth and survival outcomes, but its therapeutic utility has yet to be confirmed. In this study, we show how its genetic amplification in PTEN-deficient melanomas may rationalize the use of CDK2 inhibitors as a therapeutic strategy. Analysis of array-CGH data revealed that PTEN deficiency is coupled tightly with genomic amplification encompassing the NUAK2 locus, a finding strengthened by immunohistochemical evidence that phospho-Akt overexpression was correlated with NUAK2 expression in clinical specimens of acral melanoma. Functional studies in melanoma cells showed that inactivation of the PI3K pathway upregulated p21 expression and reduced the number of cells in S phase. NUAK2 silencing and inactivation of the PI3K pathway efficiently controlled CDK2 expression, whereas CDK2 inactivation specifically abrogated the growth of NUAK2-amplified and PTEN-deficient melanoma cells. Immunohistochemical analyses confirmed an association of CDK2 expression with NUAK2 amplification and p-Akt expression in melanomas. Finally, pharmacologic inhibition of CDK2 was sufficient to suppress the growth of NUAK2-amplified and PTEN-deficient melanoma cells in vitro and in vivo. Overall, our results show how CDK2 blockade may offer a promising therapy for genetically defined melanomas, where NUAK2 is amplified and PTEN is deleted. Cancer Res; 75(13); 2708–15. ©2015 AACR.

We generated a severe immunodeficient NOD/Shi-scid-IL-2Rγnull (NOG) mouse substrain expressing the transgenic human IL-2 gene (NOG–IL-2 Tg). Upon transfer of human cord blood–derived hematopoietic stem cells (HSCs), CD3−CD56highCD16+/− cells developed unexpectedly, predominantly in the NOG–IL-2 Tg (hu-HSC NOG–IL-2 Tg). These cells expressed various NK receptors, including NKp30, NKp44, NKp46, NKG2D, and CD94, as well as a diverse set of killer cell Ig-like receptor molecules at levels comparable to normal human NK cells from the peripheral blood, which is evidence of their maturity. They produced levels of granzyme A as high as in human peripheral blood–derived NK cells, and a considerable amount of perforin protein was detected in the plasma. Human NK cells in hu-HSC NOG–IL-2 Tg produced IFN-γ upon stimulation, and IL-2, IL-15, or IL-12 treatment augmented the in vitro cytotoxicity. Inoculation of K562 leukemia cells into hu-HSC NOG–IL-2 Tg caused complete rejection of the tumor cells, whereas inoculation into hu-HSC NOG fully reconstituted with human B, T, and some NK cells did not. Moreover, when a CCR4+ Hodgkin’s lymphoma cell line was inoculated s.c. into hu-HSC NOG–IL-2 Tg, the tumor growth was significantly suppressed by treatment with a therapeutic humanized anti-CCR4 Ab (mogamulizumab), suggesting that the human NK cells in the mice exerted active Ab-dependent cellular cytotoxicity in vivo. Taken together, these data suggest that the new NOG–IL-2 Tg strain is a unique model that can be used to investigate the biological and pathological functions of human NK cells in vivo.

This study aimed to evaluate the feasibility of and immune response to Wilms tumor gene 1 (WT1) peptide-pulsed dendritic cell vaccination combined with gemcitabine (DCGEM) as a first-line therapy among patients with advanced pancreatic cancer. Ten HLA-A*2402 patients were treated with WT1 peptide-pulsed DC vaccination (1 × 10(7) cells) on days 8 and 22 and gemcitabine (1000 mg/m(2) ) on days 1, 8 and 15. Induction of a WT1-specific immune response was evaluated using the delayed-type hypersensitivity (DTH) skin test, interferon-γ enzyme-linked immunospot and HLA tetramer assays, along with assays for various immunological factors. DCGEM was well-tolerated, and the relative dose intensity of gemcitabine was 87%. Disease control associated with a low neutrophil/lymphocyte ratio was observed in all three patients with DTH positivity; it was also correlated with a low percentage of granulocytic myeloid derived suppressor cells in the pretreatment peripheral blood (P = 0.017). Patients with liver metastases and high levels of inflammatory markers such as C-reactive protein and interleukin-8 (IL-8) showed poor survival even though a WT1-specific immune response was induced in them. WT1 peptide-pulsed DCGEM is feasible and effective for inducing anti-tumor T-cell responses. Our results support future investigations for pancreatic cancer patients with non-liver metastases and favorable immunological conditions. This trial was registered with the University hospital Medical Information Network (UMIN) Clinical Trials Registry (http://www.umin.ac.jp/ctr/ number: UMIN-000004855).

Purpose This study aimed to clarify the mechanisms and assess the characteristics of the chronic graft-versus-host disease (cGVHD) fibrosis in the lacrimal gland (LG) of mice. Methods Histopathology of LG tissues was examined by immunohistochemistry and electron microscopy. Cultured fibroblasts derived from the LG were analyzed by phase-contrast microscopy, immunocytochemistry, flow cytometry, proliferation assay, and invasion and migration assays. Results Cultured murine LG fibroblasts in cGVHD were spindle-shaped and relatively small, whereas those from syngeneic controls were polygon-shaped and relatively large. Flow cytometric analysis showed that the LG fibroblasts in cGVHD had elevated HSP47 levels. The LG fibroblasts in cGVHD also showed increased expression of major histocompatibility complex class II. Furthermore, the proportion of Sca-1PDGFR-α cells among the LG fibroblasts in cGVHD was considerably increased compared with controls. Cell counting kit-8 assays demonstrated that the LG fibroblasts in cGVHD were highly proliferative, and cell invasion assays indicated that they were highly invasive with high migration ability. Conclusions LG fibroblasts in cGVHD can be aberrantly activated, thereby eliciting fibrosis by producing excessive extracellular matrix, leading to LG dysfunction in mice.

Antibodies to programmed cell death-1 (PD-1), such as nivolumab, have shown promising clinical activity in patients with advanced non-small-cell lung cancer (NSCLC), but their efficacy appears to be less pronounced in patients with such tumors harboring epidermal growth factor receptor gene (EGFR) mutations. Recent findings suggest that patients with EGFR mutation-positive NSCLC who develop resistance to tyrosine kinase inhibitors (TKIs) due to mechanisms other than acquisition of the secondary T790M mutation of EGFR are more likely to benefit from nivolumab treatment, possibly as a result of a higher level of expression of the PD-1 ligand PD-L1, than are patients who are T790M-positive. The WJOG8515L study (UMIN ID: 000021133) is a randomized phase II trial to compare nivolumab with the combination of carboplatin and pemetrexed in patients with EGFR mutation-positive nonsquamous NSCLC who have developed resistance to EGFR-TKIs due to mechanisms other than T790M. Eligible patients are those with stage IV or recurrent EGFR mutation-positive NSCLC who experience disease progression after therapy with more than 1 EGFR-TKI, including gefitinib, erlotinib, or afatinib; they must show no evidence of the T790M mutation on analysis of a tumor biopsy specimen obtained after progression on such EGFR-TKI therapy, or, if T790M is detected, they must again experience progression on subsequent treatment with a third-generation EGFR-TKI. The primary endpoint is progression-free survival (PFS), and secondary end points include overall survival (OS), objective response rate, duration of response, safety, and OS and PFS according to PD-L1 expression level. Recruitment started in May 2016 and is ongoing.

Nogo receptor type 1 (NgR1) is known to inhibit neuronal regeneration in the CNS. Previously, we have shown that lateral olfactory tract usher substance (LOTUS) interacts with NgR1 and inhibits its function by blocking its ligand binding. Therefore, LOTUS is expected to have therapeutic potential for the promotion of neuronal regeneration. However, it remains unknown whether the soluble form of LOTUS (s-LOTUS) also has an inhibitory action on NgR1 function as a candidate for therapeutic agents. Here, we show that s-LOTUS inhibits NgR1-mediated signaling by inhibiting the molecular interaction between NgR1 and its coreceptor, p75 neurotrophin receptor (p75NTR). In contrast to the membrane-bound form of LOTUS, s-LOTUS did not block ligand binding to NgR1. However, we identified p75NTRas a novel LOTUS binding partner and found that s-LOTUS suppressed the interaction between p75NTRand NgR1. s-LOTUS inhibited myelin-associated inhibitor (MAI)-induced RhoA activation in murine cortical neurons. Functional analyses revealed that s-LOTUS inhibited MAI-induced growth cone collapse and neurite outgrowth inhibition in chick DRG neurons. In addition, whereas olfactory bulb neurons oflotus-KO mice are sensitive to MAI due to a lack of LOTUS expression, treatment with s-LOTUS inhibited MAI-induced growth cone collapse in these neurons. Finally, we observed that s-LOTUS promoted axonal regeneration in optic nerve crush injury of mice (either sex). These findings suggest that s-LOTUS inhibits NgR1-mediated signaling, possibly by interfering with the interaction between NgR1 and p75NTR. Therefore, s-LOTUS may have potential as a therapeutic agent for neuronal regeneration in the damaged CNS.SIGNIFICANCE STATEMENTNogo receptor type 1 (NgR1) is a receptor well known to inhibit neuronal regeneration in the CNS. Because the membrane-bound form of lateral olfactory tract usher substance (LOTUS) antagonizes NgR1 through acis-type molecular interaction between LOTUS and NgR1, the soluble form of LOTUS (s-LOTUS) is expected to be a therapeutic agent for neuronal regeneration. In our present study, we show that s-LOTUS inhibits the interaction between NgR1 and p75NTR, NgR1 ligand-induced RhoA activation, growth cone collapse, and neurite outgrowth inhibition and promotes axonal regeneration. Our results indicate that s-LOTUS inhibits NgR1-mediated signaling through atrans-type molecular interaction between LOTUS and NgR1 and, therefore, s-LOTUS may have therapeutic potential for neuronal regeneration.

In this study, we aimed to evaluate the feasibility and efficacy of peptide-pulsed dendritic cell (DC) vaccine in combination with carboplatin and paclitaxel chemotherapy (DCCP) for patients with stage IV melanoma previously treated with dacarbazine-containing regimen. Six HLA-A24 and 3 HLA-A02 patients were treated with carboplatin (area under the curve 5) and paclitaxel (175 mg/m) on day 1 and DCs (2×10 cells) pulsed with Wilms tumor gene 1 (WT1), gp100, tyrosinase, and either MAGE-A3 (for HLA-A24) or MAGE-A2 (for HLA-A02) peptides on days 8 and 22 in 28-day cycle for up to three cycles. DCCP was well tolerated, and median progression-free survival and median overall survival were 2.3 and 12.0 months, respectively. In four of nine patients, a WT1-specific immune response (WT1-IR) was detected using the interferon-γ enzyme-linked ImmunoSpot assay and WT1/HLA tetramer assay. DCCP was more likely to elicit a WT1-IR in patients who received DCs pulsed with the HLA-A24-restricted peptide (75%) compared with patients who received DCs pulsed with the HLA-A02-restricted peptide (0%, P=0.058). Furthermore, three (75%) of four patients with a WT1-IR survived longer than 12 months, whereas only one (20%) of five patients without a WT1-IR who received the BRAF inhibitor after DCCP survived longer than 12 months. These results suggest that DCCP may be beneficial for HLA-A24 melanoma patients with a WT1-IR.

Background The purpose of the present study was to explore novel biomarkers that can predict the clinical outcome of patients before treatment or during vaccination. These would be useful for the selection of appropriate patients who would be expected to exhibit better treatment outcomes from vaccination, and for facilitating the development of cancer vaccine treatments. Methods From a single-arm, non-randomized, human leukocyte antigen (HLA)-A-status-blind phase II trial of a vaccine treatment using three HLA-A*2402-restricted peptides for advanced pancreatic cancer (PC), we obtained peripheral blood samples from 36 patients of an HLA-A*2402-matched group and 27 patients of an HLA-A*2402-unmatched group. Results Multivariate analysis (HR = 2.546; 95% CI = 1.138 to 5.765; p = 0.0231) and log-rank test (p = 0.0036) showed that a high expression level of programmed death-1 (PD-1) on CD4+ T cells was a negative predictive biomarker of overall survival in the HLA-A*2402-matched group . Moreover, a high expression level of PD-1 on CD4+ T cells was a negative predictor for the induction of cytotoxic T lymphocytes (p = 0.0007). After treatment, we found that the upregulation of PD-1 and T cell immunoglobulin mucin-3 (Tim-3) expression on CD4+ and CD8+ T cells was significantly associated with a poor clinical outcome in the HLA-A*2402-matched group (p = 0.0330, 0.0282, 0.0046, and 0.0068, respectively). In contrast, there was no significant difference for these factors in the HLA-A*2402-unmatched group. Conclusions Our results indicate that the upregulation of PD-1 and Tim-3 expression on CD4+ and CD8+ T cells may restrict T cell responses in advanced PC patients; therefore, combination immunotherapy with blockade of PD-1 and Tim-3 to restore T cell responses may be a potential therapeutic approach for advanced PC patients. Trial registration Clinical-Trail-Registration: UMIN000008082. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0509-1) contains supplementary material, which is available to authorized users.

In cancer patients, sentinel lymph nodes (SLNs) are crucial in the induction of antitumor T cells. However, in many cases, SLNs and tumors appear to be in immunosuppressive condition through mechanisms yet to be elucidated. In this study, the role of tumor-derived TGF-β1 in the generation of immunosuppressive microenvironments of tumors and SLNs was investigated. Murine colorectal carcinoma CT26 transduced with TGF-β1 cDNA (CT26-TGF-β1) showed enhanced tumor growth compared with mock-transduced CT26 (CT26-Mock) when implanted in syngeneic Balb/c mice, even though CT26-TGF-β1 shows slower growth in vitro. This enhanced growth was not observed when implanted in immunodeficient mice, suggesting that TGF-β1 enhanced tumor growth by suppressing antitumor T-cell responses. Analysis of immune cells in CT26-TGF-β1-implanted mice revealed impairment of dendritic cells (DCs), decrease of CD8 T cells, and increase of MDSCs and Tregs in the tumors. Similarly, the SLNs of these mice showed an increase of MDSCs, Tregs, and PD-L1 DCs, and decrease of T-cell stimulatory activity of DCs accompanied by decreased CD80 expression and TNF-α production. In addition, induction of tumor antigen-specific T cells from SLNs of the CT26-TGF-β1-implanted mice was significantly reduced. These results demonstrate that overproduction of TGF-β1 is critical for the generation of immunosuppressive microenvironments in both tumors and SLNs, which may result in suppression of spontaneous antitumor CD8 T-cell responses. Therefore, TGF-β1 is an attractive target for restoration of immunosuppressive condition in cancer patients.

We have developed NOD-Rag2(null) IL-2Rγ(null) (NR2G) mice similar to NOD-scidIL-2Rγ(null) (NOG) mice that are known as an excellent host to generate humanized mice. To evaluate the usefulness of NR2G mice as a host for humanized mice, the engraftment rates and differentiation of human cells after human hematopoietic stem cell (HSC) transplantation were compared among NR2G, NOG, and NOD-scid mice. For this purpose, the appropriate irradiation doses to expand the niche for human stem cells in the bone marrow were first determined. As a result, 8 and 2.5 Gy in adult, and 4 and 1 Gy in newborn NR2G and NOG mice, respectively, were found to be appropriate. Next, 5 × 10(4) human umbilical cord blood CD34(+) cells were intravenously inoculated into irradiated adult or newborn of the immunodeficient mice. These HSC transplantation experiments demonstrated that both NR2G and NOG mice showed high engraftment rates compared with NOD-scid mice, although NOG mice showed a slightly higher engraftment rate than that for NR2G mice. However, no difference was found in the human cell populations differentiated from HSCs between NR2G and NOG mice. The HSC transplantation experiments to adults and newborns of two immunodeficient mice also revealed that the HSC transplantation into newborn mice resulted in higher engraftment rate than those into adults. These results showed that NR2G mice could be used as an alternative host to NOG mice to generate humanized mice.

O1 Combinatorial CD8+ and PD-L1+ cell densities correlate with response and improved survival in non-small cell lung cancer (NSCLC) patients treated with durvalumab #### Sonja Althammer1, Keith Steele2, Marlon Rebelatto2, Tze Heng Tan1, Tobias Wiestler1, Guenter Schmidt1, Brandon Higgs2, Xia

Background Anti-PD-1 antibody nivolumab has been approved for advanced melanoma in Japan. Although there have been clinical trial reports on nivolumab treatment for advanced melanoma, real-world data on the efficacy of nivolumab in an Asian patient cohort is still lacking. The aim of this study is to obtain real-world data on the efficacy of nivolumab in Japanese patients with advanced melanoma. Methods This prospective observational study was performed on unresectable or metastatic melanoma patients who were treated with nivolumab. Primary endpoints were response rate (RR), and overall survival (OS). The secondary endpoints were progression-free survival (PFS) and the immune-related adverse events (irAEs) ratio. Results In total, 124 patients from 22 institutions in Japan were enrolled between Dec. 2015 and Dec. 2017. Mucosal melanoma (34%) was the most frequent subtype in this study, followed by acral lentiginous melanoma (20%), nodular melanoma (15%), superficial spreading melanoma (13%). We observed complete response (CR) in 2 (2%), partial response (PR) in 21 (17%), stable disease (SD) in 27 (22%), and progressive disease (PD) in 59 patients (47.6%) (objective RR: 19%). Use of nivolumab as first-line treatment showed a tendency toward higher response than when used as second-line treatment (objective RR, 23% vs 11%, P = 0.12). IrAEs comprised skin reactions and endocrine-related adverse effects. The median PFS in patients with skin reactions (or skin-related irAEs) was 8.61 months compared to 2.14 months without skin-related irAEs, and 8.99 months (p Conclusions The results of this interim analysis showed a lower RR than that reported in recent phase 2 trials conducted in Japan. The differences in the proportions of melanoma subtypes, with a higher proportion of mucosal and acral melanoma, and use of nivolumab as second-line treatment will probably lead to a lower RR to nivolumab in Japan. As the results of this study showed that the occurrence of irAEs had a significant impact on therapeutic efficiency, it is critical to take appropriate measures to manage the occurrence of irAEs. Legal entity responsible for the study The authors. Funding This study was sponsored by Public Health Research Foundation under the funding support from Ono Pharmaceutical Co., Ltd. and Bristol-Myers Squibb K.K. Disclosure N. Yamazaki: Honoraria (self), Honoraria (institution), Non-remunerated activity/ies: ONO Phamaceuticals; Honoraria (self), Honoraria (institution), Non-remunerated activity/ies: Bristol-Myers Squibb; Honoraria (self), Honoraria (institution), Non-remunerated activity/ies: Novartis Pharma K.K.; Honoraria (self), Honoraria (institution), Non-remunerated activity/ies: MSD K.K.; Honoraria (self), Honoraria (institution): Merck Serono Co., Ltd.; Honoraria (self): Takeda Pharmaceutical Co., Ltd. K. Namikawa: Honoraria (institution): Public Health Research Foundation; Honoraria (self): Ono Pharmaceutical; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Merck Sharp & Dohme. T. Takenouchi: Advisory / Consultancy: Ono Pharmaceutical Co., Ltd.; Advisory / Consultancy: MSD KK; Advisory / Consultancy: Chugai Pharmaceutical Co., LTD.; Advisory / Consultancy: Novartis Pharma KK. Y. Nakamura: Honoraria (self): ONO pharmaceuticals; Honoraria (self): MSD; Honoraria (self): Bristol Myers-Squibb; Honoraria (self): Novartis Pharma K.K.; Honoraria (self): Taisho Toyama Pharma; Honoraria (self): Maruho; Honoraria (self): Taiho Pharma. S. Kitano: Honoraria (self): AstraZeneca; Honoraria (self): Chugai; Honoraria (self): Pfizer; Honoraria (self): Sanofi; Honoraria (self): Nippon Kayaku; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Meiji Seika Pharma; Honoraria (self): Taiho. T. Fujita: Honoraria (self): Public Health Research Foundation; Advisory / Consultancy: ONO PHARMACEUTICAL CO., LTD; Advisory / Consultancy: Bristol-Myers Squibb Company. T. Yamanaka: Honoraria (self), Honoraria (institution): Takeda; Honoraria (self), Honoraria (institution): Taiho; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Chugai. Y. Kawakami: Advisory / Consultancy: Public Health Research Foundation; Advisory / Consultancy: Ono Pharmaceutical Co., LTD; Advisory / Consultancy: Bristol-Myers Squibb Company. All other authors have declared no conflicts of interest.

Background Immunoscore® is an in vitro diagnostic test that predicts the risk of relapse in patients with Colon Cancer (CC) by measuring the host immune response at the tumor site. It is an immune risk-assessment tool providing independent and superior prognostic value than traditional histopathological risk parameters, and is intended to be used as an adjunct to the TNM classification. Currently, Immunoscore plays a critical role to guide post-surgery decisions in stage II & III CC patients. In stage I, survival rates are high and adjuvant chemotherapy is not typically recommended. However, approximately 10% of stage I CC tumors will recur even after surgical resection. Methods A subgroup analysis was performed on the stage I patients (n = 451) from the Immunoscore international validation study (Pages et al. The Lancet 2018). Patients were classified by Immunoscore based on pre-defined cutoffs, either in 5 (IS 0-4) or in 3 categories: IS Low (IS0-1), Intermediate (IS 2), High (IS 3-4). Time to recurrence (TTR) was compared between Immunoscore categories. Results Immunoscore Low, Intermediate and High were observed in 14%, 47% and 39% of the cohort, respectively. Immunoscore was positively and significantly correlated with TTR. When adjusting the model with Immunoscore, age, gender, T-stage sidedness and MSI, Immunoscore remained the sole significant parameter (stratified by participating center HRlow vs high=7.82; 95% CI 1.49 − 41.01; p = 0.015). In multivariate analysis, the variable with the most important relative contribution to the risk (Chi2) was Immunoscore. The robust correlation between Immunoscore classification and TTR was further corroborated by a separate analysis of the same cohort distributed into five IS categories. In MSS stage I patients, TTR rates at 5 years were 100%, 98.1%, 93.5%, 85.4%, 87.5% for IS4, IS3, IS2, IS1, IS0, respectively. Conclusions Immunoscore® is a robust prognostic indicator of the risk of recurrence in stage I CC. This risk assessment tool reliably identifies a sub-group of patients with an increased risk of relapse for whom a more intensive surveillance program after curative resection may be recommended. Legal entity responsible for the study SITC (Society for ImmunoTherapy of Cancer) / INSERM. Funding Grants from National Institute of Health and Medical Research (INSERM), the LabEx Immuno-oncology, the Transcan ERAnet European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, INCA translationnel, Japan-AMED (P-DIRECT) and MEXT (Grants-in-aid for Scientific Research-S), and Ministry of Health of the Czech Republic (AZV CR 15-28188A) Progres Q25-LF1. Disclosure J. Galon: Advisory / Consultancy, Shareholder / Stockholder / Stock options, Co-founder : HalioDx; Research grant / Funding (institution): Perkin-Elmer; Advisory / Consultancy, Research grant / Funding (institution): IObiotech; Advisory / Consultancy, Research grant / Funding (institution): MedImmune; Research grant / Funding (institution): Janssen; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Imcheck Therapeutics; Advisory / Consultancy: BMS; Advisory / Consultancy: Northwest Biotherapeutics; Advisory / Consultancy: Amgen; Advisory / Consultancy: Gilead; Advisory / Consultancy: CatalYm GmbH; Advisory / Consultancy: Sanofi; Licensing / Royalties, Patent holder: INSERM. F. Hermitte: Shareholder / Stockholder / Stock options, Full / Part-time employment, Co-founder : HalioDx. B. Mlecnik: Licensing / Royalties, Patent holder: INSERM. F. Pages: Licensing / Royalties, Patent holder: INSERM. All other authors have declared no conflicts of interest.

556 Background: Immune check points blockade with specific antibodies can accelerate anti-tumor immunity, resulting in a clinical response in patients with various types of cancer. Thus, a wide variety of treatment combinations based on PD-L1/ PD-1 pathway blockage are under development to enhance the therapeutic effect. Here, the effects of the combination treatment of PRI-724, a selective inhibitor of the CBP/β-catenin, with anti-PD-L1 antibody were examined in a mouse model of the liver metastasis of colon cancer. Methods: Mice were inoculated with SL4 colon cancer cells into the spleen to produce metastatic liver tumors. The animals were intraperitoneally injected with or without PRI-724 and/or anti-PD-L1 antibody (10F.9G2) 3 times a week. A part of mice treated with PRI-724 and anti-PD-L1 antibody was administrated with anti-mouse CD4 or CD8 antibody 3 times a week. First, to evaluate anti-tumor effect in those mice, we analyzed liver histology and survival rates after treatment. Next, to examine immune response in the liver, intrahepatic lymphocytes were analyzed by FACS for CD8 memory phenotype, Treg cells, macrophages, and dendritic cells, and the cytokine production from these cells (TNFa, IFNg etc.). Furthermore, inflammatory cytokines and chemokines mRNAs levels and PCR array concerned to Wnt signaling in the liver and serum cytokines levels were also analyzed. Results: The combination of the treatments resulted in regression of tumor growth, whereas monotherapy of each treatment did not show any anti-tumor activity. PRI-724 increased T lymphocytes recruitment, including CD8+ T cells, in the tumor, which may have been induced by inflammatory chemokines and a change of the macrophage property to the cytotoxic phenotype in the liver. Anti-PD-L1 antibody induced CD69+-activated T lymphocytes in the PRI-724-treated livers of mice inoculated with SL4. Administration of anti-CD8 antibody canceled the anti-tumor effects of the combination treatments of PRI-724 and anti-PD-L1 antibody. Conclusions: Targeting CBP/β-catenin combined with PD-1/PD-L1 immune check points blockade shows potential as a new therapeutic strategy for treating the liver metastasis of colon cancer.

487 Background: Immunoscore Colon is an IVD test predicting the risk of relapse in early-stage colon cancer (CC) patients, by measuring the host immune response at the tumor site. It is a risk-assessment tool providing independent and superior prognostic value than the usual tumor risk parameters and is intended to be used as an adjunct to the TNM classification. Risk assessment is particularly important to decide when to propose an adjuvant (adj.) treatment for stage (St) II CC patients. High-risk stage II patients defined as those with poor prognostic features including T4, lymph nodes < 12, poor differentiation, VELIPI, bowel obstruction/perforation can be considered for adj. chemotherapy (CT). However, additional risk factors are needed to guide treatment decisions. Methods: A subgroup analysis was performed on the St II untreated patients (n = 1130) from the Immunoscore international validation study (Pagès The Lancet 2018). The high-risk patients (with at least 1 clinico-pathological high-risk feature) were classified in 2 categories using pre-defined cutoffs: Low Immunoscore versus High Immunoscore and their five-year time to recurrence (5Y TTR) was compared to the TTR of the low-risk patients (without any clinico-pathological high-risk feature). Results: Among the patients with high-risk features (n = 630), 438 (69.5%) had a High Immunoscore with a corresponding 5Y TTR of 87.4 (95% CI 83.9-91.0), statistically similar (logrank pv not stratified p > 0.42, wald pv stratified by center p > 0.20) to the TTR 89.1 (95% CI 86.1-92.1) observed for the 500 low-risk patients (with no clinico-pathological feature). Furthermore, 5Y TTR for these patients were statistically similar to those of St II patients with high-risk features and a High Immunoscore (n = 438), who received adj. CT (n = 162) (5Y TTR of 83.4 (95% CI 77.6-89.9). Conclusions: These data show that despite the presence of high-risk features that usually trigger adj. treatment, when not treated with CT, a significant part of these patients (69.5%) have a recurrence risk similar to the low risk patients. Therefore, the Immunoscore test could be a good tool for adj. treatment decision in St II patients.

Bone metastasis greatly deteriorates the quality of life in patients with cancer. Although mechanisms have been widely investigated, the relationship between cancer bone metastasis and antitumor immunity in the host has been much less studied. Here, we report a novel mechanism of bone metastasis mediated by FSTL1, a follistatin-like glycoprotein secreted by Snail+ tumor cells, which metastasize frequently to bone. We found that FSTL1 plays a dual role in bone metastasis—in one way by mediating tumor cell invasion and bone tropism but also in a second way by expanding a population of pluripotent mesenchymal stem-like CD45−ALCAM+ cells derived from bone marrow. CD45−ALCAM+ cells induced bone metastasis de novo, but they also generated CD8low T cells with weak CTL activity in the periphery, which also promoted bone metastasis in an indirect manner. RNA interference-mediated attenuation of FSTL1 in tumor cells prevented bone metastasis along with the parallel increase in ALCAM+ cells and CD8low T cells. These effects were accompanied by heightened antitumor immune responses in vitro and in vivo. In clinical specimens of advanced breast cancer, ALCAM+ cells increased with FSTL1 positivity in tumor tissues, but not in adjacent normal tissues, consistent with a causal connection between these molecules. Our findings define FSTL1 as an attractive candidate therapeutic target to prevent or treat bone metastasis, which remains a major challenge in patients with cancer. Cancer Res; 73(20); 6185–93. ©2013 AACR.

T-helper 17 (Th17) cells are characterized by producing interleukin-17 (IL-17, also called IL-17A), IL-17F, IL-21, and IL-22 and potentially TNF-αand IL-6 upon certain stimulation. IL-23, which promotes Th17 cell development, as well as IL-17 and IL-22 produced by the Th17 cells plays essential roles in various inflammatory diseases, such as experimental autoimmune encephalomyelitis, rheumatoid arthritis, colitis, and Concanavalin A-induced hepatitis. In this review, we summarize the characteristics of the functional role of Th17 cells, with particular focus on the Th17 cell-related cytokines such as IL-17, IL-22, and IL-23, in mouse models and human inflammatory diseases.

Eggs of spiny dogfish Squalus acanthias are an indispensable and effective component of the compound diets currently used for larvae of the Japanese eel Anguilla japonica. However, this fish species, a natural resource, is becoming more scarce, suggesting that suitable diets should be developed using more sustainable components. In the search for appropriate diets without shark eggs, we have designed test diets in which shark eggs were replaced with general feed materials, such as fish protein hydrolysate (FPH), with the notable improvement that the FPH had been pre-digested with integral enzymes from frozen krill. Feeding trials were conducted to assess the effects of the test diets on survival and growth of Japanese eel larvae at early developmental stages. Larvae fed on the test diets were found to be able to survive up to 65 days after hatching, the maximum total length and body depth were 14.1 and 1.5 mm, respectively. Although larvae fed on the test diets still exhibited a low survival rate and poor growth, the FPH-based diets had a nutritional value that promoted substantial larval growth based on the significant difference between the initial and final body sizes. Our results suggest that the development of FPH-based diets not based on shark eggs but rather on general feed ingredients is promising but that there is much room for improvement.

Immunological status in tumor tissues varies among patients. Infiltration of memory-type CD8(+) T cells into tumors correlates with prognosis of patients with various cancers. However, the mechanism of the differential CD8(+) T cell infiltration has not been well investigated. In general, tumor-associated microenvironments, including tumor and sentinel lymph nodes, are under immunosuppressive conditions such that the immune system is not able to eliminate cancer cells without immune-activating interventions. Constitutive activation of various signaling pathways in human cancer cells triggers multiple immunosuppressive cascades that involve various cytokines, chemokines, and immunosuppressive cells. Signaling pathway inhibitors could inhibit these immunosuppressive cascades by acting on either cancer or immune cells, or both. In addition, common signaling mechanisms are often utilized for multiple hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression). Therefore, targeting these common signaling pathways may be an attractive strategy for cancer therapy including immunotherapy.

Antitumor functions of the host immune system are frequently compromised in patients with malignancies. In the current study, we evaluated the relationship between expression ratio of mRNAs for the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax (the Bcl-2/Bax ratio) in peripheral blood mononuclear cells and clinical outcomes in patients with head and neck carcinomas. The overall survival (OS) time of patients with Bcl-2/Bax ratios ≥ 1.2 tended to be longer than that of patients with Bcl-2/Bax ratios < 1.2 but not significantly so (P = .084, n = 61). Disease-free survival (DFS) of patients with Bcl-2/Bax ratios ≥ 1.2 was statistically significantly longer than that of patients with Bcl-2/Bax ratios < 1.2 (P = .001, n = 76). All of the patients whose Bcl-2/Bax ratio is ≥ 2.0 were alive after 36 months and survived without any evidence of disease for 24 months (Bcl-2/Bax ≥ 2.0 versus Bcl-2/Bax < 2.0; P = .035, n = 61 in OS, P < .001, n = 76 in DFS, respectively). In 56 patients who received immunochemoradiotherapy using UFT and OK-432 in combination with radiotherapy, a statistically significant relationship between the Bcl-2/Bax ratio and the therapeutic effect estimated using Response Evaluation Criteria in Solid Tumors was observed, as well as a relation with interferon-γ (IFN-γ) induction in response to the therapy [P = .002 in complete response versus partial response + stable disease; P = .046 in IFN-γ(+) versus IFN-γ(-)]. In addition, there were significant correlations of the Bcl-2/Bax ratio with both the absolute number of CD4+ T cells and the rate of CD4+ T cell and natural killer cell activity. These findings strongly suggest that the balance of expression of Bcl-2 and Bax genes in circulating immune cells has a high prognostic value in head and neck cancer patients.

Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4(+) HNSCC cells, but not CCR4(-) cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206(+) M2-like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206(high) M2-like macrophages increased the cell motility of CCR4(+) HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4(+) cancer cells, but not CCR4(-) cells, metastasized to lymph nodes which contained CCL22 producing M2-like macrophages. These results demonstrate that lymph node metastasis of CCR4(+) HNSCC is promoted by CCL22 in an autocrine or M2-like macrophage-dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.

Primordial germ cells (PGC) are the only cell type in developing embryos with the potential to transmit genetic information to the next generation. In this study, PGC of Japanese eel (Anguilla japonica) were visualized by injection of mRNA synthesized from a construct carrying the green fluorescent protein (GFP) gene fused to the 3' untranslated region of the Japanese eel nanos gene. We investigated the feasibility of cryopreserving Japanese eel PGC by vitrification of dechorionated whole somite stage embryos. The GFP-labeled PGC were rapidly cooled using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (methanol, dimethyl sulfoxide, and glycerol for 10 min and ethylene glycol for 10, 20, and 30 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose for 1, 5, and 10 min. Ethylene glycerol is an effective cryoprotectant for embryonic cells and shows no evidence of ice formation after thawing. Vitrified and thawed PGC were transplanted into blastula stage embryos from zebrafish (Danio rerio). The GFP-labeled PGC migrated toward the host gonadal ridge, suggesting maintenance of their normal migration motility. These techniques may assist in achieving inter- and intraspecies germ-line chimers using donor Japanese eel PGC.

Chronic graft-versus-host disease (cGVHD) is a notorious complication of allogeneic hematopoietic stem cell transplantation and causes disabling systemic inflammation and fibrosis. In this novel study, we focused on a relationship between endoplasmic reticulum (ER) stress and cGVHD, and aimed to create effective treatment of cGVHD. A series of experiments were conducted using a mouse model of cGVHD. Our data suggested (1) that ER stress was elevated in organs affected by cGVHD and (2) that 4-phenylbutyric acid (PBA) could reduce cGVHD-induced ER stress and thereby alleviate systemic inflammation and fibrosis. Because fibroblasts are thought to be implicated in cGVHD-elicited fibrosis and because macrophages are reported to play a role in the development of cGVHD, we investigated cGVHD-triggered ER stress in fibroblasts and macrophages. Our investigation demonstrated (1) that indicators for ER stress and activation markers for fibroblasts were elevated in cGVHD-affected lacrimal gland fibroblasts and (2) that they could be reduced by PBA. Our work also indicated that splenic macrophages from PBA-dosed mice exhibited the lower levels of ER stress and M2 macrophage markers than those from cGVHD-affected mice. Collectively, this study suggests that the reduction of ER stress utilizing PBA can be a clinically translatable method to treat systemic cGVHD.

Kinesin family member 20A (KIF20A), an ideal cancer-testis antigen, was reported to be a promising immunotherapeutic target for pancreatic cancers. Clinical trials of KIF20A peptide vaccine immunotherapy have been conducted against pancreatic cancers. To demonstrate the efficacy of KIF20A as a candidate molecular target for gliomas, we analyzed the expression and function of KIF20A in gliomas. Western blot and quantitative PCR analyses showed that KIF20A expression in glioma cell lines and glioma tissues was high compared with that found in a normal brain. KIF20A immunostaining of glioma cells and glioma tissues demonstrated that KIF20A was involved in spindle formation and cytokinesis, and that KIF20A was highly expressed, especially in glioma cells undergoing mitosis. In silico analysis of a cancer microarray database revealed that KIF20A was highly expressed in gliomas depending on the pathological grade, and glioma patients with higher expression of KIF20A showed poorer prognosis. Down-regulating KIF20A reduced cell proliferation in glioma cells due to the failure of cytokinesis and generation of binucleated cells. Additionally, KIF20A inhibition induced significant apoptosis in SF126 glioma cells. Taken together, KIF20A is a tumor-associated antigen involved in the glioma cell growth and cell survival, suggesting that KIF20A is an oncoantigen of gliomas. Thus, KIF20A is a candidate novel immunotherapeutic target for gliomas.

Background T-cell infiltration in tumors has been used as a prognostic tool in non-small-cell lung cancer (NSCLC). However, the influence of smoking habit and histological type on tumor-infiltrating lymphocytes (TILs) in NSCLC remains unclear. Patients and methods We evaluated the prognostic significance of TILs (CD4+, CD8+, CD20+, and FOXP3+) according to histological type and smoking habit using automatic immunohistochemical staining and cell counting in 218 patients with NSCLC. Results In multivariate survival analyses of clinical, pathological, and immunological factors, a high ratio of FOXP3+ to CD4+ T cells (FOXP3/CD4) [hazard ratio (HR): 4.46, P Conclusions A low number of CD8+ T cells in non-AD, a high FOXP3/CD4 ratio in smokers with AD, and a low number of CD20+ B cells in non-smokers with AD were identified as independent unfavorable prognostic factors in resected NSCLC. Evaluating the influence of histological type and smoking habit on the immunological environment may lead to the establishment of immunological diagnosis and appropriate individualized immunotherapy for NSCLC.

Although evidence over the last 30 years suggests that the autonomic nervous system (ANS) mediates stress-induced allostatic and immune responses, the crucial role that it plays in the tumor micro-environment has only recently been reported. Here, we review the action of ANS signaling in this micro-environment. Emerging data suggest that primary tumors are innervated by the ANS which mediates stress-related effects on tumor progression. The activation of the sympathetic nervous system (SNS) takes advantage of neurotransmitters and neuropeptides from the innervating neural circuitry and/or hypothalamic-pituitary-adrenal axis glucocorticoids via their receptors to modulate the gene expression associated with oncogenesis, the proliferation and apoptosis of tumor cells, angiogenesis, and the tumor-associated immune response. The parasympathetic nervous system has also been implicated in some tumor types, but its contribution in the tumor micro-environment remains unclear. In addition to identifying the ANS signaling pathways involved in tumor progression, recent reports suggest that the ANS could be a potential biomarker to predict tumor progression, and have identified new pharmacological strategies, such as the use of β-adrenergic blockers, to inhibit tumor progression and metastasis by targeting this system. These findings are reviewed here.

MicroRNAs (miRNAs/miRs) regulate the levels of transcripts and serve a critical function in the regulation of tumor microenvironments. Therefore, miRNA levels in cancer tissues are thought to be potential biomarkers for immunotherapy. From a phase I trial of a vaccine treatment using 5 human leukocyte antigen (HLA)-A*2402-restricted peptides (registration no. UMIN000004948), colorectal cancer (CRC) tissues were obtained from 8 patients and normal colorectal tissues from 5 patients via surgery. From a phase II trial using the same peptides (registration no. UMIN000001791), CRC tissues were obtained from 16 patients from the HLA-A*2402-matched group and 10 patients from the HLA-A*2402-unmatched group. These tissues were used for miRNA microarray analysis. As the first step, cancer tissues from the phase I study were used and 10 candidate miRNAs were selected by comparing the miRNA expression between two groups; one with improved prognosis and the other with poor prognosis. The miRNAs were subsequently validated using the cases enrolled in the phase II study. Significantly improved prognoses were identified in 16 patients in the HLA-A*2402-matched group with high expression of miR-196b-5p and low expression of miR-378a-3p and miR-486-5p. There was no difference in prognosis in the 10 patients in the HLA-A*2402-unmatched group. Therefore, high miR-196b expression and low miR-378a-3p and miR-486-5p expression were indicated as useful biomarkers for prediction of the efficacy of vaccine treatment for patients with metastatic CRC. In a planned phase III study, expression levels of these 3 miRNAs (miR-196b-5p, miR-378a-3p and miR-486-5p) may be useful biomarkers for assessing patients who are likely to have an improved outcome following vaccination.

Primordial germ cells (PGCs) appear during early embryogenesis and differentiate into gametes through oogenesis or spermatogenesis. Teleost PGCs can be visualized by injecting RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3). Although this method has been widely applied to teleost PGCs, the visualization of PGCs in pelagic species that have eggs with a hard chorion is more problematic due to the technical difficulty of microinjection into their eggs. In this study, we developed a reliable method for microinjection of fertilized eggs in a pelagic species, the barfin flounder. Using a microneedle with a constriction "brake", we were able to introduce gfp-nos3 3'UTR mRNA into embryos and to determine the origin and migration route of PGCs. We also isolated the barfin flounder nos3 (bf-nos3) gene to compare its 3'UTR sequence with that of zebrafish. The 3'UTR of the bf-nos3 sequence was longer than that of zf-nos3. However, PGCs were also visualized after injection of gfp-bf-nos3 3'UTR mRNA both in zebrafish and barfin flounder. These results suggest that the function of nos3 is conserved between these species regardless of the sequence differences. The method developed here for labeling PGCs with gfp-nos3 mRNA will provide a means to study PGC development in the embryos of a wide range of marine fish species.

Macrophage migration inhibitory factor (MIF) plays an important role in supporting the proliferation and/or survival of murine neural stem/progenitor cells (NSPCs); however, the downstream effectors of this factor remain unknown. Here, we show that MIF increases the expression of Pax6 and Chd7 in NSPCs in vitro. During neural development, the chromatin remodeling factor Chd7 (chromatin helicase-DNA-binding protein 7) is expressed in the ventricular zone of the telencephalon of mouse brain at embryonic day 14.5, as well as in cultured NSPCs. Retroviral overexpression of Pax6 in NSPCs increased Chd7 gene expression. Lentivirally-expressed Chd7 shRNA suppressed cell proliferation and neurosphere formation, and inhibited neurogenesis in vitro, while decreasing gene expression of Hes5 and N-myc. In addition, CHD7 overexpression increased cell proliferation in human embryonic stem cell-derived NSPCs (ES-NSPCs). In Chd7 mutant fetal mouse brains, there were fewer intermediate progenitor cells (IPCs) compared to wildtype littermates, indicating that Chd7 contributes to neurogenesis in the early developmental mouse brain. Furthermore, in silico database analysis showed that, among members of the CHD family, CHD7 is highly expressed in human gliomas. Interestingly, high levels of CHD7 gene expression in human glioma initiating cells (GICs) compared to normal astrocytes were revealed and gene silencing of CHD7 decreased GIC proliferation. Collectively, our data demonstrate that CHD7 is an important factor in the proliferation and stemness maintenance of NSPCs, and CHD7 is a promising therapeutic target for the treatment of gliomas. Electronic supplementary material The online version of this article (doi:10.1186/s13041-016-0275-6) contains supplementary material, which is available to authorized users.

Cancer immunotherapies utilizing tumor-specific T-cell responses, immune-checkpoint blockade, and T-cell-based adoptive cell therapy, have recently shown durable responses in advanced patients with various cancers. However, there are still cancer types and patients who do not respond to these immunotherapies. Pretreatment immune status varies in cancer patients, and it correlates with prognosis after various cancer therapies including immunotherapy. The differential T-cell response is defined by positive (e.g., number of immunogenic mutated peptides derived from mainly passenger DNA mutations in cancer cells, polymorphisms of immune-related genes of patients) and negative (e.g., oncogene activation including driver DNA mutations) immune pathways along with environmental factors (e.g., intestinal microbiota, diet, smoking, infection history). These factors could be biomarkers for selection of the patients who are likely to respond to immunotherapy and furthermore could be therapeutic targets to improve efficacy of immunotherapy possibly by combination immunotherapy with interventions on multiple key regulation points in the antitumor T-cell responses. Personalized combination immunotherapy based on the evaluation of T-cell immune status is a promising strategy for cancer treatment.

The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2(271-279)). The CTLs induced by PEPP2(271-279) recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2'-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2'-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.

Summary In gene-banking, primordial germ cells (PGCs), which are embryonic precursor cells of germ cells, are useful for cryopreservation because PGCs have a potential to differentiate into both eggs and sperm via germ-line chimera. Here, we have established vitrification methods for PGCs cryopreservation using 12- to 17-somite stage embryos in loach, Misgurnus anguillicaudatus, which were dechorionated, removed their yolk and injected with green fluorescent protein (GFP) -nos1 3′UTR mRNA to visualize their PGCs. In order to optimize cryopreservation medium for vitrification, the toxicity of cryoprotectants was analyzed. Different concentrations (2, 3, 4, 5 m) of dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and propylene glycol (PG) as cryoprotectants were tested. Then, 5 m DMSO showed significantly-high toxicity. Based on this information, combinations called DMP (2 m (14.2% [v/v]) DMSO, 2 m (8.1% [v/v]) MeOH and 2 m (14.4% [v/v]) PG), DP (2 m (14.2% [v/v]) DMSO and 4 m (28.7% [v/v]) PG) and DE (2.1 m (15% [v/v]) DMSO and 2.7 m (15% [v/v]) EG) were evaluated for their toxicities and efficacy of PGCs cryopreservation using two types of equilibration step: direct immersion of cryopreservation media (one-step) and serial exposure to half and full concentration of cryopreservation media (two-step). Viable PGCs were obtained from post-thaw embryos which were cryopreserved by DP and DE with both 1- and 2-step equilibrations. Despite DP showing the highest toxicity, it gave the highest survival rate of embryonic cells after cryopreservation. When PGCs recovered from vitrified embryos were transplanted into host embryos at the blastula stage, the transplanted PGCs were able to migrate to a host genital ridge similarly as endogenous PGCs. It suggests that our methods could be useful to create a germ-line chimera for the production of gametes from PGCs of cryopreserved embryos.

We previously found that cancer metastasis is accelerated by immunosuppression during Snail-induced epithelial-to-mesenchymal transition (EMT). However, the molecular mechanism still remained unclear. Here, we demonstrate that CCL2 is a critical determinant for both tumor metastasis and immunosuppression induced by Snail(+) tumor cells. CCL2 is significantly upregulated in various human tumor cells accompanied by Snail expression induced by snail transduction or TGFβ treatment. The Snail(+) tumor-derived CCL2 amplifies EMT events in other cells including Snail(-) tumor cells and epithelial cells within tumor microenvironment. CCL2 secondarily induces Lipocalin 2 (LCN2) in the Snail(+) tumor cells in an autocrine manner. CCL2 and LCN2 cooperatively generate immunoregulatory dendritic cells (DCreg) having suppressive activity accompanied by lowered expression of costimulatory molecules such as HLA-DR but increased expression of immunosuppressive molecules such as PD-L1 in human PBMCs. The CCL2/LCN2-induced DCreg cells subsequently induce immunosuppressive CD4(+)FOXP3(+) Treg cells, and finally impair tumor-specific CTL induction. In murine established tumor model, however, CCL2 blockade utilizing the specific siRNA or neutralizing mAb significantly inhibits Snail(+) tumor growth and metastasis following systemic induction of anti-tumor immune responses in host. These results suggest that CCL2 is more than a chemoattractant factor that is the significant effector molecule responsible for immune evasion of Snail(+) tumor cells. CCL2 would be an attractive target for treatment to eliminate cancer cells via amelioration of tumor metastasis and immunosuppression.

Stem cell-based therapy has been proposed as a promising strategy for regenerating tissues lost through incurable diseases. Side population (SP) cells have been identified as putative stem cells in various organs. To examine therapeutic potential of SP cells in hypofunction of exocrine glands, SP cells isolated from mouse exocrine glands, namely, lacrimal and salivary glands, were transplanted into mice with irradiation-induced hypofunction of the respective glands. The secretions from both glands in the recipient mice were restored within 2 months of transplantation, although the transplanted cells were only sparsely distributed and produced no outgrowths. Consistent with this, most SP cells were shown to be CD31-positive endothelial-like cells. In addition, we clarified that endothelial cell-derived clusterin, a secretory protein, was an essential factor for SP cell-mediated recovery of the hypofunctioning glands because SP cells isolated from salivary glands of clusterin-deficient mice had no therapeutic potential, whereas lentiviral transduction of clusterin restored the hypofunction. In vitro and in vivo studies showed that clusterin had an ability to directly inhibit oxidative stress and oxidative stress-induced cell damage. Thus, endothelial cell-derived clusterin possibly inhibit oxidative stress-induced hypofunction of these glands.

Esophageal squamous cell cancer (ESCC) is one of the most common lethal tumors in the world, and development of new diagnostic and therapeutic methods is needed. In this study, cancer‐testis antigen, BORIS, was isolated by functional cDNA expression cloning using screening technique with serum IgG Abs from ESCC patients. BORIS was previously reported to show cancer‐testis antigen like expression, but its immunogenicity has remained unclear in cancer patients. BORIS was considered to be an immunogenic antigen capable of inducing IgG Abs in patients with various cancers, including four of 11 ESCC patients. Immunohistochemical study showed that the BORIS protein was expressed in 28 of 50 (56%) ESCC tissues. The BORIS expression was significantly associated with lymph node metastasis in ESCC patients with pT1 disease (P = 0.036). Furthermore, the patients with BORIS‐positive tumors had a poor overall survival (5‐year survival rate: BORIS‐negative 70.0% vs BORIS‐positive 29.9%, log‐rank P = 0.028) in Kaplan–Meier survival analysis and log‐rank test. Multivariate Cox proportional hazard model demonstrated that BORIS expression was an independent poor prognostic factor (hazard ratio = 4.158 [95% confidence interval 1.494–11.57], P = 0.006). Downregulation of BORIS with specific siRNAs resulted in decreased cell proliferation and invasion ability of ESCC cell lines. BORIS may be a useful biomarker for prognostic diagnosis of ESCC patients and a potential target for treatment including by BORIS‐specific immunotherapy and molecular target therapy.

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that regulates apoptosis sensitivity in a variety of cell types. Here we evaluate the roles of Mcl-1 in chemotherapy-associated apoptosis in gastric cancer cells. In addition, our study examined whether Mcl-1 contributed to apoptosis resistance in so-called cancer stem cell (CSC)-like populations in gastric cancer.Seven gastric cancer cell lines were used. The expression of Mcl-1 was assessed by either real-time polymerase chain reaction or Western blot analysis. Apoptosis was quantitated by morphological observation and caspase activity measurement. Adenovirus-mediated RNA interference (RNAi) technology was used to knockdown the expression of Mcl-1. The release of cytochrome c was evaluated by subcellular fractionation and immunoblot analysis. To identify and isolate the CSC-like populations, we used the CSC-associated cell surface marker CD44 and flow cytometry.Six out of the 7 gastric cancer cell lines overexpressed Mcl-1 protein. These Mcl-1-expressing cell lines were relatively resistant to chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin (CDDP). Depletion of Mcl-1 protein by RNAi technology effectively sensitized the cells to anticancer drug-induced mitochondrial cytochrome c release, caspase activation, and apoptosis. In addition, vast amounts of Mcl-1 mRNA were expressed in CD44-positive CSC-like cells. Mcl-1 suppression enhanced the apoptosis in CD44-positive cells to a level equivalent to that in CD44-negative cells, suggesting that Mcl-1 mediates chemotherapy resistance in CSC-like populations.These results suggest that Mcl-1 mediates the resistance to apoptosis in gastric cancer cells by blocking the mitochondrial pathway of cell death. Mcl-1 depletion appears to be an attractive strategy to overcome chemotherapy resistance in gastric cancer cells.

Osteosarcoma is the most frequent, nonhematopoietic, primary malignant tumor of bone. Histopathologically, osteosarcoma is characterized by complex mixtures of different cell types with bone formation. The role of environmental factors in the formation of such a complicated tissue structure as osteosarcoma remains to be elucidated. Here, a newly established murine osteosarcoma model was used to clarify the roles of environmental factors such as fibroblast growth factor-2 (Fgf2) or leukemia-inhibitory factor (Lif) in the maintenance of osteosarcoma cells in an immature state. These factors were highly expressed in tumor environmental stromal cells, rather than in osteosarcoma cells, and they potently suppressed osteogenic differentiation of osteosarcoma cells in vitro and in vivo. Further investigation revealed that the hyperactivation of extracellular signal–regulated kinase (Erk)1/2 induced by these factors affected in the process of osteosarcoma differentiation. In addition, Fgf2 enhanced both proliferation and migratory activity of osteosarcoma cells and modulated the sensitivity of cells to an anticancer drug. The results of the present study suggest that the histology of osteosarcoma tumors which consist of immature tumor cells and pathologic bone formations could be generated dependent on the distribution of such environmental factors. The combined blockade of the signaling pathways of several growth factors, including Fgf2, might be useful in controlling the aggressiveness of osteosarcoma. Mol Cancer Res; 10(3); 454–68. ©2012 AACR.

The feasibility of cryopreserving common carp (Cyprinus carpio) primordial germ cells (PGC) by vitrification of whole embryos at the 22- to 28-somite stage was investigated. Green fluorescent protein (GFP)-labeled PGC were cooled rapidly using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (ethylene glycol or dimethyl sulfoxide, 30 or 50 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose (5, 10, 20, or 30 min). Embryonic cells that were pretreated for 30 min and vitrified for 20 min with ethylene glycol had the greatest rate of survival of embryonic cells (68.6%; P < 0.01), an optimal highest percentage of viable PGC (73.8 to 74.9%; P < 0.05), and no evidence of ice formation after thawing. The vitrified/thawed PGC were transplanted into blastula-stage embryos from goldfish (Carassius auratus). The PGC maintained their motility and moved to the gonadal ridge of the host embryo. Thus, the combination of vitrification and transplantation to produce germ-line chimeras is a powerful tool for the artificial production of next-generation offspring.

STAT3 signaling constitutes an important negative feedback mechanism for the maintenance of immune homeostasis, a suppressive signal for the Th1 immune response in murine macrophages, and a cancer immune evasion signal in various immune cells. The strategy for STAT3 signal inhibition should be considered, because these features could impede effective cancer immunotherapy. We have evaluated the effects of STAT3 inactivation in dendritic cells (DCs) on immune responses in mice and humans. DCs derived from LysMcre/STAT3flox/flox mice displayed higher cytokine production in response to TLR stimulation, activated T cells more efficiently, and were more resistant to the suppression of cytokine production by cancer-derived immunosuppressive factors compared with DCs from control littermates. Antitumor activities of STAT3-depleted and control DCs were compared by intratumoral administration of gp70 Ag peptide-pulsed DCs in the therapeutic MC38 tumor model. Intratumoral administration of STAT3-depleted DCs significantly inhibited MC38 tumor growth of both injected and nontreated remote tumors. The inhibition was accompanied by an increase in gp70-specific T cell response as well as in systemic Th1 immune response. STAT3-depleted human DCs with adenoviral STAT3 short hairpin RNA were also capable of producing more cytokines with TLR stimulation and more resistant to cancer-derived factors, and they induced tumor Ag-specific T cells more efficiently than control DCs. The identified role of DC STAT3 signaling in both in vivo therapeutic tumor models in mice and in vitro-specific T cell induction in humans indicates that STAT3-inactivated DCs may be a promising approach for cancer immunotherapy.

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. We previously visualized the PGCs of several teleostean embryos by injecting RNA synthesized from constructs encoding green fluorescent protein (GFP) fused to the 3'UTR of the zebrafish ( Danio rerio ) nanos 1 gene ( nos 1). However, this technique was not always suitable for visualizing PGCs in embryos from all teleost species. In this study, we compared the visualization of PGCs in common carp ( Cyprinus carpio ) embryos using two artificial constructs containing GFP fused to the 3'UTR of nanos from either common carp or zebrafish. Visualization was better using GFP fused to the 3'UTR of the nanos gene from common carp, compared with that from zebrafish. The visualized PGCs successfully migrated toward the gonadal ridge after transplantation into goldfish host embryos, suggesting that they maintained normal migratory motility. These techniques could be useful for the production of inter-specific germline chimeras using common carp donor PGCs.

Cancer-induced immunosuppression is a major problem as it reduces the anti-tumor effects of immunotherapies. In cancer tissues, cancer cells, immune cells, and other stromal cells interact and create an immunosuppressive microenvironment through a variety of immunosuppressive factors. Some cancer subpopulations such as cancer cells undergoing epithelial-mesenchymal transition and cancer stem-like cells have immunosuppressive and immunoresistant properties. The production of immunosuppressive factors by cancer cells is mechanistically attributed to oncogenic signals frequently activated in cancer cells, including the STAT3, MAPK, NF-κB, and Wnt/β-catenin signals, which are upstream events leading to immunosuppressive cascades. Moreover, some of these signals are also activated in immunosuppressive immune cells stimulated by cancer-derived factors and contribute to their immunosuppressive activities. Therefore, targeting these signals both in cancer cells and immunosuppressive immune cells may result in the restoration of immunocompetence in cancer patients and improve current immunotherapy.