Sharif Uddin Ahmed

Expression of Toll-Like Receptor 4 on Dendritic Cells Is Significant for Anticancer Effect of Dendritic Cell-Based Immunotherapy in Combination with an Active Component of OK-432, a Streptococcal Preparation

A lipoteichoic acid-related molecule OK-PSA is an active component of OK-432, a Streptococcus-derived anticancer immunotherapeutic agent. In the present study, we first examined the effect of OK-PSA on the maturation of dendritic cells (DCs) in vitro by using the DCs derived from 5 healthy donors and 10 patients with head and neck cancer with or without expression of toll-like receptor 4 (TLR4) or MD-2 mRNA. OK-PSA treatment effectively increased the surface expression of MHC class II, CD80, CD83, and CD86. OK-PSA-stimulated DCs secreted the cytokines that can induce helper T-cell 1 (Th1)-type T-cell response, and stimulated allogeneic T cells to produce IFN-γ and to elicit an allogeneic antigen-specific cytotoxicity. These activities almost depended on expression of TLR4 and MD-2 genes. We next investigated the in vivo anticancer effect of intratumoral administration of syngeneic DCs followed by OK-PSA against established tumors in mice. C57BL/6 mice, which express wild-type TLR4, and C57BL/6-derived TLR4-knockout (TLR4−/−) mice were used. Although OK-PSA accelerated the antitumor effect of intratumoral DC administration in wild-type mice bearing syngeneic tumors, the antitumor effect of OK-PSA as well as of the combination therapy with DCs and OK-PSA was not significant in TLR4−/− mice. Interestingly, an administration of wild-type-mouse-derived DCs followed by OK-PSA exhibited a marked antitumor effect even in the TLR4−/− mice. These findings suggest that OK-PSA may be a potent adjuvant for local DC therapy, and that DC therapy followed by OK-PSA is able to elicit anticancer activity even in a TLR4-deficient host when TLR4 is expressed only in DCs injected intratumorally.

Mechanism of anticancer host response induced by OK-432, a streptococcal preparation, mediated by phagocytosis and Toll-like receptor 4 signaling

It has previously been reported by our group that Toll-like receptor (TLR) 4 is involved in anticancer immunity induced by OK-432, a Streptococcus-derived immunotherapeutic agent. However the detailed mechanism of the OK-432-induced immune response via TLR4 remained uncertain, because it may not be possible for OK-432, which consists of whole bacterial bodies, to bind directly to TLR4. In the current study, we conducted in vitro and in vivo experiments to investigate the hypothesis that OK-432 may first be captured and dissolved by phagocytes and that the active components released by the cells may then induce host responses via TLR4. TS-2 monoclonal antibody, which recognizes an active component of OK-432 designated OK-PSA was used in the current study. First, it was observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited in vitro by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining using TS-2 demonstrated that OK-432 was captured and dissolved by phagocytes. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by enzyme-linked immunosorbent assay using TS-2. Supernatants from OK-432-treated DC culture increased nuclear factor (NF)-κB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-κB in these cells. In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B. In this case, the amount of OK-PSA, an active component of OK-432, in the sera was also reduced by cytochalasin B. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the immune effect of OK-432.

Anti-tumor effect of an intratumoral administration of dendritic cells in combination with TS-1, an oral fluoropyrimidine anti-cancer drug, and OK-432, a streptococcal immunopotentiator: Involvement of toll-like receptor 4

The authors investigated the in vivo anti-tumor effect of intratumoral administration of bone marrow-derived dendritic cells (DCs) after chemotherapy using an oral fluoropyrimidine anti-cancer drug TS-1, and followed by immunotherapeutic agent OK-432, in two syngeneic tumor-bearing mouse models. Both in Meth-A fibrosarcoma-bearing BALB/c mice and in SCCVII-bearing C3H/HeN mice, 1 week of oral administration of TS-1 effected partial eradication of established tumors. Intratumoral injection of DCs and OK-432 caused only slight inhibition of the tumor growth. However, TS-1 administration followed by DCs and OK-432 resulted in a marked inhibition in the tumor growth and also contributed to a greater prolongation of survival. By the injection of DCs and OK-432 after TS-1 administration, a significant infiltration of immune cells, especially CD8+ T cells, was observed. Furthermore, the cytotoxic activities of tumor-infiltrating lymphocytes and draining lymph node cells against inoculated tumor cells were significantly increased by the therapy, while activities against nonspecific target cells were not. Cytotoxic memory T cells were also induced; the main effectors were MHC class I-restricted, CD8+ T cells. The same therapy was also applied to SCCVII-bearing C3H/HeJ mice in which the Toll-like receptor (TLR) 4 is mutated and its function impaired; no immunotherapeutic effect was observed in the TLR4-deficient mouse model. These findings suggest that the local DC therapy in combination with TS-1 and OK-432 may be a useful strategy for the treatment of solid tumors, and that TLR4 signaling is involved in the success of this therapy.

Enhancement of anti-cancer immunity by a lipoteichoic-acid-related molecule isolated from a penicillin-killed group A Streptococcus

Abstract. We isolated the lipoteichoic-acid-related molecule (OK-PSA) from OK-432, a streptococcal preparation, by affinity chromatography on CNBr-activated Sepharose-4B-bound monoclonal antibody TS-2, which neutralizes the interferon (IFN)-γ-inducing activity of OK-432. We have previously reported that OK-PSA is a potent inducer of Th1-type cytokines in human peripheral blood mononuclear cells in vitro. In this study, we conducted an animal experiment to examine whether OK-PSA exhibits an anti-tumor effect in vivo by acting as a Th1 inducer in syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. It was found that OK-PSA induced Th1-type cytokines [IFN-γ, tumor necrosis factor-α, interleukin (IL)-2, IL-12 and IL-18] in BALB/c mice bearing Meth-A tumor and caused a marked anti-tumor effect. Although it was suggested by an in vitro study, using spleen cells derived from the animals, that IL-18 plays the greatest role in the induction of the Th1-dominant state and tumor cell killing induced by OK-PSA, the in vivo experiments demonstrated that both IL-12 and IL-18 are essential in the anti-tumor effect exhibited by OK-PSA. These findings strongly suggest that OK-PSA is a major effector molecule of OK-432 and may be a useful immunotherapeutic agent, as a potent Th1 inducer, for cancer patients with a Th2-dominant state.

Toll-Like Receptor 4 Mediates the Antitumor Host Response Induced by a 55-Kilodalton Protein Isolated from Aeginetia indica L., a Parasitic Plant

ABSTRACT A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-κB-dependent reporter plasmid, AILb-A induced NF-κB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-κB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.

Anti-tumor immune response induced by the fractions derived from OK-432, a streptococcal preparation, by using a monoclonal antibody TS-2 that neutralizes the interferon-γ-inducing activity of OK-432: comparison between the TS-2-binding and TS-2-unbinding fraction

We have previously isolated a lipoteichoic acid (LTA)-related molecule (OK-PSA) from OK-432, a streptococcal agent, by affinity chromatography on a CNBr-activated Sepharose 4B bound TS-2 monoclonal antibody (mAb) that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. In the current study, we compared the cytokine-inducing and anti-tumor activities of OK-PSA, a TS-2-binding fraction, with those of OK-PTF, a TS-2-unbinding fraction, in order to determine the efficacy of OK-PSA for clinical use in affinity chromatography using TS-2. In the in vitro experiments using human peripheral blood mononuclear cells (PBMCs), OK-PSA markedly induced Th1-type cytokines, while interleukin (IL)-6 and IL-10, Th2-type cytokines, were induced by OK-PTF. Th1-cytokine induction by OK-PTF was not dose-dependent and was suppressed when PBMCs were treated with a high concentration of OK-PTF. In a mouse model, Th1 cytokines were also induced by OK-PSA and Th2 cytokines were induced by OK-PTF. Th2 cytokine-inducing activity of OK-PTF was accelerated in tumor-bearing mice relative to that in healthy mice. Although the anti-tumor effect of OK-PTF was statistically significant, it was much weaker than that of OK-PSA. A significant difference between the anti-tumor effect of OK-PSA and that of OK-PTF was observed (P<0.05). Finally, OK-PSA elicited its cytokine-inducing effect via Toll-like receptor (TLR) 4, whereas OK-PTF-induced signaling was mediated by both TLR2 and TLR4. These findings strongly suggested that the affinity chromatography using TS-2 is a useful strategy to separate the effective component for cancer therapy (OK-PSA) from other components.

TX‐1877, a bifunctional hypoxic cell radiosensitizer, enhances anticancer host response: Immune cell migration and nitric oxide production

We investigated in the current study the effect of TX‐1877, a bifunctional hypoxic cell radiosensitizer, in augmenting anticancer host response. In the syngeneic squamous cell carcinoma‐bearing mouse model, a single administration of TX‐1877 significantly inhibited the primary tumor growth as well as lung metastasis. TX‐1877 administration resulted in a significant infiltration of immune cells, such as CD4+T, CD8+T cells, macrophages and dendritic cells (DCs), and an increased expression of chemokines for cytotoxic T lymphocytes (CTLs), helper T‐cell 1 (Th1) cells, monocytes/macrophages and DCs, in tumor tissues. Nitric oxide (NO) production and the expression of inducible NO synthase (iNOS) and interferon‐γ, a major Th1 cytokine that plays a major role in anticancer immunity, were also enhanced. Furthermore, neutralization of NO by N‐monomethyl‐L‐arginine acetate resulted in a marked inhibition of the antitumor effect of TX‐1877. In tumor‐draining lymph nodes, MHC class I‐restricted CD8+ memory CTLs specific for inoculated cancer cells were induced by TX‐1877. In in vitro experiments, TX‐1877 induced chemokines and iNOS/NO in several types of culture cells. These findings strongly suggested that TX‐1877 induces migration of CD8+CTLs, CD4+Th1 cells, macrophage/monocytes and dendritic cells into the tumor site, and that this migration is mediated by chemokine induction. In addition, it was suggested that NO produced by several types of cells stimulated by TX‐1877 in the tumor sites plays a major role in the anticancer effect of TX‐1877. TX‐1877 was thus shown to be an effective immunopotentiator as well as a hypoxic cell radiosensitizer.

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Abstract LB-58: Spontaneous Epstein Barr virus-associated lymphomagenesis in primary human solid tumor xenografts

Xenotransplantation of primary human solid tumors into immunodeficient mice is widely utilized as a tool for the study of human cancer biology. We have generated subcutaneous xenografts from a large number of human hepatocellular carcinoma (HCC) resection specimens, and have observed that subcutaneous implantation of tumor fragments or bulk cell suspensions results in a much higher rate of primary engraftment than implantation of cells sorted to exclude CD45 + leukocytes in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. While the majority of xenografts arising from tumor fragments or bulk tumor cells retain typical characteristics of parent HCCs, we have noted the frequent development of very rapidly-growing tumors that do not share these attributes, and sought to further characterize these tumors. Histopathological analysis of these tumors revealed monomorphic populations of lymphoid cells demonstrating nuclear atypia, high mitotic index, and invasion into surrounding tissues. Flow cytometry and immunohistochemistry revealed that these tumors consisted almost completely of cells expressing the human leukocyte antigen CD45 and the human B-cell antigen CD20. In situ hybridization demonstrated strong expression of the Epstein-Barr virus (EBV)-encoded small RNA (EBER) in all tumors. Staining of tumors for mouse H2K expression was negative in all cases. These findings suggest that the originally implanted human HCC tumor fragments or bulk tumor cells were replaced by human B-cell lymphomas. No lymphomas arose from the implantation of human HCC cells that were sorted to exclude human CD45 + leukocytes. Histopathological re-examination of the parent tumors in all cases confirmed a diagnosis of HCC with no evidence of lymphoma. Analogous to post-transplant lymphoproliferative disorder (PTLD) in humans, our data suggest that these lymphomas spontaneously developed in xenografts through EBV-mediated transformation of EBV-infected passenger lymphocytes harbored in patient tumors in the permissive context of an immunodeficient host environment. Recognition of this phenomenon is important for those utilizing primary human tumor tissue in xenotransplantation assays, because the development of lymphomas may confound accurate analysis of tumor biology and may out-compete the tumor type of interest resulting in the loss of valuable experimental samples. Our observations highlight an important potential pitfall of human solid tumor xenotransplantation assays in models where primary engraftment requires the implantation of bulk cells or tumor fragments which may contain passenger lymphocytes. In such circumstances, our observations underscore the critical importance of routinely quantifying the degree of human lymphocyte “contamination” in xenograft-derived data and implementing strategies to deplete or eliminate human lymphocytes from these assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-58. doi:10.1158/1538-7445.AM2011-LB-58