Hiro Wakasugi

Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell–dependent antitumor effects

Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell-dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-gamma production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.

Vasculogenic mimicry and pseudo‐comedo formation in breast cancer

Tumors require a blood supply for growth and hematogenous metastases. Until recently, most research in this area has focused on the role of angiogenesis, the recruitment of new vessels into a tumor from preexisting vessels. Previously, in a study of breast cancer (IBC), in which we used established inflammatory breast cancer (IBC) xenografts (WIBC‐9) originating from a patient with IBC (Shirakawa et al., Cancer Res 2001:61:445–451), we reported observing vasculogenic mimicry (VM), a condition in which bloodstreams within cancer tissue are not accompanied by a lining of endothelial cells (ECs) (Shirakawa et al., Cancer Res 2002:62:560–566). In the present study, we examined 331 surgically resected breast cancer specimens for evidence of VM, using immunohistochemistry and laser‐captured microdissection (LCM) followed by nested reverse transcriptase polymerase chain reaction (RT‐PCR). Surprisingly, 7.9% (26 specimens) of the 331 specimens exhibited evidence of VM. Of these 26 VM specimens, 84.6% (22 specimens) exhibited pseudo‐comedo formation. RT‐PCR analysis of 8 microdissected typical VM specimens revealed expression of Tie‐2, Flt‐1, thrombin receptor and CD31 in 63, 50, 0 and 0% of specimens, respectively. In contrast, results of RT‐PCR analysis of 8 specimens from non‐VM tumors were negative for expression of these genes. The 26 VM cases tended to have a higher percentage of hematogenous recurrence (p = 0.059) and a lower percentage of 5‐year survival (p = 0.071) than the 305 non‐VM cases. However, there were no significant differences in tumor size, lymph node metastasis, estrogen receptors or progesterone receptors between the 2 groups (p > 0.1). Our results suggest that the existence of VM increases the likelihood of hematogenous metastases and is in inverse proportion to prognosis.

Elevated serum level of thioredoxin in patients with hepatocellular carcinoma.

Thioredoxin (TRX) is known to contain an active site with aredox-active disulfide and has various biological activities. The objectiveof the present study was to investigate whether circulating TRX levels areelevated in patients with chronic hepatitis (CH) or liver cirrhosis (LC) andhepatocellular carcinoma (HCC). An anti-TRX monoclonal antibody andpolyclonal antibodies that specifically recognize TRX, were generated andused for the development of an ELlSA system to measure TRX levels in humanserum. The geometric mean and its 95% confidence interval of serumlevel of TRX in healthy volunteers was 81.75 ng/ml (74.60-89.59 ng/ml). Theserum level of TRX in LC/CH patients without HCC was 80.87 ng/ml(69.66-93.88 ng/ml). The value was not statistically different from that inserum from normal volunteers (p=0.69). In contrast, the serum level of TRXin patients with HCC was 147.35 ng/ml (125.53-1 72.96 ng/ml), which wassignificantly higher when compared with the level in serum of normalvolunteers (p<0.001) and in serum of LC/CH patients without HCC(p<0.001). In four patients with HCC, the initially high level of serum TRX(>150 ng/ml) decreased below 150 ng/ml after surgical removal of the tumor.The data reported herein revealed that patients with HCC had a significantlyelevated serum level of TRX, suggesting that measurement of serum of TRXmight be a useful clinical parameter when HCC is suspected.

IL-4 confers NK stimulatory capacity to murine dendritic cells: a signaling pathway involving KARAP/DAP12-triggering receptor expressed on myeloid cell 2 molecules.

Dendritic cells (DC) regulate NK cell functions, but the signals required for the DC-mediated NK cell activation, i.e., DC-activated NK cell (DAK) activity, remain poorly understood. Upon acute inflammation mimicked by LPS or TNF-α, DC undergo a maturation process allowing T and NK cell activation in vitro. Chronic inflammation is controlled in part by Th2 cytokines. In this study, we show that IL-4 selectively confers to DC NK but not T cell stimulatory capacity. IL-4 is mandatory for mouse bone marrow-derived DC grown in GM-CSF (DCGM/IL-4) to promote NK cell activation in the draining lymph nodes. IL-4-mediated DAK activity depends on the KARAP/DAP12-triggering receptor expressed on myeloid cell 2 signaling pathway because: 1) gene targeting of the adaptor molecule KARAP/DAP12, a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif, suppresses the DCGM/IL-4 capacity to activate NK cells, and 2) IL-4-mediated DAK activity is significantly blocked by soluble triggering receptor expressed on myeloid cell 2 Fc molecules. These data outline a novel role for Th2 cytokines in the regulation of innate immune responses through triggering receptors expressed on myeloid cells.

Tumor‐infiltrating endothelial cells and endothelial precursor cells in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a specific type of breast tumor that generally has a poor prognosis, in spite of recent advances in treatment. In the present study, semiquantitative reverse transcriptase polymerase chain reaction examination of resected specimens showed that angiogenic factors, not lymphangiogenic factors, are overexpressed in IBC tumors, compared with non‐IBC tumors. Immunohistochemical analysis of the specimens revealed a significantly higher population of tumor‐infiltrating (TI) endothelial cells (ECs) or endothelial precursor cells (EPCs) in tumor‐associated stroma of IBC specimens than in non‐IBC specimens. In a previous study, we examined the phenotype of host cells in response to transplanted IBC cells, using an established human IBC xenograft model (WIBC‐9) (Shirakawa et al., Cancer Res 2001;61:445–51). The data obtained in that study are consistent with the findings of the present study. To explore the therapeutic potential of blocking vascular endothelial growth factor (VEGF) and angiopoietin (Ang) pathways in IBC, established vectors encoding soluble Flt‐1 (sFlt‐1) and soluble Tie2 (sTie2) were injected directly into WIBC‐9. Both vectors produced growth inhibition ratios of WIBC‐9 that were significantly higher than those of a non‐IBC xenograft (MC‐5). Also, both vectors suppressed WIBC‐9 lung metastases. The efficacy correlated with the number of TI ECs/EPCs, which was determined by fluorescence‐activated cell sorting. These ECs/EPCs incorporated acetylated lipoprotein and were integrated within a HUVEC monolayer in vitro culture on day 5.

Inflammatory breast cancer: Vasculogenic mimicry and its hemodynamics of an inflammatory breast cancer xenograft model

We recently established a new human inflammatory breast cancer (IBC) xenograft (WIBC-9) originating from a patient with IBC. The original tumor and WIBC-9 revealed invasive ductal carcinoma with a hypervascular structure of solid nests and marked lymphatic permeation in the overlying dermis. In the central part of the solid nests, vasculogenic mimicry, which showed an absence of endothelial cells, was observed. Comparison of WIBC-9 with an established non-IBC xenograft (MC-5), using time-course dynamic micro-magnetic resonance angiography analysis (with a newly developed intravascular macromolecular contrast agent for magnetic resonance imaging) demonstrated that the WIBC-9 tumor had blood flow and a vascular mimicry–angiogenesis junction.

The Role of PGE2 in the Differentiation of Dendritic Cells: How Do Dendritic Cells Influence T‐Cell Polarization and Chemokine Receptor Expression?

The role of prostaglandin E2 (PGE2) in the function of dendritic cells (DCs), T‐cell polarization, and expression of chemokine receptors was evaluated in human cells. Immature DCs were generated from peripheral blood CD14+ cells using a combination of GM‐CSF and interleukin‐4 (IL‐4) with or without PGE2. On day 6, maturation of DCs was induced by the addition of tumor necrosis factor alpha with or without PGE2. DCs harvested on day 6 (immature DCs) or day 9 (mature DCs) were examined using functional assays. In the presence of PGE2, immature and mature DCs showed, phenotypically, a lower expression of CD1a and, functionally, a higher allostimulatory capacity at a high DC/T‐cell ratio than control cells cultured in the absence of PGE2. DCs cultured in the presence of PGE2 induced the differentiation of naïve T cells toward a helper T‐cell type 1 (Th1) response, which was independent of IL‐12 secretion in the basal state despite a slightly lower interferon gamma secretion compared with control cells. However, the function of cytotoxicity‐stimulating autologous T cells was not augmented by the addition of PGE2. Immature DCs expressed the inflammatory chemokine receptors, CCR1 and CXCR4, but not CCR6, regardless of the presence or absence of PGE2. Mature DCs expressed CCR7 equally, measured using a migration test and the measurement of calcium flux with macrophage inflammatory protein‐3β and reverse transcription‐polymerase chain reaction assay in all of the groups. All of these findings suggest that PGE2 affects the DC‐promoted differentiation of naïve T cells to a Th1 response in the basal state, without affecting chemokine receptor expression on DCs.

Antithymocyte globulin affects the occurrence of acute and chronic graft-versus-host disease after a reduced-intensity conditioning regimen by modulating mixed chimerism induction and immune reconstitution

Background. There have been no detailed analyses of the induction of donor cell–type chimerism, the onset and incidence of acute and chronic graft-versus-host disease (GVHD), and the immune recovery kinetics after reduced-intensity stem cell transplantation (RIST). Methods. To address these, with particular emphasis on the impact of the use of antithymocyte globulin (ATG) in RIST, we compared 39 consecutively registered patients who underwent RIST from an HLA-matched related donor and 33 patients who underwent conventional marrow-ablative transplantation. Results. The incidences of grades II to IV acute and chronic GVHD tended to be less in RIST with ATG than in either RIST without ATG or conventional marrow-ablative transplantation. In a multivariate analysis, the predictive factors for acute and chronic GVHD included, respectively, ATG and grades II to IV acute GVHD. In a chimerism analysis, the achievement of complete donor chimera in T-cell lineage was delayed in RIST without ATG compared with RIST with ATG (P =0.038), which might explain the observed delayed onset of acute GVHD in RIST with ATG compared with the other two regimens. The ratio of type 1 and 2 dendritic cells did not affect the development of GVHD, whereas the number of naive CD4+ T cells did. No difference was observed in the incidence of clinically definitive infection, including cytomegalovirus, among the three cohorts, regardless of the use of ATG. Conclusions. We suggest that the conditioning regimen and immunosuppressive strategy after RIST should be carefully balanced against the risk of GVHD and of relapse of the basic disorder caused by the lack of a graft-versus-leukemia benefit.

Quinolone-induced differential modification of IL-1α and IL-1β production by LPS-stimulated human monocytes

Abstract We previously reported that ciprofloxacin (Cip), a quinoline-derivative antibiotic, decreases the biological activity of IL-1 released by LPS-stimulated monocytes after 24 hr of culture without affecting cell-associated IL-1 activity. To analyze further the effects of Cip on LPS-induced IL-1α and IL-1β synthesis, each species was measured in the supernatants and cell lysates of monocyte cultures over a 4-day period using IL-1α and IL-1β-specific ELISA methods. Cip had a post-transcriptional differential effect on the production of IL-1α and IL-1β, reducing the total amount of IL-1β produced by LPS-stimulated monocytes, while that of IL-1α was unaffected. In addition, the production of both species was delayed. These findings explain the discrepancy between the Cip-induced alteration of extracellular IL-1 activity and the preservation of cellassociated activity. Cip is, to our knowledge, the first pharmacological agent found to have a differential effect on the synthesis of IL-1α and IL-1β. It may form the basis for new pharmacological agents capable of selectively reducing the systemic effects of IL-1 without affecting local activity.

T-cell-conditioned medium efficiently induces the maturation and function of human dendritic cells

We present evidence that T‐cell‐conditioned media (TCCM) can efficiently induce human immature dendritic cells (DC) to express high levels of immune accessory molecules commonly found on mature DC. TCCM prepared from cell‐free supernatants of anti‐CD3‐activated T cells contained several soluble factors including CD40‐ligand (sCD40L), TNF‐α, and IFN‐γ. In contrast to moderate up‐regulation of costimulatory molecules by the addition of individual cytokines or monocyte‐conditioned medium, treatment of immature DC with TCCM induced a marked increase in the expression of costimulatory molecules in a dose‐dependent manner. The ability of TCCM to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for CD40L, TNF‐α, and IFN‐γ, indicating that these factors present in TCCM are mainly implicated in the maturation of DC. Importantly, TCCM‐treated DC can produce significantly higher levels of IL‐12 and are highly effective stimulators in allogenenic and autologous mixed‐lymphocyte reactions. Overall, these findings show that cultivation with TCCM is an efficient approach for the induction of mature DC that should be useful in eliciting antigen‐specific immune responses against cancer and viruses.