Intramuscular Transplantation Improves Engraftment Rates for Esophageal Patient-Derived Tumor Xenografts.

BACKGROUND: Recently, there has been an increase in the availability of targeted molecular therapies for cancer treatment. The application of these approaches to esophageal cancer, however, has been hampered by the relative lack of appropriate models for preclinical testing. Patient-derived tumor xenograft (PDTX) models are gaining popularity for studying many cancers. Unfortunately, it has proven difficult to generate xenografts from esophageal cancer using these models. The purpose of this study was to improve the engraftment efficiency of esophageal PDTXs. METHODS: Fresh pieces of esophageal tumors obtained from endoscopic biopsies or resected specimens were collected from 23 patients. The tumors were then coated in Matrigel and transplanted in immunocompromised mice subcutaneously (n = 6) and/or using a novel implantation technique whereby the tumor is placed in a dorsal intramuscular pocket (n = 18). They are then monitored for engraftment. RESULTS: With the novel intramuscular technique, successful engraftment was achieved for all 18 patient tumors. Among these PDTXs, 13 recapitulated the original patient tumors with respect to degree of differentiation, molecular and genetic profiles, and chemotherapeutic response. Lymphomatous transformation was observed in the other five PDTXs. Successful engraftment was achieved for only one of six patient tumors using the classic subcutaneous approach. DISCUSSION: We achieved a much higher engraftment rate of PDTXs using our novel intramuscular transplant technique than has been reported in other published studies. It is hoped that this advancement will help expedite the development and testing of new therapies for esophageal cancer.

Imaging Bradykinin B1 Receptor with 68Ga-Labeled [des-Arg10]Kallidin Derivatives: Effect of the Linker on Biodistribution and Tumor Uptake.

Bradykinin B1 receptor (B1R) that is overexpressed in cancers but minimally expressed in normal healthy tissues represents an attractive biomarker for the development of cancer imaging agents. The goal of this study was to evaluate the effect of different linkers on the pharmacokinetics and tumor uptake of a B1R-targeting radio-peptide sequence, 68Ga-DOTA-linker-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu. Four peptides, SH01078, P03034, P04115, and P04168, with 6-aminohexanoic acid, 9-amino-4,7-dioxanonanoic acid, Gly-Gly, and 4-amino-(1-carboxymethyl)piperidine, respectively, as the linker were synthesized and evaluated. In vitro competition binding assays showed that the Ki values of SH01078, P03034, P04115, and P04168 were 27.8±4.9, 16.0±1.9, 11.4±2.5, and 3.6±0.2 nM, respectively. Imaging and biodistribution studies were performed in mice bearing both B1R-positive HEK293T::hB1R and B1R-negative HEK293T tumors. All tracers showed mainly renal excretion with excellent tumor visualization and minimal background activity except for kidneys and bladder. The average uptake of 68Ga-labeled SH01078, P03034, and P04115 in HEK293T::hB1R tumor was similar (1.96-2.17%ID/g) at 1 h postinjection. 68Ga-P04168 generated higher HEK293T::hB1R tumor uptake (4.15±1.13%ID/g) and lower background activity, leading to a >2-fold improvement in HEK293T::hB1R tumor-to-background (HEK293T tumor, blood, muscle, and liver) contrasts over those of 68Ga-labeled SH01078, P03034, and P04115. Our results indicate that the choice of linker affects binding affinity, pharmacokinetics, and tumor targeting. The use of the cationic 4-amino-(1-carboxymethyl)piperidine linker improved tumor visualization, and the resulting 68Ga-P04168 might be promising for clinical application for imaging B1R-expressing tumors with positron emission tomography.

The IL-2/IL-2R system: from basic science to therapeutic applications to enhance immune regulation.

IL-2 plays a critical role in the normal function of the immune system. A trophic factor for lymphocytes, IL-2 is required for mounting and sustaining adaptive T cell responses; however, IL-2 is also critical for immune regulation via its effects on regulatory T cells (Treg cells). Over the years, we have contributed to the understanding of the biology of IL-2 and its signaling through the IL-2 receptor and helped define the key role played by IL-2 in Treg development and function. Our data show that Treg cells have a heightened sensitivity to IL-2, which may create a therapeutic window to promote immune regulation by selective stimulation of Treg cells. We are now developing new efforts to translate this knowledge to the clinical arena, through our focused interest in Type 1 diabetes as a prototypic autoimmune disease. Specifically, we aim at developing IL-2-based therapeutic regimens and incorporate means to enhance antigen-specific Treg responses, for improved and more selective regulation of islet autoimmunity. In parallel, we are pursuing studies in preclinical models of autoimmunity and transplantation to define critical factors for successful adoptive Treg therapy and develop clinically applicable therapeutic protocols.

SOX2 expression associates with stem cell state in human ovarian carcinoma.

The SRY-related HMG-box family of transcription factors member SOX2 regulates stemness and pluripotency in embryonic stem cells and plays important roles during early embryogenesis. More recently, SOX2 expression was documented in several tumor types including ovarian carcinoma, suggesting an involvement of SOX2 in regulation of cancer stem cells (CSC). Intriguingly, however, studies exploring the predictive value of SOX2 protein expression with respect to histopathologic and clinical parameters report contradictory results in individual tumors, indicating that SOX2 may play tumor-specific roles. In this report, we analyze the functional relevance of SOX2 expression in human ovarian carcinoma. We report that in human serous ovarian carcinoma (SOC) cells, SOX2 expression increases the expression of CSC markers, the potential to form tumor spheres, and the in vivo tumor-initiating capacity, while leaving cellular proliferation unaltered. Moreover, SOX2-expressing cells display enhanced apoptosis resistance in response to conventional chemotherapies and TRAIL. Hence, our data show that SOX2 associates with stem cell state in ovarian carcinoma and induction of SOX2 imposes CSC properties on SOC cells. We propose the existence of SOX2-expressing ovarian CSCs as a mechanism of tumor aggressiveness and therapy resistance in human SOC.

The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: involvement of interleukin-2 system.

Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell-cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1ß, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the ß and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth.

Aurora kinase A-specific T-cell receptor gene transfer redirects T lymphocytes to display effective antileukemia reactivity.

Aurora kinase A (AURKA) is overexpressed in leukemias. Previously, we demonstrated that AURKA-specific CD8(+) T cells specifically and selectively lysed leukemia cells, indicating that AURKA is an excellent target for immunotherapy. In this study, we examined the feasibility of adoptive therapy using redirected T cells expressing an HLA-A*0201-restricted AURKA(207-215)-specific T-cell receptor (TCR). Retrovirally transduced T cells recognized relevant peptide-pulsed but not control target cells. Furthermore, TCR-redirected CD8(+) T cells lysed AURKA-overexpressing human leukemic cells in an HLA-A*0201-restricted manner, but did not kill HLA-A*0201(+) normal cells, including hematopoietic progenitors. In addition, AURKA(207-215)-specific TCR-transduced CD4(+) T cells displayed target-responsive Th1 cytokine production. Finally, AURKA(207-215)-specific TCR-transduced CD8(+) T cells displayed antileukemia efficacy in a xenograft mouse model. Collectively, these data demonstrate the feasibility of redirected T cell-based AURKA-specific immunotherapy for the treatment of human leukemia.

Idiopathic CD4+ T lymphopenia without autoimmunity or granulomatous disease in the slipstream of RAG mutations.

A girl presented during childhood with a single course of extensive chickenpox and moderate albeit recurrent pneumonia in the presence of idiopathic CD4+ T lymphocytopenia (ICL). Her clinical condition remained stable over the past 10 years without infections, any granulomatous disease, or autoimmunity. Immunophenotyping demonstrated strongly reduced naive T and B cells with intact proliferative capacity. Antibody reactivity on in vivo immunizations was normal. T-cell receptor-Vß repertoire was polyclonal with a very low content of T-cell receptor excision circles (TRECs). Kappa-deleting recombination excision circles (KRECs) were also abnormal in the B cells. Both reflect extensive in vivo proliferation. Patient-derived CD34+ hematopoietic stem cells could not repopulate RAG2(-/-)IL2Rγc(-/-) mice, indicating the lymphoid origin of the defect. We identified 2 novel missense mutations in RAG1 (p.Arg474Cys and p.Leu506Phe) resulting in reduced RAG activity. This report gives the first genetic clue for ICL and extends the clinical spectrum of RAG mutations from severe immune defects to an almost normal condition.

A complement-dependent cytotoxicity-enhancing anti-CD20 antibody mediating potent antitumor activity in the humanized NOD/Shi-scid, IL-2Rγ(null) mouse lymphoma model.

Engineering the Fc region of monoclonal antibodies (mAb) in order to enhance effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC) is likely to a be promising approach for next-generation mAb therapy. Here, we report on such an antibody, 113F, a novel CDC-enhancing variant of rituximab, and determine the tumor-associated factors influencing susceptibility to 113F-induced CDC. The latter included the quantity of complement inhibitors present, such as CD55 and CD59. We report that compared to rituximab, 113F mediated highly enhanced CDC against primary CD20-expressing lymphoma cells in vitro. Currently, a major problem in the field of immunotherapy research is the lack of suitable small animal models to evaluate human CDC in vivo. Therefore, we established a novel human tumor-bearing NOD/Shi-scid, IL-2Rγ(null) mouse model, in which human complement functions as the CDC mediator. We demonstrated that rituximab exerted significant antitumor effects via human CDC in this humanized mouse. The finding of specific localization of human C1q on CD20-expressing tumor cell membranes was consistent with the observation that human CDC indeed contributed to the antitumor effect in this model. Moreover, 113F exerted significantly more potent antitumor effects than rituximab in this in vivo model. The detection of more abundant dense signals from C1q using 113F compared to rituximab was consistent with the concept that this reagent represented a CDC-enhancing mAb. In the near future, the efficacy of this type of CDC-enhancing antibody will be determined in clinical trials in humans.

Multiple receptor tyrosine kinases regulate HIF-1alpha and HIF-2alpha in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors.

Novel treatment approaches are needed for children with advanced neuroblastoma. Studies with neuroblastoma cells have indicated the presence of a hypoxia-driven vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-1 autocrine loop modulating hypoxia-inducible factor-1alpha (HIF-1alpha). Whether other receptor tyrosine kinases (RTKs) are capable of modulating HIF-1alpha levels and whether RTKs can regulate HIF-2alpha as well is largely unknown. We evaluated neuroblastoma cell lines for expression of various RTKs. Although cell lines were heterogeneous in the expression of VEGFR-1, -3, c-Kit and RET, most cells expressed PDGFR-alpha and -beta. Ligand-induced activation of multiple RTKs upregulated HIF-1alpha levels, whereas activation of VEGFR-1 alone upregulated HIF-2alpha. Multitargeted tyrosine kinase inhibitor sunitinib reduced hypoxia-induced rises in HIF-1alpha and HIF-2alpha through mechanisms involving effects on both mRNA levels and protein stability. In addition, sunitinib and sorafenib had direct effects on tumor cell viability in vitro. In a neuroblastoma xenograft model, tumor growth inhibition by sunitinib was associated with inhibition of angiogenesis and reduced HIF-1alpha levels. These findings show that multiple RTKs may regulate the HIF axis in normoxia and hypoxia and suggest that multikinase inhibitors may exert antiangiogenic effects not only by direct effects on endothelial cells, but also by blocking compensatory hypoxia- and ligand-induced changes in HIF-1alpha and HIF-2alpha.

Zinc signals promote IL-2-dependent proliferation of T cells.

Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T-cell activation by IL-2 was investigated in the murine cytotoxic T-cell line CTLL-2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin-3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL-2-receptor. Activation of the ERK-pathway was blocked by chelation of free zinc with N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK-specific gene expression of c-fos and IL-2-induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK-dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL-2-induced ERK signaling and proliferation of T cells.

Irrespective of CD34 expression, lineage-committed cell fraction reconstitutes and re-establishes transformed Philadelphia chromosome-positive leukemia in NOD/SCID/IL-2Rgammac-/- mice.

Stem cells of acute myeloid leukemia (AML) have been identified as immunodeficient mouse-repopulating cells with a Lin(-)CD34(+)38(-) phenotype similar to normal hematopoietic stem cells. To identify the leukemia-propagating stem cell fraction of Philadelphia chromosome-positive (Ph(+)) leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n = 3) or Ph(+) acute lymphoblastic leukemia (n = 3) into NOD/SCID/IL-2Rgammac(-/-) mice. Engrafted cells were almost identical to the original leukemia cells as to phenotypes, IGH rearrangements, and karyotypes. CD34(+)CD38(-)CD19(+), CD34(+)38(+)CD19(+), and CD34(-)CD38(+)CD19(+) fractions could self-renew and transfer the leukemia, whereas the CD34(-)CD38(+)CD19(+) fraction did not stably propagate in NOD/SCID mice. These findings suggest that leukemia-repopulating cells in transformed Ph(+) leukemia are included in a lineage-committed but multilayered fraction, and that CD34(+) leukemia cells potentially emerge from CD34(-) populations.

CD27 sustains survival of CTLs in virus-infected nonlymphoid tissue in mice by inducing autocrine IL-2 production.

Immunity to infections relies on clonal expansion of CD8+ T cells, their maintenance as effector CTLs, and their selection into a memory population. These processes rely on delivery of survival signals to activated CD8+ T cells. We here reveal the mechanism by which costimulatory CD27-CD70 interactions sustain survival of CD8+ effector T cells in infected tissue. By unbiased genome-wide gene expression analysis, we identified the Il2 gene as the most prominent CD27 target gene in murine CD8+ T cells. In vitro, CD27 directed IL-2 expression and promoted clonal expansion of primed CD8+ T cells exclusively by IL-2-dependent survival signaling. In mice intranasally infected with influenza virus, Cd27-/- CD8+ effector T cells displayed reduced IL-2 production, accompanied by impaired accumulation in lymphoid organs and in the lungs, which constitute the tissue effector site. Reconstitution of Cd27-/- CD8+ T cells with the IL2 gene restored their accumulation to wild-type levels in the lungs, but it did not rescue their accumulation in lymphoid organs. Competition experiments showed that the IL-2 produced under the control of CD27 supported effector CD8+ T cell survival in the lungs in an autocrine manner. We conclude that CD27 signaling directs the IL-2 production that is reportedly essential to sustain survival of virus-specific CTLs in nonlymphoid tissue.

Novel role for interleukin-2 receptor-Jak signaling in retrovirus transmission.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma, and it encodes a number of nonstructural proteins that are involved in virus replication and immune evasion. The viral protein p12 previously has been characterized to interfere with major histocompatibility complex class, ICAM-1, and ICAM-2 expression, and it activates STAT5. Using a previously established T-cell line immortalized with an HTLV-1 molecular clone deleted for p12, we assessed the role of p12 in regulating cellular growth and virus transmission. These cells were complemented for p12 expression by the transduction of a lentivirus vector expressing p12. We report that p12 conferred a selective growth advantage in vitro and increased the colony formation of human T cells in soft-agar assays. Consistently with previous studies, p12- and p12+ cell lines produced similar amounts of virus particles released into the supernatant of cultured cells, although we found that p12 expression greatly enhanced virus transmission. Moreover, we found that interleukin-2 (IL-2) stimulation also increased HTLV-1 transmission whether p12 was expressed or not, and inversely, that the inhibition of Jak signaling significantly reduced HTLV-1 transmission. Intriguingly, IL-2/Jak signaling was not associated with changes in viral gene expression, viral RNA encapsidation, the maturation of the virus particle, cell-cell adherence, or Gag polarization and virological synapse formation. We do demonstrate, however, that IL-2 stimulation and p12 expression significantly increased the rate of syncytium formation, revealing a novel role for IL-2 signaling and Jak activation in HTLV-1 virus transmission.

Identification and preclinical characterization of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway.

Tropomyosin-related kinases (TrkA, TrkB, and TrkC) are receptor tyrosine kinases that, along with their ligands, the neurotrophins, are involved in neuronal cell growth, development, and survival. The Trk-neurotrophin pathway may also play a role in tumorigenesis through oncogenic fusions, mutations, and autocrine signaling, prompting the development of novel Trk inhibitors as agents for cancer therapy. This report describes the identification of AZ-23, a novel, potent, and selective Trk kinase inhibitor. In vitro studies with AZ-23 showed improved selectivity over previous compounds and inhibition of Trk kinase activity in cells at low nanomolar concentrations. AZ-23 showed in vivo TrkA kinase inhibition and efficacy in mice following oral administration in a TrkA-driven allograft model and significant tumor growth inhibition in a Trk-expressing xenograft model of neuroblastoma. AZ-23 represents a potent and selective Trk kinase inhibitor from a novel series with the potential for use as a treatment for cancer.

Cannabinoid receptor 1 is a potential drug target for treatment of translocation-positive rhabdomyosarcoma.

Gene expression profiling has revealed that the gene coding for cannabinoid receptor 1 (CB1) is highly up-regulated in rhabdomyosarcoma biopsies bearing the typical chromosomal translocations PAX3/FKHR or PAX7/FKHR. Because cannabinoid receptor agonists are capable of reducing proliferation and inducing apoptosis in diverse cancer cells such as glioma, breast cancer, and melanoma, we evaluated whether CB1 is a potential drug target in rhabdomyosarcoma. Our study shows that treatment with the cannabinoid receptor agonists HU210 and Delta(9)-tetrahydrocannabinol lowers the viability of translocation-positive rhabdomyosarcoma cells through the induction of apoptosis. This effect relies on inhibition of AKT signaling and induction of the stress-associated transcription factor p8 because small interfering RNA-mediated down-regulation of p8 rescued cell viability upon cannabinoid treatment. Finally, treatment of xenografts with HU210 led to a significant suppression of tumor growth in vivo. These results support the notion that cannabinoid receptor agonists could represent a novel targeted approach for treatment of translocation-positive rhabdomyosarcoma.

Pharmacokinetics and immunodynamics of basiliximab in pediatric renal transplant recipients on mycophenolate mofetil comedication.

BACKGROUND: The aim of this substudy within a prospective, multicenter, placebo-controlled trial was to assess the pharmacokinetics and immunodynamics of basiliximab in pediatric renal transplant recipients on comedication with mycophenolate mofetil (MMF). METHODS: Eighty-two patients aged 3 to 18 years, receiving cyclosporine microemulsion, MMF, corticosteroids, and basiliximab or placebo were investigated. Basiliximab serum concentrations were determined by ELISA, CD25+, and CD122+ T lymphocytes by flow cytometry. RESULTS: Basiliximab clearance adjusted to body surface area was significantly (P<0.05) greater in children versus adults, but the relatively higher basiliximab dose given to children yielded similar exposure compared with adolescents. A cross-study comparison revealed that MMF reduced basiliximab clearance and prolonged CD25 saturation duration from approximately 5 weeks in the absence of MMF to 10 weeks in the presence of MMF. Basiliximab led to a marked reduction of CD25+ T-cell fraction during the first 8 to 10 weeks posttransplant, but did not specifically affect CD122+ T cells. The majority of biopsy-proven acute rejection episodes (BPAR) were observed after interleukin (IL) 2-R desaturation, whereas about a quarter of BPARs occurred despite adequate IL2-R blockade. CONCLUSIONS: The currently recommended basiliximab dose for pediatric patients, when used with cyclosporine microemulsion and corticosteroids, yielded adequate drug exposure in children and adolescents also under MMF comedication. The observation that about a quarter of BPARs occurred despite adequate IL2-R blockade suggests that another T-cell activation pathway independent of the IL-2/IL-2R pathway is operative, for example, the IL-15 signaling pathway.

HTLV-1 and apoptosis: role in cellular transformation and recent advances in therapeutic approaches.

A universal cellular defense mechanism against viral invasion is the elimination of infected cells through apoptotic cell death. To counteract host defenses many viruses have evolved complex apoptosis evasion strategies. The oncogenic human retrovirus HTLV-1 is the etiological agent of adult-T-cell leukemia/lymphoma (ATLL) and the neurodegenerative disease known as HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The poor prognosis in HTLV-1-induced ATLL is linked to the resistance of neoplastic T cells against conventional therapies and the immuno-compromised state of patients. Nevertheless, several studies have shown that the apoptotic pathway is largely intact and can be reactivated in ATLL tumor cells to induce specific killing. A better understanding of the molecular mechanisms employed by HTLV-1 to counteract cellular death pathways remains an important challenge for future therapies and the treatment of HTLV-1-associated diseases.