Novel immunologic tolerance of human cancer cell xenotransplants in zebrafish.

Immune deficiency or suppression in host animals is an essential precondition for the success of cancer cell xenotransplantation because the host immune system has a tendency to reject implanted cells. However, in such animals, the typical tumor microenvironment seen in cancer subjects does not form because of the lack of normal immunity. Here, we developed a novel zebrafish (Danio rerio) model based on 2 rounds of cancer cell xenotransplantation that achieved cancer-specific immunologic tolerance without immunosuppression. We irradiated human cancer cells (PC-3, K562 and HepG2) to abolish their proliferative abilities and implanted them into zebrafish larvae. These cells survived for 2 weeks in the developing host. Three months after the first implantation, the zebrafish were implanted with the same, but nonirradiated, cell lines. These cancer cells proliferated and exhibited metastasis without immune suppression. To reveal the transcriptional mechanism of this immune tolerance, we conducted dual RNA-seq of the tumor with its surrounding tissues and identified several regulatory zebrafish genes that are involved in immunity; the expression of plasminogen activator, urokinase, and forkhead box P3 was altered in response to immunologic tolerance. In conclusion, this xenograft method has potential as a platform for zebrafish-based anticancer drug discovery because it can closely mimic human clinical cancers without inducing immune suppression.

Correlation of pharmacokinetic features and tissue distribution with toxicity of Q39, a hypoxic cell cytotoxic agent.

3-(4-Bromophenyl)-2-(ethylsulfonyl)-6-methyl- quinoxaline 1,4-dioxide (Q39), is one of synthesized tirapazamine (TPZ) analogues that has been investigated preclinically as a hypoxic anticancer candidate. To date, there has not been a study to systematically evaluate the toxicity and pharmacokinetics of Q39. In the present study, we characterized the toxicity profile of Q39 in ICR mice. No toxicities were observed in mice treated with Q39 at dose levels that 168 mg/kg. LD50 value was 257.8 mg/kg (95% confidence interval = 231.1-287.5 mg/kg), which was 3.3-fold higher than that of TPZ. For the plasma pharmacokinetic assessment, a balb/c nude mice bearing K562 leukemia cell xenografted model was introduced and dosed with Q39 intravenous (i.v.)(1.0 mg/kg). Rapid resolution liquid chromatography coupled with tanderm mass spectrometry quantitative detection method (RRLC-MS/MS) was used to quantitatively determine plasma concentration. The RRLC-MS/MS method was validated within the concentration range 25-2000 ng/mL, and the calibration curves were linear with correlation coefficients of > 0.999. Following intravenous administration to nude mice (1.0 mg/kg), plasma concentrations declined rapidly from 1063.3 microg/mL at 10 min to 81.5 microg/mL at 3h. Elimination was triexponential, with T1/2 values of 1.4 h. The CL was 930.0 mL/h/kg, the V(d) was 1.88 L/kg, and the AUC(0-infinity) was 1080.5 ng/mL h. In the tissue distribution assay, concentration of Q39 was higher in the heart, liver, spleen and tumor tissues. The present study offers insights into the toxicological and pharmacologic profiles of Q39 which could help to optimize the dosage of Q39 for the future research and development.

INNO-406, a novel BCR-ABL/Lyn dual tyrosine kinase inhibitor, suppresses the growth of Ph+ leukemia cells in the central nervous system, and cyclosporine A augments its in vivo activity.

Central nervous system (CNS) relapse accompanying the prolonged administration of imatinib mesylate has recently become apparent as an impediment to the therapy of Philadelphia chromosome-positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib mesylate into the cerebrospinal fluid because of the presence of P-glycoprotein at the blood-brain barrier. To overcome imatinib mesylate-resistance mechanisms such as bcr-abl amplification, mutations within the ABL kinase domain, and activation of Lyn, we developed a dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25 to 55 times more potent than imatinib mesylate in vitro and at least 10 times more potent in vivo. The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. We found that INNO-406, like imatinib mesylate, is a substrate for P-glycoprotein. The concentrations of INNO-406 in the CNS were about 10% of those in the plasma. However, this residual concentration was enough to inhibit the growth of Ph+ leukemic cells which expressed not only wild-type but also mutated BCR-ABL in the murine CNS. Furthermore, cyclosporine A, a P-glycoprotein inhibitor, augmented the in vivo activity of INNO-406 against CNS Ph+ leukemia. These findings indicate that INNO-406 is a promising agent for the treatment of CNS Ph+ leukemia.

Effect of antisense VEGF cDNA transfection on the growth of chronic myeloid leukemia K562 cells in vitro and in nude mice.

To further elucidate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of chronic myeloid leukemia (CML), we transfected K562 cells with a VEGF(121)cDNA sense vector (S), an antisense (AS) vector or vector (V) alone. The growth of transfected cells was investigated by MTT and colony-formation assays, and apoptosis was measured by flow cytometry (FCM) of Annexin-V-FITC/PI dual labeled cells. Transfected cells were subcutaneously transplanted into nude mice and the microvessel density (MVD) of tumor masses was determined by vWF immunohistochemistry staining. We tested the supernatant of different transfected K562 cells against human bone marrow endothelial cells (BMECs), and examined the synergic effects of antisense VEGF(121)cDNA and IFNalpha or STI571 on the proliferation and apoptosis of K562 cells. We found that K562/AS transfectants exhibited a 49% reduction in VEGF secretion, whereas K562/S transfectants exhibited a 3-fold increase in VEGF secretion, all in comparison to the vector controls. K562 cells transfected with antisense VEGF(121)cDNA showed growth retardation in vitro. In transplanted nude mice in vivo, transfection of implanted cells with antisense VEGF(121)cDNA resulted in decreased tumor MVD, and increased apoptosis in the presence of IFNalpha. Taken together, these results suggest that VEGF may be involved in the pathogenesis of CML through autocrine and paracrine mechanisms, and that anti-VEGF therapy alone or in combination with conventional treatment may be beneficial for CML patients.

Antitumor activity of a human cytotoxic T-cell line (TALL-104) in brain tumor xenografts.

Malignant glioma in adults and primitive neuroectodermal tumors/medulloblastomas in children are the most common malignant primary brain tumors that either respond poorly to current treatment or tend to recur. Adoptive therapy with TALL-104 cells-an IL-2-dependent, major histocompatibility complex nonrestricted, cytotoxic T-cell line-has demonstrated significant antitumor activity against a broad range of implanted or spontaneously arising tumors. This study investigates distribution of systemically and locally administered TALL-104 cells and their efficacy in effecting survival of a rat model of human brain tumor. In vitro, TALL-104 cells showed significant cytotoxic activity when added to human glioblastoma cell lines U-87 MG, U-251 MG, and A1690; the medulloblastoma cell lines DAOY, D283 Med, and D341 Med; and the epidermoid cancer cell line A431. In brain tumor-bearing rats, the amount of fluorescent dye-labeled TALL-104 cells in brain increased after they were given by intracarotid injection as compared with i.v. cell administration. However, TALL-104 cells rapidly decreased to low levels within 1 h after intracarotid injection. This finding suggests that TALL-104 cells given systemically may not invade brain or tumor tissues, but rather may remain in the vascular system, making this approach less efficient for brain tumor treatment. In a model of athymic rats engrafted with human A431 carcinoma brain tumor, repetitive local administration of TALL-104 cells directly into the tumor bed resulted in a significant increase in survival time compared with control animals. Therefore, local therapy with TALL-104 cells may be a novel and highly effective treatment approach for malignant brain tumors.

Technology evaluation: leukemia therapy, University of Pennsylvania.

The University of Pennsylvania is developing an antisense oligonucleotide (AS ON) as a potential treatmentfor myelogenous leukemia. The 24-mer phosphorothioate (PS) ON targets the c-myb gene (codons 2 to 9), a regulator of transcription. In a pilot study, patient bone marrow was purged with the PS ON before being returned to the patient. In January 1997, it was reported that out of six evaluable patients, four demonstrated marked hematological remission with normalized white blood cell counts. A second phase I trial was initiated, in which myelogenous leukemia patients were treated with systemic infusions of the PS ON at doses of 0.3 to 2.0 mg/kg/dayfor 7 days. By January 1997, 18 patients had been treated, 12 showed stable disease and one patient in blast crisis experienced a transient reversal to the chronic phase of the disease. No dose-related toxicity was noted and c-myb mRNA and protein levels were halved. Preclinical studies in leukemic mice showed that the myb AS PS ON increased survival times 2- to 4-fold and reduced leukemic proliferation in the brain [229790]. The ON was originally developed and patented at Temple University and was being jointly developed by Lynx Therapeutics, however, this collaboration was terminated in 1996 [264351]. New phase I studies are starting in 1999, with INX-3001 (University of Pennsylvania c-myb AS PS) supported by the NIH and Inex Pharmaceuticals Corporation.

Antitumor activity of human umbilical cord blood cells: A comparative analysis with peripheral blood and bone marrow cells.

Although the hematopoietic reconstituting ability of human umbilical cord blood cells (UCBC) is well documented, their antitumor cytotoxic potential has not been well studied. Therefore, UCBC were compared to normal peripheral blood stem cells (PBSC) and bone marrow (BM) stem cell harvests for cytomorphology, antitumor cytotoxic activity before and after ex vivo cytokine manipulation, response to T and B cell mitogens, expression of adhesion molecules and immunophenotypes using flow cytometry, cytokine production and in vivo antitumor activity. BM and PBSC, but not UCBC, did not form cellular clusters in culture. More cytotoxic granules were present in the cytoplasm of UCBC than PBSC following activation in vitro. Ex vivo manipulation of UCBC with cytokines produced more cytotoxicity to K562 and Raji tumor cells than PBSC or BM (p<0.001). Most cytotoxic cells in UCBC cultures were T lymphocytes, and a correlation existed between the number of CD56+ cells and cytotoxicity levels, particularly after in vitro activation with interleukin-2. No significant difference in adhesion molecule expression was noted among UCBC, PBSC and BM cells. However, there was a significantly decreased expression of CD54 molecules (ICAM) on UCBC compared to PBSC (p<0.05). IL-2 activated UCBC showed significant antitumor effects against K562 leukemic cells grown in SCID mice. Thus UCBC contained more antitumor effector cells and precursors than cells from marrow or peripheral blood cells which might be capable of providing a therapeutic effect.