Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma

Mitigation of regulatory T cell-mediated immunosuppression and elicitation of immunogenic tumor cell death are crucial events for optimal anti-tumor immune activity in vivo. This study was designed to investigate the potential synergistic activity of the combined use of cyclophosphamide (CP) and doxorubicin (DR), both of which are known to resolve these two issues. BALB/c mice were inoculated subcutaneously with CT-26 carcinoma cells in the bilateral flank and treated with an intraperitoneal injection of a low dose of CP followed by an intratumoral injection of DR into one side of the tumor. We found that, in addition to a significant suppression of growth on the DR-treated side of the tumor, combination therapy suppressed the growth of DR-untreated remote tumors in both tumor-specific and T cell-dependent manners. Mitomycin C showed no such synergistic anti-tumor activity with CP treatment. Combination therapy increased the frequency of interferon (IFN)-γ-producing T lymphocytes specific to a CT-26-associated class I-binding tumor peptide in the tumor-draining lymph nodes. Real-time PCR analysis revealed that combination therapy led to an increase in IFN-γ and tumor necrosis factor-α mRNA expression; however, levels of Foxp3 and transforming growth factor-β within the remote tumor tissues were decreased. In addition, knock down of calreticulin expression in CT-26 cells using small interfering RNA attenuated anti-tumor vaccine effects induced by DR-treated CT-26 cells. These results provide an immunological rationale for the combined use of chemotherapeutic drugs, i.e., CP and DR, and further recommend their use with current cancer vaccines.     

IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.     

Influence of Host Cell Infiltration on the Glycolipid Content of Mouse Brain Tumors

Abstract: Previous studies showed that levels of some glycosphingolipids (GSLs) expressed in solid brain tumors grown in vivo were reduced or undetectable in cultured cells prepared from the tumors. This phenomenon has been attributed either to suppressed glycolipid synthesis from unknown forces of the tissue culture environment or to the absence of host cells that normally infiltrate the solid tumors growing in vivo. To test further the host cell hypothesis, we examined host cell markers in two experimental mouse brain tumors, the ependymoblastoma and the CT-2A, that were grown as subcutaneous solid tumors in the flank of C57BL/6J (B6) mice or as cultured cells in vitro. The markers included ganglioside N -glycolylneuraminic acid (NeuGc), GA1 (asialo-GM1), and Fc receptor-bearing cells. NeuGc-containing gangliosides, GA1, and Fc receptors are expressed by macrophages and lymphoid-type cells of the mouse host immune system but are not normally expressed by mouse neural cells. Differences in the relative content of Fc receptor-bearing cells in ependymoblastoma and CT-2A tumors grown in vivo (8.3 and 16.8%, respectively) were proportional to differences in the relative content of NeuGc-containing gangliosides (25.5 and 45.1%) and GA1 (8.5 and 13.8%), respectively. Neither cultured tumor cell line expressed Fc receptors, GA1, or NeuGc-containing gangliosides. These findings suggest that non-neoplastic host infiltrating cells (macrophages) contribute significantly to the GSL composition of solid tumors growing in vivo.     

Poly(ethylene glycol) modification of β-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs

 Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coliβ-glucuronidase (βG) was examined as a method to improve the stability and pharmacokinetics of antibody-βG conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect βG activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity. The enzyme was found to be stable in serum regardless of PEG modification. PEG-modified βG was coupled via a thioether bond to mAb RH1, an IgG_2a antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-βG-3PEG), 5.2 (RH1-βG-5PEG) or 9.8 (RH1-βG-10PEG) PEG molecules per βG with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-βG. In contrast to the rapid serum clearance of RH1-βG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-βG-3PEG and RH1-βG-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-βG. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-βG-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-βG conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective. Received: 27 February 1997 / Accepted: 6 May 1997     

Systemic p53 gene therapy of cancer with immunolipoplexes targeted by anti-transferrin receptor scFv.

BACKGROUND: A long-standing goal in genetic therapy for cancer is a systemic gene delivery system that selectively targets tumor cells, including metastases. Here we describe a novel cationic immunolipoplex system that shows high in vivo gene transfer efficiency and anti- tumor efficacy when used for systemic p53 gene therapy of cancer. MATERIALS AND METHODS: A cationic immunolipoplex incorporating a biosynthetically lipid-tagged, anti-transferrin receptor single-chain antibody (TfRscFv), was designed to target tumor cells both in vitro and in vivo. A human breast cancer metastasis model was employed to evaluate the in vivo efficacy of systemically administered, TfRscFv-immunolipoplex-mediated, p53 gene therapy in combination with docetaxel. RESULTS: The TfRscFv-targeting cationic immunolipoplex had a size of 60-100 nm, showed enhanced tumor cell binding, and improved targeted gene delivery and transfection efficiencies, both in vitro and in vivo. The p53 tumor suppressor gene was not only systemically delivered by the immunolipoplex to human tumor xenografts in nude mice but also functionally expressed. In the nude mouse breast cancer metastasis model, the combination of the p53 gene delivered by the systemic administration of the TfRscFv-immunolipoplex and docetaxel resulted in significantly improved efficacy with prolonged survival. CONCLUSIONS: This is the first report using scFv-targeting immunolipoplexes for systemic gene therapy. The TfRscFv has a number of advantages over the transferrin (Tf) molecule itself: (1) scFv has a much smaller size than Tf producing a smaller immunolipoplex giving better penetration into solid tumors; (2) unlike Tf, the scFv is a recombinant protein, not a blood product; (3) large scale production and strict quality control of the recombinant scFv, as well as scFv-immunolipoplex, are feasible. The sensitization of tumors to chemotherapy by this tumor-targeted and efficient p53 gene delivery method could lower the effective dose of the drug, correspondingly lessening the severe side effects, while decreasing the possibility of recurrence. Moreover, this approach is applicable to both primary and recurrent tumors, and more significantly, metastatic disease. The TfRscFv-targeting of cationic immunolipoplexes is a promising method of tumor targeted gene delivery that can be used for systemic gene therapy of cancer with the potential to critically impact the clinical management of cancer.     

Standardization of an orthotopic mouse brain tumor model following transplantation of CT-2A astrocytoma cells

Animal models of glial-derived neoplasms are needed to study the biological mechanisms of glioma tumorigenesis and those that sustain the disease state. With the aim to develop and characterize a suitable in vivo experimental mouse model for infiltrating astrocytoma, with predictable and reproducible growth patterns that recapitulate human astrocytoma, this study was undertaken to analyze the long-term course of a syngeneic orthotopically implanted CT-2A mouse astrocytoma in C57BL/6J mice. Intracranial injection of CT-2A cells into caudate-putamen resulted in development of an aggressive tumor showing typical features of human glioblastoma multiforme, sharing close histological, immunohistochemical, proliferative, and metabolic profiles. To simulate metastatic disease to the brain, CT-2A cells were injected through the internal carotid artery. Tumors identical to those obtained by intracranial injection were obtained. Finally, CT-2A cells were re-isolated from experimental brain tumors and transcranially re-injected into the caudate-putamen of healthy mice. These cells generated new tumors that were indistinguishable from the initial ones, suggesting in vivo self-renewal of tumor cells. Small-animal models are essential for testing novel biological therapies directed against relevant molecular targets. In a preliminary study, experimental CT-2A tumors were chronically treated with the small molecule 77427, a gastrin-releasing peptide (GRP) blocker compound that inhibits angiogenesis. Treated animals developed significantly smaller tumors than controls, suggesting an antitumor action for 77427 in glioblastomas. We conclude that the orthotopic CT-2A tumor model, as described herein, is appropriate to explore the mechanisms of glioma development and for preclinical trials of promising drugs.     

Antinucleosome antibody-modified liposomes and lipid-core micelles for tumor-targeted delivery of therapeutic and diagnostic agents

Liposomes and lipid-core micelles prepared of polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugates have been modified with nucleosome-specific monoclonal antinuclear autoantibody (ANA) 2C5 (mAb 2C5) specifically recognizing a broad variety of cancer cells through the cancer cell surface-bound nucleosomes. mAb 2C5 preserves its specific properties upon the binding with the lipid-based pharmaceutical nanocarriers, and 2C5-modified immunoliposomes and immunomicelles demonstrate an enhanced binding with tumor cells both in vitro and in vivo. We have investigated the delivery of therapeutic and diagnostic agents with such tumor-targeted immunoliposomes and immunomicelles to various tumors in vivo and in vitro. Both lipid-based nanocarriers provided enhanced tumor delivery of imaging agents ((111)In) and antitumor drugs (doxorubicin and photodynamic therapy agents) to tumor cells under different experimental settings. Pharmaceutical lipid-based nanoparticular carriers modified with mAb 2C5 could represent universal systems for tumor-specific delivery of various soluble and insoluble pharmaceuticals.     

A herpes simplex virus recombinant that exhibits a single-chain antibody to HER2/neu enters cells through the mammary tumor receptor, independently of the gD receptors

The human epidermal growth factor receptor 2/neuregulin (HER2/neu) receptor is overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis. It is a target for therapy; humanized monoclonal antibodies to HER2 have led to increased survival of patients with HER2/neu-positive breast cancer. As a first step in the design of an oncolytic herpes simplex virus able to selectively infect HER2/neu-positive cells, we constructed two recombinants, R-LM11 and R-LM11L, that carry a single-chain antibody (scFv) against HER2 inserted at residue 24 of gD. The inserts were 247 or 256 amino acids long, and the size of the gD ectodomain was almost doubled by the insertion. We report the following. R-LM11 and R-LM11L infected derivatives of receptor-negative J or CHO cells that expressed HER2/neu as the sole receptor. Entry was dependent on HER2/neu, since it was inhibited in a dose-dependent manner by monoclonal antibodies to HER2/neu and by a soluble form of the receptor. The scFv insertion in gD disrupted the ability of the virus to enter cells through HVEM but maintained the ability to enter through nectin1. This report provides proof of principle that gD can tolerate fusion to a heterologous protein almost as large as the gD ectodomain itself without loss of profusion activity. Because the number of scFv's to a variety of receptors is continually increasing, this report makes possible the specific targeting of herpes simplex virus to a large collection of cell surface molecules for both oncolytic activity and visualization of tumor cells.     

Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes

Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.     

Gene-linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma

Brain tumor growth and progression is dependent upon vascularity, and is associated with altered ganglioside composition and distribution. In this study, we examined the influence of gangliosides on growth and vascularity in a malignant mouse astrocytoma, CT-2A. Ganglioside distribution was altered in CT-2A tumor cells using an antisense construct to beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T), a key enzyme that uses the simple ganglioside GM3 as a substrate for the synthesis of the more complex gangliosides, GM2, GM1 and GD1a. GalNAc-T gene expression was significantly lower in CT-2A cells stably transfected with the antisense GalNAc-T plasmid, pcDNA3.1/TNG (CT-2A/TNG) than in either non-transfected CT-2A or mock-transfected (CT-2A/V) control tumor cells. GM3 was elevated from 16% to 58% of the total ganglioside distribution, whereas GM1 and GD1a were reduced from 17% and 49% to 10% and 17%, respectively, in CT-2A/TNG tumor cells. Growth, vascularity (blood vessel density and Matrigel assay) and vascular endothelial growth factor (VEGF) expression was significantly less in CT-2A/TNG tumors than in control CT-2A brain tumors. In addition, the expression of VEGF, hypoxia-inducible factor 1alpha (HIF-1alpha) and neuropilin-1 (NP-1) was significantly lower in CT-2A/TNG tumor cells than in control CT-2A tumor cells. These data suggest that gene-linked changes in ganglioside composition influence the growth and angiogenic properties of the CT-2A astrocytoma.     

Intra-tumoral injection of CpG results in the inhibition of tumor growth in murine Colon-26 and B-16 tumors

Direct tumor injections of CpG (ODN #1826) into murine tumors markedly suppressed the tumor growth and increased the survival of the mice. Tumor growth was reduced by 60–67% in Colon Tumor 26 (CT-26) and B-16 melanoma tumors treated with CpG as compared to untreated one. In CT-26 and B-16 tumors treated with CpG, the average survival of the animals were prolonged to 26 and 28 d as compared to 16 and 18 d in control respectively. Long-term surviving animals in CT-26 tumor groups were also protected from a subsequent injection of a lethal dose of tumor cells. In the present study, effect of CpG was mediated through CD8⁺ T cells, as their depletion resulted in the abrogation of the therapeutic effects of the CpG. It suggests that direct tumor injection might be a simple means of achieving a clinical response in cancer patients.     

新型多胺代谢酶抑制剂SI-4650对结肠癌CT-26细胞增殖的影响及其机制

目的:探讨本实验室新发现的新型多胺代谢酶小分子抑制剂SI-4650对结肠癌CT-26细胞增殖、自噬和凋亡的影响。方法:体外培养CT-26细胞,以0μmol·L^-1SI-4650处理48 h细胞为正常对照组,单独2.5 mmol·L^-13-MA处理细胞为自噬抑制对照组,40、80μmol·L^-1SI-4650处理48 h细胞以及3-MA联合40、80μmol·L^-1SI-4650处理48 h细胞为4个实验组,化学发光法检测CT-26细胞中多胺代谢酶SMO和APAO酶活性的变化,HPLC法检测细胞中多胺含量的变化,CCK8法检测CT-26细胞增殖能力变化; PI单染结合流式细胞术分析细胞周期; Western blot法分析细胞自噬; PI/FITC-Annexin V双染、JC-1荧光探针和Fluo-3 AM钙离子荧光探针分别结合流式细胞术以及Western blot法分析细胞凋亡。结果:与正常对照组比较,40、80μmol·L^-1SI-4650实验组细胞生长抑制率分别为36.9...     

Expression of theJE/MCP-1 gene suppresses metastatic potential in murine colon carcinoma cells

The purpose of this study was to determine whether the expression of theJE/MCP-1 gene encoding for the monocyte chemottractant protein, MCP-1 (also known as monocyte chemotactic and activating factor MCAF, TDCF, and SMC-CF) can influence the metastatic properties of tumor cells. The highly metastatic murine colon carcinoma CT-26 cells, syngeneic to BALB/c mice that do not produce endogenous JE/MCP-1 protein, were transfected with a BCMGS-Neo expression vector (control) or a vector containing full-lengthJE cDNA. CT-26 parental cells, CT-26 Neo, and CT-26 JE/MCP-1-positive cells were injected into syngeneic or nude mice. The CT-26 JE/MCP-1-positive cells produced significantly fewer lung metastases. The decrease in incidence of metastasis was not due to the inability of the transfected cells to arrest in the lung vasculature or to differences in cell cycle time. CT-26 cells producing JE/MCP-1 were highly susceptible to lysis by syngeneic macrophages treated with subthreshold concentrations of lipopolysaccharide. In addition, culture supernatants of JE/MCP-1-expressing cells plus lipopolysaccharide synergistically activated tumoricidal properties in syngeneic macrophages. This activity was blocked by anti-JE/MCP-1 antibodies, indicating the involvement of the JE/MCP-1 molecule in this process. Moreover, purified JE/MCP-1 added to lipopolysaccharide-containing medium resulted in significant activation of macrophages against parental CT-26 cells. These data suggest that, in addition to its chemotactic properties, JE/MCP-1 can synergize with bacterial endotoxins to activate macrophages to become tumoricidal and, hence, could suppress metastasis.     

抗卵巢癌单链免疫细胞因子真核表达载体的构建及表达

为将细胞因子IL 2靶向卵巢癌局部 ,提高肿瘤局部细胞因子的浓度 ,构建并表达了抗卵巢癌单链免疫细胞因子IL 2 1 83B2scFv .通过基因工程将两段基因IL 2和 1 83B2scFv开放读码框架的编码序列克隆在一起 ,在CHO细胞内人巨细胞病毒启动子的作用下表达可溶性融合蛋白 .酶联免疫吸附法检测其免疫学活性 ,并检测其促淋巴细胞增殖活性 .构建成功的抗体细胞因子融合蛋白能够在哺乳动物细胞内表达并能分泌到细胞外 ,且融合蛋白既能与卵巢癌相关抗原OC1 83B2很好地结合 ,又能刺激IL 2依赖细胞株的增殖 ,为其进一步临床应用打下实验基础     

Nuclear estrogen receptor targeted photodynamic therapy: selective uptake and killing of MCF-7 breast cancer cells by a C17alpha-alkynylestradiol-porphyrin conjugate

We hypothesized that over-expression of estrogen receptor (ER) in hormone-sensitive breast cancer could be harnessed synergistically with the tumor-migrating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill the tumor cells upon exposure to red light. In the present work we synthesized four (4) conjugates of C17-alpha-alkynylestradiol and chlorin e6-dimethyl ester with varying tether lengths, and showed that all these conjugates specifically bound to recombinant ER alpha. In a cellular uptake assay with ER-positive MCF-7 and ER-negative MDA-MB 231 human breast cancer cell-lines, we observed that one such conjugate (E17-POR, XIV) was selectively taken up in a dose-dependent and saturable manner by MCF-7 cells, but not by MDA-MB 231 cells. Furthermore, MCF-7 cells, but not MDA-MB 231 cells, were selectively and efficiently killed by exposure to red light after incubation with E17-POR. Therefore, the combination approach, including drug and process modalities has the potential to be applied clinically for hormone-sensitive cancers in organs where ER is significantly expressed. This could potentially be carried out either as monotherapy involving a photo-induced selective destruction of tumor cells and/or adjuvant therapy in post-surgical treatment for the destruction of residual cancer cells in tissues surrounding the tumor.     

A recombinant scFv-FasLext as a targeting cytotoxic agent against human Jurkat-Ras cancer

Background

Targeted therapy of human cancers is an attractive approach and has been investigated with limited success. We have developed novel cytotoxic agents for targeted therapy of human cancers based on the extracellular cytotoxicity domain of CD178 (FasL) and the specificity offered by single chain antibodies (scFv) against dominant human tumor Ag TAG-72 (cc49scFv) and TAL6 (L6scFv).

Results

The cc49scFv-FasL_ext is highly effective in in vitro killing of human TAG-72⁺ Jurkat-Ras tumor cells with a 30,000 fold greater cytotoxicity as compared to soluble FasL (sFasL). On the other hand, L6scFv-FasL_ext only increased cytotoxicity 500-fold as compared with sFasL against TAL6⁺ HeLa cells in in vitro assays. The high specificity and strong cytotoxicity of cc49scFv-FasL_ext made it feasible to cure IP-implanted Jurkat-Ras tumors in SCID mice.

Conclusion

Our study demonstrated that scFv-FasL_ext with a strong cytotoxicity against sensitive human tumor targets may be useful as effective chemotherapeutic agents.     

Identification of MAGE-C1 (CT-7) epitopes for T-cell therapy of multiple myeloma

Multiple myeloma is incurable with standard therapies but is susceptible to a T-cell-mediated graft versus myeloma effect after allogeneic stem cell transplantation. We sought to identify myeloma-specific antigens that might be used for T-cell immunotherapy of myeloma. MAGE-C1 (CT-7) is a cancer-testis antigen that is expressed by tumor cells in >70% of myeloma patients and elicits a humoral response in up to 93% of patients with CT-7⁺ myeloma. No CD8⁺ T-cell epitopes have been described for CT-7, so we used a combination of reverse immunology and immunization of HLA-A2 transgenic mice with a novel cell-based vaccine to identify three immunogenic epitopes of CT-7 that are recognized by human CD8⁺ T-cells. CT-7-specific T-cells recognizing two of these peptides are able to recognize myeloma cells as well as CT-7 gene-transduced tumor cells, demonstrating that these epitopes are naturally processed and presented by tumor cells. This is the first report of the identification of immunogenic CD8⁺ T-cell epitopes of MAGE-C1 (CT-7), which is the most commonly expressed cancer-testis antigen found in myeloma, and these epitopes may be promising candidate targets for vaccination or T-cell therapy of myeloma or other CT-7⁺ malignancies.     

In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate.

To target disseminated tumors in vivo, transgenes [beta-galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV-TK)] were conjugated to transferrin (Tf) by a biotin-streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf-R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf-beta-galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf-R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK-1) whereas in normal diploid cells (HEL), which express low levels of Tf-R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf-HSV-TK gene conjugate for massively metastasized k562 tumors in severe combined immune-deficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tumors.     

Changes in gastric endocrine cells in Balb/c mice bearing CT-26 carcinoma cells: an immunohistochemical study

The distribution and density of gastric endocrine cells in Balb/c mice bearing CT-26 carcinoma cells were studied immunohistochemically employing specific antisera against serotonin, somatostatin, glucagon, gastrin, cholecystokinin (CCK)-8 and human pancreatic polypeptide (hPP). The animals were divided into two groups, a non-implanted sham group and a CT-26 carcinoma cell-implanted group. Samples were collected from two regions of the stomach (fundus and pylorus) at 28 days after implantation of the medium or the CT-26 cells (1x10(5) cells/mouse). Five of the 6 types of immunoreactive (IR) cells were identified, with only the hPP IR cells not being detected. The regional distribution of the gastric endocrine cells in the CT-26 implanted group was similar to that of the non-implanted sham group. However, the endocrine cells were significantly decreased in the CT-26-implanted group as compared to those of the non-implanted sham group. Serotonin- and somatostatin-IR cells in the fundus and pylorus , and gastrin- and CCK-8-IR cells in the pylorus of the CT-26 implanted groups were significantly decreased compared to those of the sham group. In addition, glucagon-IR cells were restricted only to the fundus of the sham animals. hPP-IR cells were not detected in either the T-26 implanted- or the non-implanted group. Since endocrine cells are the anatomical units responsible for the production of gut hormones, a change in their density may reflect a change in their capacity to produce such hormones. Implantation of the tumor cell mass induced severe quantitative changes in gastric endocrine cell density, an abnormality which may contribute to the development of gastrointestinal symptoms, such as anorexia and indigestion, frequently encountered in cancer patients.