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Peritoneal dissemination is the most common mode of metastasis in gastric cancer. We previously reported the importance of milky spots (MS), peritoneal lymphoid tissues, as selective sites of cancer implantation in peritoneal dissemination. In the present study, we first demonstrated that intraperitoneal injection of adenovirus vector encoding the GFP gene into tumor-free nude mice resulted in GFP expression at omental and mesenteric MS; MS macrophages were target cells for adenovirus infection. We confirmed that intraperitoneal injection of adenovirus vector encoding the NK4 gene (AdNK4) resulted in NK4 production localized to the peritoneal cavity, especially the omentum. Adenovirus vector-mediated MS-selective transgene expression was markedly impaired in tumor-bearing mice whose MS had already been replaced by infiltrating cancer cells. However, prior injection of AdNK4 successfully inhibited MS-selective cancer cell implantation, resulting in suppression of peritoneal dissemination and prolongation of survival. Adenovirus vector-mediated MS-selective delivery of a therapeutic gene may prevent peritoneal dissemination of gastric cancer.

Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and the E1a gene are driven by the L-plastin tumor-specific promoter (AdLpCDIRESE1a). A similar vector driven by the CMV promoter has also been constructed (AdCMVCDIRESE1a) as a control. We have carried out in vitro cytotoxicity in carcinomas of the breast, ovary and colon, and in vivo efficacy studies with these vectors in an animal model of colon cancer. While the addition of the AdLpCDIRESE1a vector to established cancer cell lines showed significant cytotoxicity in tumor cells derived from carcinomas of the breast (MCF-7), colon (HTB-38) and ovary (Ovcar 5), no significant toxicity was seen in explant cultures of normal human mammary epithelial cells (HMEC) exposed to this vector. The addition of 5-fluorocytosine (5FC) significantly increased the cytotoxicity in an additive fashion of both the AdLpCDIRESE1a and AdCMVCDIRESE1a vectors as well as that of the AdLpCD replication incompetent vector to established tumor cell lines. However, no significant cytotoxicity was observed with the addition of 5FC to explant cultures of normal human mammary epithelial cells that had been exposed to the L-plastin-driven vectors. Studies with mixtures of infected and uninfected tumor cell lines showed that the established cancer cell lines infected with the AdLpCDIRESE1a vector generated significant toxicity to surrounding uninfected cells (the "bystander effect") even at a ratio of 0.25 of infected cells to infected + uninfected cells in the presence of 5FC. The injection of the AdLpCDIRESE1a vector into subcutaneous deposits of human tumor nodules in the nude mice was potentiated by administering 5-FC by intraperitoneal injection. This treatment resulted in a decreased tumor size and a decreased tumor cell growth rate. The mice treated with a combination of the AdLpCDIRESE1a vector intratumoral injection and intraperitoneal 5FC injections lived much longer than the other experimental groups exposed to the viral vector alone or to the combination of the intratumoral AdLpCD replication incompetent vector injections plus intraperitoneal 5-FC injections. These encouraging results with our newly constructed AdLpCDIRESE1a vector suggest a need for further study of its utility in a preclinical model of intracavitary therapy of pleural or peritoneal carcinomatosis.

Herpesvirus saimiri (HVS) is capable of establishing a persistent infection in a variety of human carcinoma cell lines, by virtue of episomal maintenance. Moreover, the viral episome provides expression of a transgene in both in vitro and in vivo environments. At present, HVS vectors utilize heterologous promoters such as the IE hCMV promoter. However, this promoter maybe unsuitable for long-term expression in vivo, as promoter silencing has been observed in this and other herpesvirus-based vector systems. Ideal regulatory regions would be functional when the herpesvirus genome is maintained as a latent episome. We have previously shown that gene expression in an HVS-persistently-infected human carcinoma cell line is limited to an adjacent set of genes encoding ORFs 71-73. These genes are transcribed as a polycistronic mRNA species from a common regulatory region upstream of the ORF 73 gene. In this report, we assess the potential of the ORF 73 regulatory region to provide heterologous gene expression in a wide variety of human cancer cell lines. We demonstrate, utilizing transient transfection assays, that the ORF 73 regulatory region can provide transgene expression in a variety of human carcinoma cell lines, although levels of transgene expression are not as high as achieved under the control of heterologous promoters such as the IE hCMV promoter. Furthermore, incorporation of the minimal ORF 73 regulatory region in a recombinant HVS-based vector provides sustained expression of the green fluorescent protein in both in vitro and in vivo environments. These results suggest that the ORF 73 regulatory region may be suitable for use in HVS-based cancer gene therapy applications.

Interleukin-10 (IL-10) is a T helper type 2 (Th2) cytokine that suppresses Th1-mediated, cell-mediated immune responses and reciprocally enhances antibody-mediated responses. Previous studies, however, demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. We then examined whether tumor-derived IL-10 could modulate systemic immune responses. Murine colon carcinoma (Colon 26) cells that were retrovirally transduced with the murine IL-10 gene (Colon 26/IL-10) were inoculated in syngeneic immunocompetent or T cell-defective nude mice. Growth of Colon 26/IL-10 tumors was augmented in immunocompetent and, to less extent, in nude mice compared with that of wild-type tumors developed in respective mice. Growth of wild-type tumors was accelerated to the same level as that of Colon 26/IL-10 tumors when wild type and Colon 26/IL-10 cells were respectively inoculated in different flanks of the same immunocompetent mice. This enhanced growth of wild-type tumors was not observed in nude mice. Immunocompetent mice that had rejected IL-2- or IL-12-secreting Colon 26 cells developed protective immunity and became completely resistant to wild-type Colon 26 cells subsequently challenged. However, some of the mice that had rejected IL-2 or IL-12 producers developed Colon 26/IL-10 tumors inoculated thereafter. The present study showed that production of IL-10 from tumor cells impaired T cell- and non-T cell-mediated systemic antitumor immunity in hosts.

Orthotopic implantation of human bladder cancer cells into immunodeficient mice is an important tool for studying the biology and effects of therapy. Nevertheless, the incidence of tumor implantation and growth by transurethral instillation of the human bladder cancer cells into murine bladders has been low or not reproducible. However, using a modified intravesical technique and the human bladder cancer cell lines, KU-7 and UM-UC-2, we have been able to obtain a high and reproducible incidence of superficial bladder tumors. Furthermore, intravesical administration of the LacZ adenovirus vector resulted in significant beta-galactosidase expression in these bladder tumors as well as the normal urothelium, which was associated with the removal of the glycosoaminoglycan layer. Because this modified technique produces a high incidence of superficial human tumor growth and allows the efficacy of gene transfer to be evaluated, it should be a useful model for the study of intravesical gene therapy for human bladder cancer.

BLK mouse fibroblasts (H-2b) were genetically engineered to express costimulatory B7.1 and interleukin-2 (BLK/IL2/B7.1). The BLK/IL2/B7.1 cells were then pulsed with an ovalbumin (OVA) epitope as a model antigen (Ag) (BLK/IL2/B7.1/OVA), and tested for the induction of OVA-specific cytotoxic T lymphocytes (CTLs) in C57BL/6 mice (H-2b). The genetically engineered fibroblasts lacking one or two of three factors (interleukin-2, B7.1, and OVA) were constructed and used as controls. Immunization with the BLK/IL2/B7.1/OVA cells induced strong cytotoxic activities against OVA-expressing EL4 (EG7) tumor cells, but not against other H-2b tumor cells, such as EL4, C1498 and B16F1 cells. The magnitude of the cytotoxic response in mice with the BLK/IL2/B7.1/OVA cells was significantly higher than the response in mice immunized with any other cell constructs. CD8+ T cells with OVA-specific cytotoxic activities were predominant in mice immunized with the BLK/IL2/B7.1/OVA cells. Furthermore, immunization with the BLK/IL2/B7.1/OVA cells significantly prolonged the survival of mice, compared with any other cell constructs, when the mice were challenged with EG7 tumor cells at 2 weeks postimmunization. Induction of antitumoral CTL immunity by the BLK/IL2/B7.1/OVA cells was independent of host Ag-presenting cells and of CD4+ T-cell and natural killer 1.1+ cell help. These results suggest that fibroblasts can be genetically modified to efficient Ag-presenting cells for the induction of an Ag-specific CTL response.

Vaccination using well-characterized allogeneic tumor cell lines expressing standardized doses of immunostimulatory cytokines is an attractive alternative for autologous gene-transfected tumor cell vaccines. In the present study, we show that vaccination with irradiated allogeneic K1 735 (H-2k) or B16F10 (H-2b) melanoma cells induces a moderate degree of cross-protection against the M-3 melanoma (H-2d) in DBA/2 mice. Cross-protection against the syngeneic tumor was markedly improved when the allogeneic vaccines were transfected with the interleukin-2 (IL-2) gene. The IL-2 gene-modified allogeneic vaccines were effective for prophylactic vaccination against subsequent tumor challenge and for therapeutic vaccination against pre-existing tumor deposits, with efficacies that were comparable with that of the IL-2 gene-modified syngeneic vaccines. Cross-protection correlated with the cytotoxic activity of splenocytes against M-3 targets. Allogeneic vaccination was not effective in another model, against the B16F10 melanoma in C57BL/6 mice, irrespective of genetic modification with the IL-2 or granulocyte-macrophage colony-stimulating factor genes.

Cancer gene therapy approaches are often designed as single-agent treatments; however, greater therapeutic effect might be obtained if combined with an established conventional treatment regimen such as chemotherapy. In this context, conditional promoters are useful tools, because they may be induced by therapeutic modalities. The human multidrug resistance gene (mdr1) promoter is inducible by cytostatic drugs and can be employed for the chemotherapy-regulated expression of therapeutic genes. In this in vivo study, the human mdr1 promoter fragment (-207 to +158) was used for drug-inducible expression of human tumor necrosis factor-alpha (TNF-alpha) in the vector construct pM3mdr-p-hTNF. The single doxorubicin and vincristine treatment of nude mice xenografted with pM3mdr-p-hTNF-transduced MCF-7 mammary tumors resulted in drug-induced and time-dependent elevation of intratumoral TNF-alpha expression at the mRNA and protein level. The highest drug induction was achieved at 2 days after drug application, as reflected by a maximum 25-fold increase in TNF-alpha secretion in the tumor. This drug-induced TNF-alpha expression is more effective in inhibiting tumor growth compared with the growth of tumors transduced with constitutively TNF-alpha-expressing vectors in combination with chemotherapy.

Immunization with modified immunoglobulin (Ig) idiotypes (Ids) of B-cell lymphomas is an attractive approach of experimental tumor immunotherapy. We show here that B-lymphoma cells can be gene-modified by homologous recombination at the Ig heavy chain locus. Although it has been demonstrated previously that a protein vaccine containing a mouse/human chimeric Ig had no immunostimulatory effect, we show that a xenogeneic Fc segment attached to the Id by gene targeting in autologous murine tumor cells can serve as an immunogenic carrier and is capable of inducing tumor protection. A prerequisite for successful vaccination is the delivery of tumor cells that have been engineered to express the Id in the chimeric form rather than administration of the soluble chimeric protein. Also DNA vaccination with plasmids encoding chimeric Ids was reported to induce an anti-idiotypic response, suggesting that there might be related mechanisms such as enhanced antigen presentation. Immunization with engineered lymphoma cells is a very potent protocol: in the cell-based setting, minute levels of expression in the gene-targeted cells are sufficient to confer tumor immunity. Because the titers of anti-Id antibodies induced do not reflect the degree of tumor protection, the immune mechanisms responsible for tumor rejection cannot be ascribed exclusively to a humoral response.

Insulin-like growth factor-1 (IGF-1) is expressed in many tumor cell lines and has a role in both normal cell proliferation and in the growth of cancers. Tumor cells transfected with a vector encoding an IGF-1 antisense cDNA transcriptional cassette driven by the mouse metallothionein-1 promoter become immunogenic and lose their tumorigenicity in syngeneic animals. The enhanced immunogenicity is associated with an up-regulation in the expression of major histocompatibility complex class I molecule on cell surfaces. Blockade of the expression of IGF-1 in tumor cells by the IGF-1 antisense RNA approach is not uniformly effective in the induction of antitumoral protective immunity in low and nonimmunogenic tumor model systems. Here, we report that the immunogenicity of hepa 1-6 hepatoma and SMCC-1 colon carcinoma cells, which are poorly immunogenic and unresponsive to antisense IGF-1 gene transfer, can be induced by cotransfection with genes encoding antisense IGF-1 and mouse B7.1 molecules. The tumor cells modified in this manner become strongly immunogenic and can be used as a cellular vaccine to induce a protective immune response in vivo. Immunization with the transfected tumor cells also results in regression of the established hepa 1-6 hepatoma and SMCC-1 colon cancer. The immunity is tumor-specific and is mediated by CD3+ CD8+ T cells. Cytotoxic T lymphocytes generated in vitro by priming naive spleen cells and in vivo by immunizing mice with the double-transfected tumor cells specifically lysed autologous tumors cells and were effective in adoptive immunotherapy. The data suggest that modification of tumor cells in vitro by cotransfection with genes encoding antisense IGF-1 and B7.1 molecules may open a new avenue for cancer immunogene therapy.

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.

We investigated the antitumor effects induced by the production of interleukin-12 (IL-12) or IL-18, which influence the function of T helper type 1 cells, in murine colon carcinoma cells (Colon 26). Retrovirally transduced cells with IL-12 genes that encoded both p35 and p40 (Colon 26/IL-12) lost their tumorigenicity when inoculated subcutaneously or intraperitoneally into syngeneic immunocompetent mice. Moreover, the mice that had rejected the Colon 26/IL-12 cells generated protective immunity to wild-type (wt) cells when subsequently challenged. Colon 26 cells transduced with the IL-18 gene (Colon 26/IL-18) could not form subcutaneous tumors in immunocompetent mice, and the mice became resistant to inoculated wt cells. Immunohistochemical analysis revealed that the numbers of blood vessels in Colon 26/IL-12 or Colon 26/IL-18 tumors were markedly reduced, and that the expression of adhesion molecules such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 increased on the endothelium in the stroma of Colon 26/IL-12 tumors. The loss of tumorigenicity of Colon 26/IL-12 or Colon 26/IL-18 cells was not observed in immunocompromised mice. However, the survival days of the immunocompromised mice inoculated with Colon 26/IL-12 but not Colon 26/IL-18 cells were significantly longer than those inoculated with wt cells. The secretion of cytokines that stimulate T helper type 1 cells from tumor cells can thereby induce an antitumor response. However, the effector cells involved in these antitumor effects could differentially migrate to the tumors, and the inhibition of angiogenesis may partially contribute to the antitumor responses observed.

We tested an adenovirus vector that carries a beta-D-galactosidase marker gene for its ability to transduce normal oral mucosa and oral carcinoma cells. Topical application of adenovirus to normal oral mucosa of mice at 1 x 10(10) plaque-forming units (pfu)/mL for 1 minute did not result in transduction of epithelial cells. Similarly, topical application to human oral mucosa ex vivo was not successful. However, systemic administration by intracardiac injection of hamsters did transduce normal oral mucosa effectively. To evaluate transduction of carcinomas, the Tu138 human oral cancer cell line was used. A single application of virus at 1 x 10(8) pfu/mL in vitro resulted in 30% of oral carcinoma cells expressing the marker gene, and 2 x 10(8) pfu/mL transduced 60% of cells. After two applications of virus at 2 x 10(8) pfu/mL with an interval of 18 hours, 100% of oral carcinoma cells expressed the marker gene. Topical application to a raft culture of Tu138 cells for 1 hour produced gene expression that penetrated up to four layers of cells. To emulate the effect of treating a carcinoma, Tu138 cells were implanted subcutaneously in nude mice, allowed to grow to a tumor 1 cm in diameter, and then injected directly with virus. This produced diffuse transduction of around 30% of cells in the tumor, and expression was seen in cells at a significant distance from the injection site. Adenovirus vectors are therefore capable of transferring novel genetic information to both normal and malignant oral mucosa. They may have potential for use in gene therapy in the prevention or treatment of oral squamous carcinomas.

CMS5, a murine fibrosarcoma, was transduced with the IL-2 gene to stimulate the host antitumor response. Previously, we described that splenocytes isolated from parental tumor bearing (PTB) mice and IL-2-secreting tumor bearing (ITB) mice differed significantly in their proliferative responses when restimulated with IL-2-secreting tumor cells. In this report we extend these results by showing that the inability of PTB cells to proliferate when stimulated with parental CMS5 cells is not corrected by providing a source of costimulation. Furthermore, we demonstrate that T cells from PTB animals exhibit defective signaling through the T-cell receptor, defined by decreased protein tyrosine phosphorylation, that correlates with the impairment of functions attributed to both CD4+ and CD8+ T cells. In contrast, T cells from ITB animals are normal in this regard. The results suggest that immunosuppression underlies functional differences between PTB and ITB splenocytes and that defects in signal transduction may be responsible for the lack of normal functional responses.

Tumor cells transduced with the IL-4 gene demonstrate reduction of growth associated with macrophage and eosinophilic infiltrates, generation of cytotoxic T-cells, and protective immunity. Using murine IL-4 retroviral vectors, murine fibroblasts and tumors that produce from 50 to 5000 U of IL-4/10(6) cells per 24 hours as determined by ELISA and bioassay were successfully transduced. In blinded studies using C57BL/6 and BALB/c mice, we have shown that tumor growth can be inhibited (mean delay of 10 days compared with controls; P < .05) and in some cases, completely suppressed by the coinjection of viable tumor with IL-4-producing fibroblasts (tumor free > 100 days; P < .001). Animals that are able to reject an initial tumor inoculate can also completely reject subsequent parental tumor challenge of 10(5) cells (P < .001) while challenge of 10(6) parental tumor cells results in a significant delay of tumor induction (P < .05). In addition, immunization with IL-4 transduced fibroblasts and irradiated tumor cells resulted in complete suppression of parental tumor challenge in animals that received the high-dose IL-4 delivery. Finally coadministration of systemic IL-2 led to enhancement of IL-4 gene therapy resulting in a 20-day delay of preestablished tumor growth compared with controls (P < .05).

Infection of CMS5 tumor cells with retroviral constructs containing interleukin-2 (IL-2) cDNA and selection in medium supplemented with G418 resulted in the isolation of clones which secreted IL-2. Whereas injection of parental tumor cells resulted in progressive tumor growth, tumor cells secreting high levels of IL-2 were rejected. Furthermore, in animals vaccinated with IL-2-secreting cells, the immunosuppression associated with the inoculation of parental tumor cells did not develop, and these animals resisted a challenge with viable tumor cells. To better understand the functional differences in the anti-tumor responses of immune and tumor-bearing mice which are at the basis for these diverse responses, we used an in vitro model to analyze interactions between splenic lymphocytes and tumor cells. Spleen cells isolated from either tumor-bearing or immune mice proliferated vigorously when cultured alone for 6 days, but much less in the presence of parental tumor cells. This effect could not be transferred with supernatant from tumor cell lines. Spleen cells from tumor-bearing mice remained unresponsive, while those from immune mice proliferated well in response to IL-2-secreting tumor cells. Only spleen cells from immune animals were able to develop cytotoxicity against CMS5 cells following in vitro restimulation. These results are consistent with the interpretation that exposure to parental tumor cells inhibited cell-mediated anti-tumor responses by a mechanism that involved cell-to-cell contact.(ABSTRACT TRUNCATED AT 250 WORDS)