Transferrin lipoplex-mediated suicide gene therapy of oral squamous cell carcinoma in an immunocompetent murine model and mechanisms involved in the antitumoral response.

Suicide gene therapy has been used for the treatment of a variety of cancers. We reported previously the in vitro efficacy of the Herpes Simplex Virus Thymidine kinase (HSV-tk)/ganciclovir (GCV) system to mediate cytotoxicity in oral squamous cancer cells, using transferrin (Tf)-lipoplexes, prepared from cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and cholesterol. In the present study, we evaluated the antitumoral efficacy mediated by this lipoplex formulation in two suicide gene therapy strategies, HSV-tk/GCV and cytosine deaminase (CD)/5-fluorocytosine (5-FC), using a syngeneic, orthotopic murine model for head and neck squamous cell carcinoma. The cellular and molecular events associated with the antitumoral response elicited by both the therapeutic approaches were investigated by analyzing tumor cell death, tumor-infiltrating immune cells and tumor cytokine microenvironment. Significant tumor reduction was achieved upon intratumoral delivery of HSV-tk or CD genes mediated by Tf-lipoplexes, followed by intraperitoneal injection of GCV or 5-FC, respectively. Enhanced apoptosis, the recruitment of NK cells, CD4 and CD8 T-lymphocytes and an increase in the levels of several cytokines/chemokines were observed within the tumors. These observations suggest that suicide gene therapy with lipoplexes modifies the tumor microenvironment, and leads to the recruitment of immune effector cells that can act as adjuvants in reducing the tumor size.

Cellular and molecular events associated with the antitumor response induced by the cytosine deaminase/5-fluorocytosine suicide gene therapy system in a rat liver metastasis model.

The bacterial cytosine deaminase (CD) gene converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil. We have previously shown, in a rat liver metastasis model from colon carcinoma, that intratumoral injection of a CD-expressing plasmid into the animals followed by 5-FC treatment results in the regression of the treated tumor as well as distant uninjected tumors. The aim of this study was to further analyze the mechanisms associated with tumor regression induced upon application of suicide CD/5-FC strategy. Tumor regression was associated with an increased apoptosis, the recruitment of natural killer cells, CD4- and CD8 T lymphocytes within the tumors and an increased expression of several cytokines/chemokines mRNAs. These data indicate that the CD/5-FC suicide strategy is associated with the triggering of cellular and molecular events leading to an efficient antitumor immune response involving both innate and acquired immunity.

Subcutaneous or intrahepatic injection of suicide gene modified tumour cells induces a systemic antitumour response in a metastatic model of colon carcinoma in rats.

BACKGROUND: Suicide gene therapy consists of the transfer into tumour cells of a "suicide" gene that can convert a non-toxic compound into a lethal drug. Expression of the cytosine deaminase gene leads to the conversion of the non-toxic compound 5-fluorocytosine to 5-fluorouracil. We have recently shown that "suicide cell based vaccination" consisting of intrahepatic injection of cytosine deaminase expressing colon cancer cells followed by 5-fluorocytosine treatment induces regression of a distant wild-type liver tumour in rats. AIMS: This study was conducted to test if (i) a distant bystander effect on a liver tumour can be induced after subcutaneous suicide cell based vaccination and (ii) suicide cell based vaccination is efficient in limiting tumour dissemination to extrahepatic compartments. METHODS: An aggressive variant of rat colon carcinoma cells was selected after successive passages in vitro. Rats carrying an experimental liver "metastasis" generated by injection of these cells were vaccinated by subcapsular or subcutaneous injection of cytosine deaminase expressing cells followed by 5-fluorocytosine treatment. RESULTS: Subcutaneous and subcapsular vaccination induced 70% regression in the median volume of the pre-established liver tumour (p=0.001) and abolished tumour dissemination compared with control animals. CONCLUSIONS: This study has compared for the first time the efficiency of subcutaneous and intrahepatic suicide cell based vaccination in a metastatic colorectal carcinoma model in rats. The results indicate that both modes of vaccination are equally efficient in inducing a systemic antitumour response, suggesting that this strategy is a powerful approach against the development and dissemination of metastatic colon carcinoma.

[Vaccination by suicide gene therapy against a model of hepatic metastasis from colon cancer in the rat]. / Vaccination par thérapie génique suicide contre un modèle de métastases hépatiques de cancer colique chez le rat.

UNLABELLED: Suicide gene therapy consists of transferring into tumor cells a viral or bacterial gene encoding for an enzyme which converts a non-toxic product into a lethal drug. STUDY AIM: To analyze the therapeutic potential of vaccination with tumor cells expressing the bacterial cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) treatment in a rat liver metastasis model. MATERIAL AND METHOD: We used a rat colon carcinoma cell line which, after subcapsular or intraportal injection in syngenic animals, generates single or multiple experimental liver metastases, respectively. We have shown that introduction of a vector expressing the CD gene in this colon carcinoma cell line results in 5-FC sensitivity (PRObCD). RESULTS: Intrahepatic subcapsular injection of PRObCD tumor cells, followed by 5-FC treatment, induces total regression of a wild-type tumor pre-established in the contralateral liver lobe in 45% of animals with a 96% decrease in mean volume (p < 0.0001), demonstrating the existence of a distant bystander effect. This vaccination significantly increased the survival of rats with single (log-rank p < 0.0001) or multiple (log-rank p = 0.01) liver metastasis CONCLUSIONS: These results suggest that suicide gene-modified tumor cells can act as potent therapeutic vaccines against liver metastasis from colon carcinoma.

Regression of experimental liver tumor after distant intra-hepatic injection of cytosine deaminase-expressing tumor cells and 5-fluorocytosine treatment.

Cytosine deaminase (CD) gene of E. coli converts the non-toxic compound 5-fluorocytosine (5-FC) into 5-fluorouracil. We have introduced a vector expressing the CD gene in a rat colon carcinoma cell line. Expression of the CD gene confers 5-FC sensitivity to these cells in vitro and in vivo. In a bifocal model consisting in a simultaneous engrafment of a CD+ tumor on one lobe of the liver and a wild-type parental tumor on the opposite lobe, treatment with 5-FC results in regression of both type of tumors, indicating the existence of a distant bystander effect.

Cytosine deaminase/5-fluorocytosine-based vaccination against liver tumors: evidence of distant bystander effect.

BACKGROUND: The cytosine deaminase gene of Escherichia coli converts the nontoxic compound 5-fluorocytosine into 5-fluorouracil (5-FU), thereby acting as a suicide gene when introduced into cancer cells, killing the cells when they are exposed to 5-fluorocytosine. We analyzed the efficacy of using cytosine deaminase-bearing cancer cells as an autologous tumor vaccine in a rat model that mimics liver metastasis from colon carcinoma. METHODS: We introduced a plasmid vector containing the E. coli cytosine deaminase gene into a BDIX rat colon carcinoma cell line. Intrahepatic injection of the modified cells in syngeneic animals generates a single experimental liver "suicide tumor." We then analyzed the effect of 5-fluorocytosine treatment in terms of regression of cytosine deaminase-expressing cells in vivo as well as protection against wild-type cancer cells. RESULTS: Treatment with 5-fluorocytosine induced regression of cytosine deaminase-expressing (CD+) tumors, with seven of 11 treated animals being tumor free at the end of 30 days and a statistically significant difference in tumor volumes between treated and control animals (two-sided P<.0001). Intrahepatic injection of CD+ cells followed by 5-fluorocytosine treatment rendered the treated animals resistant to challenge with wild-type tumor cells, with no (zero of seven) treated animals developing wild-type tumors in contrast to all (four of four) control animals. Moreover, in animals with established wild-type liver tumors, injection of CD+ tumor cells followed by 5-fluorocytosine treatment produced a statistically significant increase in survival time (two-sided P<.0001). In vivo immunodepletion and immunohistologic analysis of experimental tumors indicate that natural killer cells are the major immune component involved in this antitumor effect. CONCLUSIONS AND IMPLICATIONS: Taken together, these results suggest the potential use of suicide gene-modified tumor cells as therapeutic vaccines against liver metastasis from colon carcinoma.

Mucosal immunity and tolerance: relevance to vaccine development.

The mucosal immune system of mammals consists of an integrated network of lymphoid cells which work in concert with innate host factors to promote host defense. Major mucosal effector immune mechanisms include secretory antibodies, largely of immunoglobulin A (IgA) isotype, cytotoxic T cells, as well as cytokines, chemokines and their receptors. Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination or natural immunization by a mucosal route can effectively induce immune suppression. The diverse compartments located in the aerodigestive and genitourinary tracts and exocrine glands communicate via preferential homing of lymphocytes and antigen-presenting cells. Mucosal administration of antigens may result in the concomitant expression of secretory immunoglobulin A (S-IgA) antibody responses in various mucosal tissues and secretions, and under certain conditions, in the suppression of immune responses. Thus, developing formulations based on efficient delivery of selected antigens/tolerogens, cytokines and adjuvants may impact on the design of future vaccines and of specific immunotherapeutic approaches against diseases associated with untoward immune responses, such as autoimmune disorders, allergic reactions, and tissue-damaging inflammatory reactions triggered by persistent microorganisms.