Naked DNA injection for liver metastases treatment in rats

Abstract

The cytosine deaminase (CD) gene converts the nontoxic prodrug, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU). We previously showed that injection of CD-bearing cancer cells followed by 5-FC treatment can act as an autologous tumor vaccine in a syngenic liver metastasis model in rats. In the present work, we analyzed the antitumor efficiency of a direct intratumoral injection of a CD-expressing plasmid. In rats bearing microscopic or macroscopic metastases in right and left liver lobes, an injection of a CD-expressing plasmid was performed in the left lobe tumor, followed by 5-FC treatment of the animals. A significant regression of the DNA-injected tumor was observed in 5-FC-treated rats, both in microscopic (P ‫؍‬ .007) or advanced (P < .0001) tumor models. Moreover, this treatment also induced a potent distant bystander effect on untreated controlateral liver tumors and extrahepatic metastases, resulting in an increased survival compared with control animals in both tumor models (P < .05). In conclusion, these data suggest that direct intratumoral injection of a CD-expressing plasmid, associated to 5-FC administration, can constitute a powerful and innocuous alternative treatment for unresectable liver metastases from colon carcinoma. C olon carcinoma represents one of the most frequent cancers in Westernized countries and is associated with a high mortality, as a result of the appearance of metastases preferentially located in the liver. Resection of liver metastases constitutes the only curative treatment. Because resection is applicable in only 10% to 20% of patients, many efforts are dedicated to the development of alternative treatments. Suicide-gene therapy consists of the transfer into tumor cells of a "killer gene" that converts a systemically administered nontoxic compound into a lethal drug. This strategy has proven to be effective in several tumor models in rodents. 1 The bacterial cytosine deaminase (CD) gene encodes an enzyme capable of converting cytosine into uracil. Mammalian cells possess no endogenous CD gene. When the bacterial CD gene is expressed in mammalian cells, the enzyme transforms the nontoxic antifungal agent, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU), a widely used chemotherapeutic drug. Consequently, expression of the CD gene within cancer cells results in a specific tumortargeted chemotherapy. Although bypassing the toxic side effects of a systemic chemotherapy is, by itself, an interesting point, the key effects of this strategy rely on the existence of two associated "bystander effects." The first one, termed « local bystander effect, » is known to induce complete tumor regression even if only a small proportion of cells in a tumor express the suicide gene. 2,3 The second effect, or « distant bystander effect, » results in the regression of distant, unmodified tumors, and is mediated by the triggering of an immune reaction following destruction of the tumor expressing the suicide gene. [4][5][6][7][8][9][10][11] Previously, we developed a vaccination strategy ...

Plasmidic CpG sequences induce tumor microenvironment modifications in a rat liver metastasis model

“…Although plasmid DNA injection without prodrug treatment was not sufficient to induce a therapeutic effect in this model (4), plasmid DNA could be involved in the potent antitumoral response observed via the presence of unmethylated CpG sequences in the plasmid backbone. Indeed, plasmid DNA, as bacterial DNA, contains unmethylated cytosine-phosphateguanine (CpG) sequences that are considered immunostimulatory.…”

“…Freshly prepared rat splenocytes without erythrocytes were used for measurement of in vitro IFN-γ production. Briefly, rats were sacrificed, and single-cell suspensions were prepared by mechanical dissociation of spleen and erythrocyte lysis by incubating the cells for 10 min at 4˚C in buffer (155 mM NH 4 Cl, 10 mM NaHCO 3 , and 0.1 mM EDTA, pH 7.4).…”

“…We previously demonstrated, in a syngeneic rat bifocal liver metastasis model, that injection of a CD-expressing plasmid in one of the tumors, followed by 5-FC treatment of the animals, resulted in the regression of both lesions due to the triggering of a strong distant bystander effect (4). Although plasmid DNA injection without prodrug treatment was not sufficient to induce a therapeutic effect in this model (4), plasmid DNA could be involved in the potent antitumoral response observed via the presence of unmethylated CpG sequences in the plasmid backbone.…”

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“…Flt3 Ligand (Flt3L) is an activator of dendritic cells and NK cells. Intratumoural injections of an adenovirus encoding Flt3Lproduced a synergistic effect in association with 5-FUtherapy in subcutaneous models of hepatocarcinoma and colon cancer and induced long-term immunity against further parental cancer cell injections 77 . In a recent study, cultured human colorectal cancer cells and tumour xenograft were Recently, the effect of miRNA targeting c-myc was assessed in vitro and in vivo.…”

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

“…20 We have previously demonstrated, in rats bearing experimental metastases in the right and left liver lobes, that intratumoral injection of a CD-expressing plasmid into one of the tumors followed by 5-FC treatment results in regression of both lesions. 21 Moreover, this treatment induced a potent distant bystander effect on extrahepatic metastases, resulting in an increased survival compared to control animals.…”