Demonstration of anti-tumour bystander killing with prodrug-preloaded suicide gene-engineered tumour cells: a potential improvement for cancer therapeutics.

BACKGROUND: Therapeutic approaches for cancer rely on careful consideration of finding the optimal way of delivering the pro-drug for cellular-based cancer treatment. Cell lines and cell cultures have been used in these studies to compare the in vitro and in vivo efficacy of autologous vs. allogeneic tumour cellular gene therapy. Here we have investigated and are reporting for the first time the effect of prodrug ganciclovir (GCV)-preloading (pre-treatment) in suicide gene therapy of cancer. METHODS: This study examines the effect of GCV-preloading (pre-treatment) on a range of tumour cell lines in conjunction with suicide gene therapy of cancer. To determine the efficacy of this modality, a series of in vitro and in vivo experiments were conducted using genetically modified and unmodified tumour cell lines. RESULTS: Following co-culture of herpes simplex virus thymidine kinase (HSV-TK) modified tumour cells and unmodified tumour cells both in vitro and in vivo, GCV-preloading (pre-treatment) of TK-modified human and mouse mesothelioma cells and ovarian tumour cells allowed them to mediate efficiently bystander killing of neighbouring unmodified tumour cells in vitro. In contrast, GCV-preloading of TK-modified human and mouse mesothelioma cells and ovarian tumour cells abolished their in vivo ability to induce bystander killing of unmodified tumour cells, although there was some tumour regression compared to control groups but this was not statistically significant. These results suggest that preloading TK modified tumour cells with GCV needs further study to define the most effective strategy for an in vivo application to retain their bystander killing potential after exposure to lethal doses of GCV in vitro. CONCLUSIONS: This study highlights the promising possibility of improving the efficacy of pro-drug system to prevent any damage to the immune system and enhancing this type of suicide gene therapy of cancer, as well as the need for further studies to explore the discrepancies between in vitro and in vivo results.

The impact of γ-irradiation on the induction of bystander killing by genetically engineered ovarian tumor cells: implications for clinical use.

BACKGROUND: Cellular based therapeutic approaches for cancer rely on careful consideration of finding the optimal cell to execute the cellular goal of cancer treatment. Cell lines and primary cell cultures have been used in some studies to compare the in vitro and in vivo efficacy of autologous vs allogeneic tumour cell vaccines. METHODS: This study examines the effect of γ-irradiation on a range of tumor cell lines in conjunction with suicide gene therapy of cancer. To determine the efficacy of this modality, a series of in vitro and in vivo experiments were conducted using genetically modified and unmodified tumor cell lines. RESULTS: Following co-culture of HSV-TK modified tumor cells and unmodified tumor cells both in vitro and in vivo we observed that the PA-STK ovarian tumor cells were sensitive to γ-irradiation, completely abolishing their ability to induce bystander killing of unmodified tumor cells. In contrast, TK-modified human and mouse mesothelioma cells were found to retain their in vitro and in vivo bystander killing effect after γ-irradiation. Morphological evidence was consistent with the death of PA-STK cells being by pyknosis after γ-irradiation. These results suggest that PA-STK cells are not suitable for clinical application of suicide gene therapy of cancer, as lethal γ-irradiation (100 Gy) interferes with their bystander killing activity. However, the human mesothelioma cell line CRL-5830-TK retained its bystander killing potential after exposure to similarly lethal γ-irradiation (100 Gy). CRL-5830 may therefore be a suitable vehicle for HSV-TK suicide gene therapy. CONCLUSIONS: This study highlights the diversity among tumor cell lines and the careful considerations needed to find the optimal tumor cell line for this type of suicide gene therapy of cancer.

Rofecoxib has different effects on chemokine production in colorectal cancer cells and tumor immune splenocytes.

Cyclooxygenase-2 (COX-2) is overexpressed in colon tumors. Its main product is the immunosuppressive prostaglandin PGE2 that aids tumor immune escape. In this study, we analyzed mechanisms of action of the COX-2 inhibitor rofecoxib on the immune response to colorectal cancer in an animal model. The murine colorectal cancer cell line MC26, and splenocytes from BALB/c mice immune to irradiated MC26 cells, were incubated with rofecoxib or PGE2. In MC26 cells, 100 nM rofecoxib caused a complete abrogation of PGE2 production and inhibited cell proliferation. Splenocytes from tumor immune mice showed a 300% (P<0.01) increase in proliferation in response to irradiated MC26 cells, amplified to 450% (P<0.01) by 1 microM rofecoxib (n=3). MC26 cells incubated with 1 microM rofecoxib showed increased gene expression of CCL3, CCL5, and CCL20 (P<0.01). enzyme-linked immunosorbent assay tests also showed increased production of CCL5 and CCL20 (P<0.01). PGE2 reversed this effect causing a 40% reduction in chemokine gene expression (n=3). In contrast, splenocytes from naive BALB/c mice stimulated with irradiated MC26 cells had only a marginal chemokine response to rofecoxib. PGE2 caused a 50% down-regulation of CCL5 and CCL20 at the gene level (n=2) and 30% and 40% reduction of CCL3, CCL4, CCL5, and CCL20 at the protein level (n=2). Hence rofecoxib has a 2-fold effect upon the immune response to MC26 cells, by enhancing production of chemokines chemotactic for dendritic cells and also reducing PGE2-mediated inhibition of lymphoproliferation. Together, these may be sufficient for an effective TH1-mediated antitumor response. Rofecoxib may have potential as an addition to existing immunotherapy strategies.

Both soluble and membrane-bound forms of Flt3 ligand enhance tumor immunity following "suicide" gene therapy in a murine colon carcinoma model.

In prodrug-activated ("suicide") gene therapy, tumor cells are transfected with the gene for an enzyme that converts an inactive prodrug, such as ganciclovir (GCV), to a toxic compound. Transfected cells are killed on administration of GCV, as also are untransfected "bystander" cells. The ability of the dendritic cell stimulatory cytokine Flt3 ligand (Flt3-L) to modulate prodrug-activated gene therapy has been investigated. Transfectants of the murine colon carcinoma MC26 were generated expressing soluble (FLS) and membrane-bound forms of Flt3-L. They were inoculated together with wild-type MC26 cells and cells expressing herpes simplex virus-1 (HSV1) thymidine kinase into BALB/c mice, which were then administered GCV. Expression of Flt3-L or FLS prevented regrowth of tumor in most mice, which was comparable to the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), while tumors recurred in all mice receiving "suicide" gene therapy alone. Recurring tumor cells were resistant to direct killing by GCV but sensitive to "bystander" killing in vitro. Mice without tumor recurrence were rechallenged with unmodified MC26 cells. Of those mice given transfectants expressing GM-CSF, Flt3-L, or FLS, approximately 50% were immune to rechallenge. These mice also showed cytotoxic and proliferative responses to MC26 cells. These experiments show that both soluble and membrane-bound forms of Flt3-L were able to induce a protective immune response to colon carcinoma cells in a fashion similar to GM-CSF.