A transfectant mosaic xenograft model for evaluation of targeted radiotherapy in combination with gene therapy in vivo.

UNLABELLED: For gene therapy to be efficacious in the treatment of cancer, therapeutic transgenes must be limited in their expression to tumor cells and must be expressed at sufficiently high transcriptional levels. Moreover, the inadequacy of gene delivery must be overcome by induction of toxicity to neighboring nontargeted cells. Combining targeted radionuclide therapy with gene therapy using human telomerase promoters has shown promise in these respects, and the efficacy of this scheme has been assessed in vitro using transfectant mosaic tumor spheroids. To enable the evaluation of targeted radiotherapy combined with gene transfer in vivo, we have developed a transfectant mosaic xenograft (TMX) model. METHODS: Human telomerase promoters were used to drive expression of the noradrenaline transporter (NAT) transgene in 2 human cell lines (UVW and EJ138). Promoter activity was assessed in xenografts in nude mice by determination of the uptake of the radiopharmaceutical (131)I-metaiodobenzylguanidine ((131)I-MIBG) and by measurement of tumor growth. The efficacy of (131)I-MIBG treatment was also assessed in TMXs to determine the delay in growth of tumors composed of various proportions of NAT-expressing cells-a likely clinical scenario after gene delivery in vivo. RESULTS: In terms of induction of the capacity for active uptake of (131)I-MIBG and the resultant inhibition of tumor growth in vivo, both telomerase promoters (hTR and hTERT) were similar in potency to the CMV (cytomegalovirus) promoter as controlling elements for the expression of the NAT transgene. In TMXs derived from UVW and EJ138 cells, (131)I-MIBG uptake was proportional to NAT gene expression (r(s) = 0.910, P < 0.001 for UVW; r(s) = 0.971, P < 0.001 for EJ138). Inhibition of the growth of these tumors correlated with the fraction of NAT-transfected cells (r(s) = 0.910, P < 0.001 for UVW; r(s) = 0.971, P < 0.001 for EJ138), and substantial tumor growth delay was observed when 5% of the xenograft was composed of NAT-positive cells. CONCLUSION: TMXs constitute a suitable model to measure the efficacy of cancer gene therapy strategies when <100% of the tumor mass can be targeted to express the therapeutic transgene.

Radiation-induced biologic bystander effect elicited in vitro by targeted radiopharmaceuticals labeled with alpha-, beta-, and auger electron-emitting radionuclides.

UNLABELLED: Recent studies have shown that indirect effects of ionizing radiation may contribute significantly to the effectiveness of radiotherapy by sterilizing malignant cells that are not directly hit by the radiation. However, there have been few investigations of the importance of indirect effects in targeted radionuclide treatment. Our purpose was to compare the induction of bystander effects by external beam gamma-radiation with those resultant from exposure to 3 radiohaloanalogs of metaiodobenzylguanidine (MIBG): (131)I-MIBG (low-linear-energy-transfer [LET] beta-emitter), (123)I-MIBG (potentially high-LET Auger electron emitter), and meta-(211)At-astatobenzylguanidine ((211)At-MABG) (high-LET alpha-emitter). METHODS: Two human tumor cell lines-UVW (glioma) and EJ138 (transitional cell carcinoma of bladder)-were transfected with the noradrenaline transporter (NAT) gene to enable active uptake of MIBG. Medium from cells that accumulated the radiopharmaceuticals or were treated with external beam radiation was transferred to cells that had not been exposed to radioactivity, and clonogenic survival was determined in donor and recipient cultures. RESULTS: Over the dose range 0-9 Gy of external beam radiation of donor cells, 2 Gy caused 30%-40% clonogenic cell kill in recipient cultures. This potency was maintained but not increased by higher dosage. In contrast, no corresponding saturation of bystander cell kill was observed after treatment with a range of activity concentrations of (131)I-MIBG, which resulted in up to 97% death of donor cells. Cellular uptake of (123)I-MIBG and (211)At-MABG induced increasing recipient cell kill up to levels that resulted in direct kill of 35%-70% of clonogens. Thereafter, the administration of higher activity concentrations of these high-LET emitters was inversely related to the kill of recipient cells. Over the range of activity concentrations examined, neither direct nor indirect kill was observed in cultures of cells not expressing the NAT and, thus, incapable of active uptake of MIBG. CONCLUSION: Potent toxins are generated specifically by cells that concentrate radiohalogenated MIBG. These may be LET dependent and distinct from those elicited by conventional radiotherapy.

[131I]meta-iodobenzylguanidine and topotecan combination treatment of tumors expressing the noradrenaline transporter.

PURPOSE: Both [(131)I]meta-iodobenzylguanidine ([(131)I]MIBG) and the topoisomerase I inhibitor topotecan are effective as single-agent treatments of neuroblastoma. The aim of this study was to investigate the efficacy of [(131)I]MIBG in combination with topotecan in vitro and in vivo. EXPERIMENTAL DESIGN: The cell lines used were SK-N-BE(2c) (human neuroblastoma) and UVW/NAT (glioma cell line transfected with the noradrenaline transporter gene). Three different treatment schedules were assessed: topotecan given before (schedule 1), after (schedule 2), or simultaneously (schedule 3) with [(131)I]MIBG. DNA strand breakage was evaluated by comet assay, and cytotoxicity was determined by clonogenic survival. Efficacy was also measured by growth delay of tumor xenografts in nude mice. RESULTS: Combination schedules 2 and 3 caused more cytotoxicity than schedule 1. Similarly, significant DNA damage was observed following treatment schedules 2 and 3 (P < 0.005) but not schedule 1. The mean number of days for a doubling in volume of SK-N-BE(2c) tumors and a 10-fold increase in volume of UVW/NAT tumors were 10.4 and 18.6 (untreated), 19.7 and 25.3 (topotecan alone), 22.8 and 31.9 ([(131)I]MIBG alone), 26.3 and 37.1 (combination schedule 1), 34.3 and 49.7 (combination schedule 2), and 53.2 and >71 (combination schedule 3), respectively. The highest rate of cure of both xenografts was observed following treatment with combination schedule 3. CONCLUSIONS: The combination of topotecan and [(131)I]MIBG compared with either treatment alone gave rise to greater than additive DNA damage, clonogenic cell kill, and tumor growth delay. These effects were dependent on the scheduling of the two agents.

Comparison of radiohaloanalogues of meta-iodobenzylguanidine (MIBG) for a combined gene- and targeted radiotherapy approach to bladder carcinoma.

Targeted radiotherapy using radiolabelled meta-iodobenzylguanidine (MIBG) is a promising treatment option for bladder cancer, restricting the effects of radiotherapy to malignant cells thereby increasing efficacy and decreasing morbidity of radiotherapy. We investigated the efficacy of a combined gene therapy and targeted radiotherapy approach for bladder cancer using radiolabelled MIBG. The effectiveness of alternative radiohalogens and alternative preparations of radiolabelled MIBG for this therapeutic strategy were compared. Bladder cancer cells, EJ138, were transfected with a gene encoding the noradrenaline transporter (NAT) under the control of a tumour specific telomerase promoter, enabling them to actively take up radiolabelled MIBG. This resulted in tumour-specific cell kill. Uptake and retention of radioactivity in cells transfected with the NAT gene were compared with that obtained in cells transfected with the sodium iodide symporter (NIS) gene. Substantially greater uptake and longer retention of radioactivity in NAT-transfected cells was observed. Carrier-added (c.a.) [131I]MIBG, no-carrier added (n.c.a.) [131I]MIBG, and [211At]-labelled benzylguanidine (i.e. [211At] meta-astatobenzylguanidine (MABG)) were compared with respect to efficiency of induction of cell kill. N.c.a[(131)I]MIBG was more cytotoxic than c.a.[131I]MIBG. However, the alpha-emitter [211At]MABG was, by three orders of magnitude, more effective in causing tumour cell kill than the beta-emitter [131I]MIBG. We conclude that NAT gene transfer combined with the administration of n.c.a.[131I]MIBG or [211At]MABG, is a promising novel treatment approach for bladder cancer therapy.

An efficient targeted radiotherapy/gene therapy strategy utilising human telomerase promoters and radioastatine and harnessing radiation-mediated bystander effects.

BACKGROUND: Targeted radiotherapy achieves malignant cell-specific concentration of radiation dosage by tumour-affinic molecules conjugated to radioactive atoms. Combining gene therapy with targeted radiotherapy is attractive because the associated cross-fire irradiation of the latter induces biological bystander effects upon neighbouring cells overcoming low gene transfer efficiency. METHODS: We sought to maximise the tumour specificity and efficacy of noradrenaline transporter (NAT) gene transfer combined with treatment using the radiopharmaceutical meta-[(131)I]iodobenzylguanidine ([(131)I]MIBG). Cell-kill was achieved by treatment with the beta-decay particle emitter [(131)I]MIBG or the alpha-particle emitter [(211)At]MABG. We utilised our novel transfected mosaic spheroid model (TMS) to determine whether this treatment strategy could result in sterilisation of spheroids containing only a small proportion of NAT-expressing cells. RESULTS: The concentrations of [(131)I]MIBG and [(211)At]MABG required to reduce to 0.1% the survival of clonogens derived from the TMS composed of 100% of NAT gene-transfected cells were 1.5 and 0.004 MBq/ml (RSV promoter), 8.5 and 0.0075 MBq/ml (hTR promoter), and 9.0 and 0.008 MBq/ml (hTERT promoter), respectively. The concentrations of radiopharmaceutical required to reduce to 0.1% the survival of clonogens derived from 5% RSV/NAT and 5% hTERT/NAT TMS were 14 and 23 MBq/ml, respectively, for treatment with [(131)I]MIBG and 0.018 and 0.028 MBq/ml, respectively, for treatment with [(211)At]MABG. CONCLUSIONS: These results indicate that the telomerase promoters have the capacity to drive the expression of the NAT. The potency of [(211)At]MABG is approximately three orders of magnitude greater than that of [(131)I]MIBG. Spheroids composed of only 5% of cells expressing NAT under the control of the RSV or hTERT promoter were sterilised by radiopharmaceutical treatment. This observation is indicative of bystander cell-kill.