Lentivirus-mediated CDglyTK gene-modified free flaps by intra-artery perfusion show targeted therapeutic efficacy in rat model of breast cancer

Abstract

Background: Free flap-mediated gene therapy in the tumor bed following surgical resection is a promising approach in cancer targeted treatment of residual disease. We investigated the selective killing efficacy of a lentivirus-mediated cytosine deaminase-thymidine kinase (CDglyTK) gene in transplanted breast cancer delivered into a free flap by intra-artery perfusion.

Methods: Proliferation, apoptosis, and cell cycle of rat SHZ-88 breast cancer cells transfected with a lentivirus-mediated CD/TK gene were measured following treatment with ganciclovir and 5-flucytosine in vitro. A model of residual disease of breast cancer in a rat superficial inferior epigastric artery (SIEA) flap model was used to study the therapeutic potential of a double suicide CD/TK and prodrug system in vivo.

Results: Killing efficacy of the double suicide CD/TK and prodrug system on SHZ-88 cells was mediated by increased apoptosis and cell cycle arrest at the G1 phase with significant bystander effect. Following recombinant lentivirus transfection of rat SIEA flap by intra-artery perfusion, CD/TK gene expression was limited to the flap, and the volume and weight of transplanted tumors were significantly reduced without observable toxicity.

Conclusions: SIEA flaps transfected with a lentivirus-mediated CDglyTK gene by intra-artery perfusion effectively suppress transplanted breast tumor growth without obvious systemic toxic effects in rats.

Keywords: Breast cancer; CDglyTK gene; Free flap; Intra-artery perfusion; Lentiviral vector; Suicide gene therapy.

Fig. 1

Construction of superficial inferior epigastric (SIEA) flaps transfected with LV-CMV-CDglyTK in a Sprague-Dawley rat model. (upper left panel) The SIEA flap was designed with a 2.5 × 2.5-cm skin paddle. (upper middle panel and upper right panel) The SIEA flap was dissected carefully as described previously. (lower left panel) After the flap pedicle was divided, the afferent artery to the flap was catheterized with a 0.4 mm micro cannula. (lower middle panel) After perfusion of recombinant virus or phosphate-buffered saline through the afferent artery and incubated, the native vessels of the flap were re-anastomosed. (lower right panel) The flap was oriented in its original position in the groin and the skin incisions were closed

Fig. 2

Transduction efficiency and expression of lentivirus-mediated CDglyTK gene in SHZ-88 cells. a Lentivirus-delivered gene transfer efficiencies in SHZ-88 cells. b The effect of the recombined lentivirus on growth of transfected cells. c Reverse transcriptase polymerase chain reaction analysis of CD/TK fusion gene expression in transfected cells. d Protein expression of the CD/TK double suicide gene by western blot in transfected cells. Data are presented as the mean ± SD, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. Three replicates were done in each experiment. LV-CD/TK, SHZ-88 cells transfected with lentivirus-mediated CDglyTK gene; LV-GFP, SHZ-88 cells transfected with empty lentivirus; Control, non-transfected SHZ-88 cells; CD, cytosine deaminase; TK, thymidine kinase

Fig. 3

Effects of the double suicide CD/TK on the selective killing efficacy on SHZ-88 cells. a The cytotoxic effects of ganciclovir (GCV) and 5-flucytosine (5-FC) to transgenic cells. b The cytotoxic effects of different prodrugs to transgenic cells. c Cell apoptosis assay of 5-FC + GCV on transgenic cells. d Cell cycle analysis of 5-FC + GCV on transgenic cells. e Bystander effect analysis of the CD/TK fusion gene and prodrug system on SHZ-88 cells. Data are expressed as the mean ± SD, n = 3. & P < 0.001 compared to the LV-GFP group; #P < 0.001 compared to the control group; **P < 0.01; ***P < 0.001; LV-CD/TK, CDglyTK fusion gene transfected group; LV-GFP, empty lentivirus transfected group; Control, the untransfected group. Three replicates were done in each experiment

Fig. 4

Expression of a CDglyTK fusion gene in gene-modified superficial inferior epigastric (SIEA) flaps. a Position of the pedicle, flap caudal, and flap cephalic in the free SIEA flap. b Reverse transcriptase polymerase chain reaction analysis of CD/TK fusion gene in SIEA flaps transfected with different recombinant lentivirus. c Protein expression of CD/TK double suicide gene by western blot in transfected flaps. Data was shown as the mean ± SD of three independent samples. LV-CDglyTK, flaps transfected with lentivirus-mediated CDglyTK gene; LV-GFP, flaps transfected with empty lentivirus; Control, non-transfected flaps

Fig. 5

The lentivirus-mediated CDglyTK fusion gene by intra-artery perfusion effectively suppresses transplanted breast cancer in vivo. (upper left panel) Tumor growth curve. (upper middle panel) Appearance and (upper right panel) weight of transplanted tumors at 42 days after treatment. (lower panel) Histology analysis of the transplanted tumors by hematoxylin and eosin (H&E) staining. Scale bar: 100 μm. Data was shown as the mean ± SD of six independent samples. **P < 0.01; ***P < 0.001. LV-CDglyTK, flaps transfected with lentivirus-mediated CDglyTK gene; LV-GFP, flaps transfected with empty lentivirus; Control, non-transfected flaps

Fig. 6

The apoptosis assay in vivo tumor tissues caused by active drug converted from prodrug using TUNEL assay kit. a Cell apoptosis assay of the transplanted tumors by TUNEL assay. Red arrowhead indicated the positive apoptosis cells in the tumor tissue. b The percentage of apoptotic cells in tumor tissue was analyzed by Image-pro plus 6.0. Data was shown as the mean ± SD of six independent samples. Scale bar: 50 μm. *P < 0.05; **P < 0.01. LV-CD/TK, flaps transfected with lentivirus-mediated CDglyTK gene; LV-GFP, flaps transfected with empty lentivirus; Control, non-transfected flaps

Fig. 7

The lentivirus-mediated CDglyTK gene by intra-artery perfusion causes no discernable systemic toxic effects in vivo. a Reverse transcriptase polymerase chain reaction analysis of CD/TK fusion gene expression in the underlying flap bed and major internal organs. Analysis of serum b ALT and c AST over a 42-day post-operative period. d Assessment of the inflammatory response in major internal organs in the experimental group by hematoxylin and eosin (H&E) staining. Scale bar: 100 μm. Data was shown as the mean ± SD of six independent samples. LV-CDglyTK, flaps transfected with lentivirus-mediated CDglyTK gene; LV-GFP, flaps transfected with empty lentivirus; Control, non-transfected flaps