Multiple treatment cycles of liposome-encapsulated adenoviral RIP-TK gene therapy effectively ablate human pancreatic cancer cells in SCID mice

Abstract

Background: Adenoviral gene therapy has been applied widely for cancer therapy; however, transient gene expression as result of humoral immunoneutralization response to adenovirus limits its effect. The purpose of this study is to determine whether DOTAP:cholesterol liposome could shield adenovirus from neutralizing antibody and permit the use of multiple cycles of intravenous liposome encapsulated serotype 5 adenoviral rat insulin promoter directed thymidine kinase (L-A-5-RIP-TK) with ganciclovir (GCV) to enhance its effect.

Methods: The effect of multiple cycles of systemic L-A-5-RIP-TK/GCV therapy was evaluated in grouped PANC-1 SCID mice treated with different numbers of cycles. Humoral immune response to A-5-RIP-TK or L-A-5-RIP-TK was assessed using C57/B6J mice challenged with adenovirus or liposome adenovirus complex.

Results: The minimal residual tumor burden (3.2 ± 0.6 mm(3)) and greatest survival time (153.0 ± 6 days) were obtained in the mice receiving 4 and 3 cycles of therapy, respectively. Toxicity to islet cells associated with RIP-TK/GCV therapy was observed after 4 cycles. DOTAP:chol-encapsulated adenovectors were able to protect adenovectors from the neutralization of high titer of anti-adenoviral antibodies induced by itself.

Conclusion: Multiple treatment cycles of L-A-5-RIP-TK/GCV ablate human PANC-1 cells effectively in SCID mice; however, the mice become diabetic and have substantial mortality after the 4th cycle. Liposome-encapsulated adenovirus is functionally resistant to the neutralizing effects of anti-adenoviral antibodies, suggesting feasibility of multiple cycles of therapy. Liposome encapsulation of the adenovirus may be a promising strategy for repeated delivery of systemic adenoviral gene therapy.

FIG. 1

Adenoviral type 5 RIP-TK expression in PANC-1 tumors. A-5-RIP-TK was intravenously administered to SCID mice 2 mo after inoculation with PANC-1 human pancreatic cancer cells. L-A-5-RIP-TK was repeated administrated on day 22, 43 after initial delivery, three gene deliveries in total. Mice were sacrificed on day 7, 14, 21d after each A or L-A-5-RIP-lacZ administration, respectively. The tumor tissues were processed and sectioned as usual. Immunostaining was performed using anti-HSV-TK antibody. Red staining cells indicate strong expression of HSV-TK in tumor cells. The figure showed the TK gene expression profile in control group (top row), first gene delivery (next to top row), second delivery (third row from top) and third delivery (bottom row) (x200)

FIG. 2

Multiple treatment cycles of A or/and L-A-5-RIP-TK/GCV treatment suppresses growth of human pancreas cancer xenografts. Two weeks after i.p. injection of PANC-1 cells, SCID mice were divided into five groups (30 animals/group) and treated as follows: (1) 4 cycles of GCV, (2) 1 cycle of A-5-RIP-TK/GCV, (3) 1 cycle of A-5-RIP-TK/GCV+1 cycles of L-A-5-RIP-TK/GCV, (4) 1 cycle of A-5-RIP-TK/GCV+2 cycles of L-A-5-RIP-TK/GCV, (5) 1 cycle of A-5-RIP-TK/GCV+3 cycles of L-A-RIP-TK/GCV. At 4 months after treatment, at least five mice were sacrificed. Percentages of tumor-free animals in different groups were compared using χ2-test significance (A)Tumor size was measured and compared using Student's t-test (B) with P< 0.05 representing significance. Tumor growth was significantly suppressed in all treated mice compared with controls.

FIG. 3

Multiple treatments with A or/and L-A-5-RIP-TK/GCV prolongs survival of PANC-1 tumor bearing mice. PANC-1 tumor mice were grouped to receive (1) 4 cycles of GCV, (2) 1 cycle of A-5-RIP-TK/GCV, (3) 1 cycle of A-5-RIP-TK/GCV+1 cycles of L-A-5-RIP-TK/GCV, (4) 1 cycle of A-5-RIP-TK/GCV+2 cycles of L-A-5-RIP-TK/GCV, (5) 1 cycle of A-5-RIP-TK/GCV+3 cycles of L-A-RIP-TK/GCV. Mice survival was estimated by using the Kaplan-Meier and log rank tests. All the treatment group had significant prolonged survival as compared to control group treated with GCV. Three cycle of A and L-A-5-RIP-TK/GCV treatment obtained longest survival among all of groups (P<0.05).

FIG. 4

Multiple cycles of L-A-5-RIP-TK/GCV treatments cause diabetic. Fasting serum was collected on day 7 after each L-A-RIP-TK/GCV gene therapy. The glucose (green line) and insulin (red line) levels are shown at different L-A-5-RIP-TK/GCV treatment cycles. Glucose levels increased while insulin levels decreased (A). The changes of insulin and glucose levels were correlation with islet cell apoptosis in pancreas of treated mice (B)

FIG. 5

Repeated L-A-5-RIP-TK administration induced anti-adenovirus antibodies. C57BL/6J mice without prior exposure to adenovirus were treated with naked A-5-RIP-TK or L-A-5-RIP-TK. Repeated naked A-5-RIP-TK or L-A-5-RIP-TK was administrated in a 3 weeks interval, 3 doses in total. 7days after each administration, the serum was collected for measurement of neutralized antibody. Either A-5-RIP-TK or L-A-5-RIP-TK induced high titer of anti adenoviral antibody. Repeated administration of adenovirus resulted in increased antibody titers.

FIG. 6

Liposome shield adenovirus from neutralizing antibody. C57BL/6J mice without prior exposure to adenovirus were treated with naked adenoviral LacZ (A-5-CMV-lacZ) and liposome encapsulated adenoviral lacZ (L-A-5-CMV-lacZ). Repeated A-5-CMV-lacZ or L-A-5-CMV-LacZ was administrated on day 21 after initial injection of naked A-5-RIPTK or L-A-RIP-TK vector. The β- gal expression was evaluated at 7, 21 and 30d following initial adenoviral lacZ administration and day 7 after repeated adenovirus administration by x-gal staining for liver cells as previous described. Blue staining cells indicate positive for β-gal expression. (X200)