Conditionally replicative adenoviral vectors for imaging the effect of chemotherapy on pancreatic cancer cells

Abstract

Pancreatic cancer has a poor prognosis after complete macroscopic resection combined with chemotherapy. Even after neoadjuvant chemotherapy, R0 resection is often not possible. Moreover, current imaging techniques cannot reliably distinguish viable cancer cells from scar tissue at the resectional margin. We investigated the use of a conditionally replicative adenovirus (CRAd), Ad5/3Cox2CRAd-ΔE3ADP-Luc, for imaging the effects of chemotherapy. The CRAd infectivity of pancreatic cancer cells was enhanced by a chimeric Ad5/3 fiber, E1A expression was under the control of the Cox2 promoter, and the luciferase gene was inserted adjacent to the adenovirus death protein (ADP) gene. Subcutaneous xenografts of the pancreatic cancer cell line MiaPaCa-2 were established in 24 BALB/c nu/nu mice. When xenografts reached a diameter of 4-6 mm (day 1), the mice were injected i.p. with either PBS (group A; n = 12) or 1000 mg/kg gemcitabine (group B; n = 12), weekly. On days 19, 26, 33, and 40, CRAd were injected intratumorally into three mice in groups A and B. Bioluminescence was imaged 72 h after CRAd injection, and gross tumor volumes were measured then tumors were removed for ex vivo histopathology using H&E and Ki-67 staining. Correlations between gross tumor volume, pathological evaluation of the percentage of viable tumor area, and CRAd bioluminescence were analyzed. Bioluminescence correlated closely with the percentage of viable tumor area (R = 0.96), but not with gross tumor volume (R = 0.31). Therefore, CRAds might be reliable imaging tools for monitoring chemotherapy in pancreatic cancer, and could improve our ability to distinguish viable tumor cells from scar tissue.

Figure 1

Structure of the conditionally replicative adenovirus Ad5/3‐Cox2CRAd‐ΔE3‐ADP‐Luc. A chimeric Ad5/3 fiber enhances infectivity, a luciferase reporter gene is inserted at the E3 region deletion, and an adenovirus death protein (ADP) gene is in its native location. L‐ITR, left inverted terminal repeat; R‐ITR, right inverted terminal repeat.

Figure 2

Cytocidal effect of gemcitabine (GEM) and tegafur/gimeracil/oteracil (TS‐1) on pancreatic cell lines. MiaPaCa‐2, Panc‐1, and BxPC‐3 cells were plated into 96‐well plates at 2 × 10⁴ cells/mL in 100 μL serum‐containing medium per well. After 24 h, an additional 100 μL medium containing 0.01 μg/mL GEM or 0.5 μg/mL TS‐1 was added, and MTT assays were carried out after 24, 48, 72, and 96 h.

Figure 3

Protocol for assessing the use of conditionally replicative adenovirus (CRAd), Ad5/3‐Cox2CRAd‐ΔE3‐ADP‐Luc, as an in vivo imaging diagnostic, showing the timing of i.p. gemcitabine (GEM) injection, intratumoral injection of CRAd, bioluminescent imaging, and ex vivo histopathology.

Figure 4

Each cell line was infected with conditionally replicative adenovirus Ad5/3‐Cox2CRAd‐ΔE3‐ADP‐Luc at 10 viral particles per well. Luciferase activity was analyzed daily for 7 days.

Figure 5

Bioluminescent imaging of conditionally replicative adenovirus (CRAd) Ad5/3‐Cox2CRAd‐ΔE3‐ADP‐Luc replication after intratumoral injection into MiaPaCa‐2 xenografts. BALB/c nu/nu mice were inoculated with MiaPaCa‐2 cells, and when tumor nodules achieved a diameter of 6–10 mm, 1.0 × 10¹⁰ viral particles CRAd(Ad5/3‐Cox2CRAd‐ΔE3‐ADP‐Luc) were injected into tumors. The luciferase signal from the tumors was monitored from day 1.

Figure 6

Control results for the in vivo use of conditionally replicative adenovirus (CRAd) Ad5/3‐Cox2CRAd‐ΔE3‐ADP‐Luc to monitor the pancreatic cancer cell response to gemcitabine. Established MiaPaCa‐2 xenografts in BALB/c nu/nu mice were injected i.p. with PBS weekly, then on days 33 (a) and 40 (b), 1.0 × 10¹⁰ viral particles CRAd(Ad5/3‐Cox2CRAd‐ΔE3‐ADP‐Luc) in 50 μL PBS were injected intratumorally. On day 36 (a) and day 42 (b), tumors (upper left panel) and bioluminescence were measured (lower left panel), and tumors were removed and stained with H&E (upper right panel) and Ki‐67 (lower right panel). (c) Gross tumor volume and CRAd bioluminescence were closely correlated (R = 0.94).

Figure 7

In vivo use of conditionally replicative adenovirus (CRAd) Ad5/3‐Cox2CRAd‐ΔE3‐ADP‐Luc to monitor the pancreatic cancer cell response to gemcitabine. Established MiaPaCa‐2 xenografts in BALB/c nu/nu mice were injected i.p. with gemcitabine weekly, then on days 33 (a) and 40 (b), 1.0 × 10¹⁰ viral particles CRAd(Ad5/3‐Cox2CRAd‐ΔE3‐ADP‐Luc) in 50 μL PBS were injected intratumorally. On day 36 (a) and day 42 (b), tumors (upper left panel) and bioluminescence were measured (lower left panel), and the tumors were removed and stained with H&E (upper right panel) and Ki‐67 (lower right panel). (c) Xenograft growth was monitored by gross tumor volume, by% viable tumor area and CRAd bioluminescence. The correlations between these measurements were analyzed using Pearson's correlation coefficient. Bioluminescence correlated closely with% viable tumor area (R = 0.96) but not with gross tumor volume (R = 0.31).