Introduction

Pancreatic carcinoma (PC) is the fourth leading cause of cancer mortality in the United States, with more than 31,000 deaths attributed to the disease each year¹. Moreover, the incidence of PC has been steadily increasing in the United States, rising nearly threefold since 1920^2,3. Despite aggressive surgical and chemotherapeutic treatment regimens, PC remains one of the deadliest malignancies, with an overall mortality of 90%^4,5. Although systemic chemotherapy is the main treatment for liver and peritoneal metastases, it is only marginally effective, and the median survival for patients with Stage IV disease is less than 3 to 5 months⁶. Clearly, more effective therapeutic approaches are needed.

PC is known to be strongly associated with genetic abnormalities, including activation of the oncogene K-ras and downregulation of multiple tumor suppressor genes such as p53, p16/CDK2, and DPC4^7,8,9. Recently, several pilot gene therapy strategies targeting specific molecular defects have been evaluated for PC^10,11. These studies confirm the potential of gene therapy for the treatment of pancreatic cancer and suggest that one of the major limitations for the clinical application of gene therapy in this setting is the inability to deliver therapeutic genes efficiently to PC in vivo. This limitation underscores the importance of developing clinically relevant animal models to study the efficacy and mechanisms of PC gene therapy in vivo.

We have focused on the gene encoding the cancer-associated Sm-like protein (CaSm)¹². CaSm is overexpressed in the majority (87.5%) of pancreatic cancers^12,13 and codes for a 1.2-kb mRNA transcript, with the largest open reading frame encoding a 133-amino-acid polypeptide that contains two Sm motifs found in the LSm (like-Sm) family of proteins with a predicted molecular weight of 15 kDa. In previous studies, we have demonstrated that CaSm functions as an oncogene and that CaSm overexpression is required to maintain a neoplastic phenotype¹². Currently, the normal intracellular function of human CaSm remains to be elucidated, but its homology to yeast Sm-like protein 1 (Lsm1) suggests a potential role for CaSm in messenger RNA stability^14,15,16. Of particular interest, downregulation of CaSm expression by adenoviral vector-mediated delivery of CaSm antisense RNA (Ad-CaSm) can reduce the in vivo tumor growth of PC¹⁷. We found that downregulation of CaSm expression in PC cells resulted in reduced proliferation, primarily due to disruption of cell cycle progression, with minimal effects on apoptosis^18,19.

To evaluate rigorously the therapeutic potential of CaSm antisense gene therapy in PC, we have established a preclinical model of advanced PC based on the PC cell line Panc02 (CaSm overexpression) challenge in syngeneic mice via the portal circulation²⁰. This model consistently results in the establishment of peritoneal and hepatic metastases. We report here that systemic administration of Ad-CaSm results in successful delivery of CaSm antisense to peritoneal and hepatic metastases, resulting in a significant reduction in tumor burden and prolonged survival. Furthermore, we provide evidence that the antitumor activity elicited by CaSm downregulation is a result of both direct and bystander mechanisms. These data support the concept of CaSm downregulation as a novel gene therapy approach for the treatment of PC.

Results

Intratumoral injection of Ad-CaSm inhibits the growth of Panc02 PC in a subcutaneous tumor challenge model

We established Panc02 tumors in female C57BL/6 mice by subcutaneous injection. We monitored tumor growth closely, and when the tumors reached a volume of 100 mm³, we injected them three times (days 0, 3, and 6) with 10⁹ pfu of Ad-CaSm or Ad-LacZ or gave them the mock treatment. We monitored changes in tumor volume over a 5-week period. Both mock-treated and Ad-LacZ-injected tumors had identical growth profiles, with both groups rapidly increasing in size (Fig. 1A). In contrast, tumors treated with Ad-CaSm gradually decreased in size initially over a period of 20 days, at which point they began to increase slowly in volume (Fig. 1A). Overall, the ability of Ad-CaSm to inhibit Panc02 sc tumor growth was very significant (P < 0.001). X-gal staining revealed that cells transduced by Ad-LacZ treatments typically clustered around needle tracks (Fig. 1B), indicating relatively limited penetration of the vector into the tumor mass. Quantitative assessment of the total number of X-gal-stained cells in sections of Ad-LacZ-treated tumors indicated that approximately 20% of tumor cells were transduced under our experimental conditions (data not shown). These results suggest that the antitumor efficacy of Ad-CaSm therapy is greater than might be expected given the transduction efficiency following intratumoral injection. The possibility that this may represent a bystander effect is explored in more detail below.

Figure 1.

Treatment of subcutaneous Panc02 tumors with Ad-CaSm significantly inhibits growth, despite only partial transduction. Subcutaneous tumors were established by injection of 5 10⁵ Panc02 cells into C57BL/6 mice. When tumors reached volumes of approximately 100 mm³, mice were treated with intratumoral injection of 100 l of PBS, 100 l of PBS containing 1 10⁹ pfu of Ad-CaSm, or 100 l of PBS containing 1 10⁹ pfu of Ad-LacZ as indicated. (A) Treatment effect on tumor growth was measured and tumor volumes are reported as means SEM (n = 10). ^**P < 0.001 compared with PBS or Ad-LacZ treatment. (B) Efficiency of intratumoral delivery was evaluated by extracting Ad-LacZ-injected tumors 2 days postinjection and staining with X-gal to detect cells transduced with Ad-LacZ. Scale bar, 250 m.

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Systemic administration of Ad-CaSm decreases the number of hepatic metastases and enhances survival in a preclinical model of advanced PC

Peritoneal and/or hepatic metastases represent the most common sites of disease progression in patients with PC²¹. To determine whether adenoviral-mediated delivery of CaSm antisense can be effective against advanced PC, we used a recently developed preclinical model of PC²⁰. In this model, metastases are established by portal circulation tumor challenge with 5 10⁶ Panc02 cells. PC metastases can be detected in the liver as early as day 3 following tumor challenge. Animals are moribund and require euthanasia as early as day 10 following tumor challenge, with a 50% mortality typically seen by day 16. One hundred percent mortality is typically seen between days 21 and 24. Postmortem examinations indicate extensive metastases throughout the liver, as well as in the spleen and pancreas. In addition, large tumors form within the omentum.

Following portal circulation tumor challenge, animals received three tail-vein injections of 1 10⁹ pfu of Ad-CaSm or Ad-LacZ on days 3, 6, and 9. We sacrificed the mice on day 19 and determined the number of hepatic metastases. Ad-CaSm treatment resulted in a significant reduction in the number of hepatic metastases compared to treatment with a control vector (23 8 vs 109 10) (Figs. 2A and 2B). Two of six Ad-CaSm-treated animals had no detectable hepatic metastases, and there was no significant difference in the number of hepatic metastases seen between saline- and control vector-treated animals (Figs. 2A and 2B).

Figure 2.

Systemic administration of Ad-CaSm results in reduced numbers of hepatic metastases and prolonged survival. Panc02 metastases were established by intrasplenic injection, and tumor-bearing mice were injected (iv) with 1 10⁹ pfu of Ad-CaSm, Ad-LacZ, or PBS on days 3, 6, and 9. (A) Phase-contrast dissecting microscopy images of the hepatic metastases (indicated by arrows) on the surface of the livers from mice sacrificed on day 19. Scale bar, 1 mm. (B) Quantitation of the number of hepatic metastases from mice sacrificed on day 19. The numbers of hepatic metastases were counted with a dissecting microscope and are indicated as means SEM (n = 6). ^**P < 0.001 compared with PBS or Ad-LacZ treatment. (C, D) Survivorship profiles of mice bearing Panc02 metastatic tumors and treated with Ad-CaSm and control mice at early stage (n = 12 in each group) and later stage (n = 10 in each group), respectively (arrows indicate injection). ^*P < 0.05 compared with PBS or Ad-LacZ treatment.

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We monitored the survival of the remaining mice over time. All of the saline- and Ad-LacZ control vector-treated animals died, or were moribund requiring euthanasia, by days 22–24 (n = 12 in each group). Ad-CaSm-treated animals had a median survival of 35 days, with one mouse surviving until day 53 (n = 12) (Fig. 2C). Encouraged by these initial results, we also evaluated the efficacy of Ad-CaSm treatment given at delayed time points, to approximate more closely PC patients carrying an extensive tumor burden. Hence, we treated mice with Ad-CaSm or control vector on days 10, 13, and 16 following portal circulation tumor challenge. Survival of both saline- and Ad-LacZ-treated mice was the same as for the early treatment protocol—with a median survival between 17 and 24 days. Treatment with Ad-CaSm prolonged the maximum survival to 34 days and median survival to 26 days (n = 10 in each group) (Fig. 2D). Animals did not exhibit any obvious negative physiological responses or toxicity to any of the treatments.

Systemic administration of Ad-CaSm results in enhanced transduction of tissues containing Panc02 metastases

To determine the transduction efficiency following systemic administration of Ad-CaSm, we treated animals with tail-vein injection of 1 10⁹ pfu of Ad-LacZ or Ad-GFP on days 3, 6, and 9 following portal circulation tumor challenge. We sacrificed the mice on day 12 and examined the organs from the abdominal cavity (including the omentum) for the presence of metastases by hematoxylin and eosin (H&E) staining and for transduction efficiency by X-gal staining. We saw significantly higher transduction efficiencies in hepatic tissues containing Panc02 metastases compared with normal livers, with X-gal staining specifically appearing to be focally increased in the PC metastases (Fig. 3A). We also observed elevated transduction efficiencies in omental tissues containing PC metastases (Fig. 3A). Quantitative assessment of the total numbers of positively stained cells (mixture of hepatocytes and pancreatic tumor cells) revealed that 37% of cells in tumor-bearing livers were transduced, compared to 18% in normal livers (Fig. 3B). In omental tumors, transduced cell numbers (15 to 17%) were very similar to those in normal liver (Fig. 3B) and significantly higher than in any other tissues, such as lung or kidney (<5% transduction). We confirmed these findings by a quantitative analysis of GFP expression in different tissues following injection of Ad-GFP virus. We saw the highest GFP fluorescence levels per gram of tissue in diseased livers containing PC metastases, followed by normal livers and diseased omentum (Fig. 3C). Globally, we noted increased GFP fluorescence in diseased tissues containing PC metastases compared to normal tissues (Fig. 3C). While these results do indicate some specificity of targeting to tissues containing PC, they suggest that direct targeting of individual tumor cells may not be the only mechanism contributing to the antitumor efficacy of CaSm antisense gene therapy. The possibility of a bystander effect mediated by CaSm antisense gene therapy is explored below.

Figure 3.

Systemic administration of Ad5-based vectors results in enhanced gene delivery to Panc02 metastases relative to normal tissues. C57BL/6 mice received intrasplenic injections of 5 10⁶ Panc02 cells or PBS. Animals were injected three times (iv) with 1 10⁹ pfu of (A and B) Ad-LacZ or (C) Ad-GFP on days 3, 6, and 9. Gene expression was evaluated on day 12. (A) Cryosections of livers with or without Panc02 metastases and of a large Panc02 metastatic tumor established in the omentum stained with H&E and with X-gal. Scale bar, 250 m. (B) Estimation of cells positively stained with X-gal from five fields of the indicated tissues. Percentage of X-gal-positive cells is reported as means SEM (n = 3). (C) Quantification of Ad-vector-mediated delivery by determining GFP fluorescence in various tissues. Li, liver; Om, omental tumor; Pn, pancreas; Sp, spleen; Kd, kidney; Lu, lung; Hr, heart. Background fluorescence (calculated by determining fluorescence of same tissues from control mice injected with PBS) was subtracted from experimental values, and reported values are mean relative fluorescence units (RFU) per gram of tissue SEM (n = 3). ^*P < 0.05 or ^**P < 0.001 compared with same tissue without tumor.

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Bystander effect contributes to the antitumor efficacy of CaSm antisense gene therapy

The data presented above suggest that the antitumor efficacy of CaSm antisense gene therapy is significantly greater than would be expected based upon the transduction efficiencies observed. One hypothesis to explain these results is that CaSm downregulation induces a local bystander effect, inhibiting the growth of untransduced cells. To address this hypothesis, we performed tumor challenge experiments with mixtures of transduced and untransduced Panc02 PC cells. First, we evaluated transducibility of Panc02 cells with Ad-LacZ with a dose–response experiment (Fig. 4A). Subsequently, cultured cells were transduced at a multiplicity of infection (m.o.i.) of 100, resulting in transduction of approximately 90% of Panc02 cells (Fig. 4A). Initial experiments indicated that tumor volumes at the time of analysis were proportional directly to the number of viable cells used to establish the tumor and that pretreatment of up to 50% of the cells with Ad-LacZ had no impact on the efficiency of tumor formation (Fig. 4B). Panc02 cells pretreated with Ad-LacZ established tumors with an efficiency similar to that of untreated cells and grew at the same rate. On the other hand, pretreatment with Ad-CaSm resulted in complete loss of the ability to establish tumors (Fig. 4B). Next, we examined the impact of mixing a constant number (5 10⁵) of untreated Panc02 cells with different numbers of cells treated with Ad-CaSm, which do not contribute to tumor establishment and growth (Fig. 4B). Pretreatment of 50% of Panc02 cells with Ad-CaSm inhibited tumor growth of the mixed tumor cell population up to 80% compared to tumors initiated by untransduced Panc02 cells alone (Fig. 4C) or by a mixture of untransduced and Ad-LacZ-transduced Panc02 cells (Fig. 4B). The efficiency with which tumor growth was suppressed correlated with the percentage of Panc02 cells pretreated with Ad-CaSm in the tumor-initiating population (Fig. 4C).

Figure 4.

Ad-CaSm-transduced Panc02 cells suppress the tumorigenic potential of untransduced cells. (A) Cultured Panc02 cells are efficiently transduced by Ad vectors. Cells were transduced at the indicated m.o.i. with Ad-LacZ and stained with X-gal 36 h later. Transduced and untransduced cells were scored as described under Materials and Methods. Percent transduction is reported as the mean SEM (n = 3). (B) Tumor growth is proportional to the number of untreated cells in the establishing population, and tumor growth is unaffected by preinfection with Ad-LacZ but eliminated by preinfection with Ad-CaSm. C57BL/6 mice were subcutaneously injected on day 0 with Panc02 cells that were either untreated or pretreated (m.o.i. of 100) 16 h previously, as indicated. Tumor volumes on day 15 are reported as means SEM (n = 6). ^**P < 0.001 compared with untreated or Ad-LacZ-pretreated cells. (C) Tumor volume decreases with the increasing numbers of Ad-CaSm-treated cells in the establishing population. Tumors were established and evaluated as described for B. ^*P < 0.05 compared with 5 10⁵ untreated cells only.

Full figure and legend (84K)

These results supported the hypothesis that the antitumor efficacy of CaSm antisense gene therapy in vivo was being enhanced by a bystander effect. To characterize this effect further, we wanted to determine if we could reproduce this phenomenon in vitro and assess whether it was being mediated by a soluble factor. We collected conditioned medium (CM) from cultured Panc02 cells that were transduced with Ad-CaSm or appropriate controls. Then, we treated the CM with adenovirus-neutralizing antibody 1D6.14 to negate the effect of any potential vector carryover. Antibody 1D6.14 has previously been shown to inhibit adenoviral infection by binding to the fiber knob and inhibiting attachment to the coxsackie adenovirus receptor²². We then mixed the CM with fresh medium at the concentration indicated and added it to untransduced Panc02 cells. We measured the cell viability by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) assay after 96 h. As shown in Fig. 5A, CM from Ad-CaSm-treated Panc02 cells resulted in a dose-dependent decrease in the number of viable untransduced Panc02 cells. Undiluted Ad-CaSm CM (100% CM) resulted in an 80% reduction in the number of viable cells, compared to a 4% reduction resulting from 100% culture with conditioned medium from the Ad-LacZ-transduced Panc02 cells (Fig. 5A). To investigate further the mechanism responsible for this loss of cell viability, we analyzed the levels of apoptosis in untransduced Panc02 cells following treatment with CM. After 72 h of culture in the CM, Ad-CaSm CM was able to induce apoptosis in untransduced cells in a dose-dependent manner (Fig. 5B), and the levels of apoptosis were comparable to the losses of cell viability at each level of CM treatment. This effect was unlikely to be caused by Ad-CaSm carryover to fresh Panc02 cells, since under our experimental conditions (including treatment with Ad-neutralizing antibody 1D6.14), less than 2% of cells were transduced when treated with 100% CM from Ad-LacZ-transduced cells (Fig. 5C).

Figure 5.

The bystander effect induced in Ad-CaSm-transduced Panc02 cells is mediated by a soluble factor. Conditioned media were prepared from Panc02 cells transduced with Ad-CaSm, Ad-LacZ, or untransduced cells. (A) Panc02 cells were treated for 96 h with (lane 1) 100% CM from Ad-LacZ-infected cells or CM from Ad-CaSm-infected cells at (lane 2) 25%, (lane 3) 50%, or (lane 4) 100%. Cell viability was determined using the MTT assay. Data represent % of viability as means SEM (n = 3). ^*P < 0.05 or ^**P < 0.001 compared with 100% CM from Ad-LacZ-infected cells. (B) Panc02 cells were treated for 72 h with 100% CM from Ad-LacZ- or Ad-CaSm-infected cells as indicated in (A). The % of cells undergoing apoptosis was determined by flow cytometry with propidium iodide staining. Data points represent % of cells with a sub-G1 DNA content as means SEM (n = 3). ^*P < 0.05 compared with 100% CM from Ad-LacZ-infected cells. (C) The extent of Ad vector carryover as determined by X-gal staining of Panc02 cells treated with 100% CM from Ad-LacZ-transduced cells and with Ad fiber knob-binding (neutralizing) 1D6.14 antibody or mouse IgG_2a (isotype control; BD Biosciences). ^*P < 0.05 compared with isotype control antibody.

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Discussion

In a previous study, we demonstrated that mCaSm mRNA and protein levels were significantly decreased in cultured Panc02 cells after Ad-CaSm treatment²⁰. Here, we show that in a syngeneic subcutaneous tumor model, intratumoral injections of Ad-CaSm (but not control vector) effectively reduced the growth rate of the otherwise rapidly growing tumors, despite the fact that we could confirm transduction of only one in five tumor cells. These initial findings indicated that downregulation of CaSm may be inducing a bystander effect in vivo, with the implication that it may not be necessary to achieve 100% delivery to reduce the growth of PC effectively in vivo.

The efficacy of CaSm antisense gene therapy was also evaluated in a more clinically relevant model of advanced PC, based on the portal circulation tumor challenge model that we have established. In this model, metastases are initiated by intrasplenic injection of Panc02 cells and are consistently detected in the liver, spleen, pancreas, and omentum. Disease progression is rapid, with mice dying or requiring euthanasia within 24 days of tumor challenge. This model is well suited to investigate one of the major obstacles to successful implementation of cancer gene therapy—the inefficient delivery of currently available vectors to the target cancer cells. This is especially true for metastatic disease, in which the only conceptually viable approach is systemic administration of the vector. The vector must travel via the circulatory system to the site of tumor growth, transverse the blood vessel wall, and transduce tumor cells at a sufficiently high efficiency and with enough relative specificity to be able to elicit a beneficial antitumor response. Previous studies have demonstrated that vectors based on Ad2 and Ad5 are rapidly cleared from the bloodstream by the reticular endothelial system, and the majority of detectable virus expression is in the liver, due to the highly permeable nature of hepatic vasculature and the high receptor expression levels on hepatocytes^{23,24,25,26,27,28}. Delivery to other organs and tissues is much less efficient, and the dose of administration is limited by the fact that adenovirus capsid proteins are hepatotoxic at high concentrations²⁸. However, many types of tumors also have a vasculature that is far more permeable than that of normal tissue^29,30, which suggests the possibility that systemic delivery of Ad vectors may at least semipreferentially target metastatic PC. Indeed, we found that Panc02 metastases in liver, omentum, and pancreas could be transduced with Ad vectors with an efficiency that was significantly higher than that of the corresponding normal tissues. However, we did not ascertain the specific identity of the transduced cells, which may be part of tumor vasculature rather than Panc02 in origin. Nevertheless, systemic Ad-CaSm therapy produced a significant reduction in the number of hepatic metastases, as well as an impressive increase in median survival in this model. Our findings are consistent with both the reasonably effective targeting of Panc02 metastases by the Ad-CaSm vector and the significant antitumor activity of antisense CaSm in Panc02-derived tumors. This is noteworthy for several reasons. First, from a clinical perspective, patients with PC commonly have liver metastases, and a therapeutic agent that has enhanced efficacy against metastases at this site is very desirable. Second, no treatment-related toxicity or adverse events related to Ad-CaSm therapy were seen in mice treated with the vector within the scope of our experimental setup. This observation agrees with previous reports that found that antisense CaSm expression had no negative impact on cells expressing either no or very low levels of CaSm in vitro (²⁰ and Y. Yan, unpublished data). Interestingly, following multiple systemic Ad vector deliveries, there was no apparent toxicity associated with inflammation due to adenovirus capsid proteins or significant T cell infiltration in the liver, despite the immunocompetent status of these mice. Finally, a significant antitumor effect was observed following systemic administration, despite the fact that less than half of the tumor cells were transduced. This suggested that downregulation of CaSm in transduced cells elicited a bystander effect, whereby untransduced cells in proximity to those infected with Ad-CaSm were also negatively affected. A bystander effect can result from a number of different mechanisms, both direct and indirect^31,32,33. In some cases, the use of gene therapy vectors can lead to the overexpression of cytotoxic molecules, such as Fas ligand or ceramide, on targeted cells, which can then affect neighboring cells³⁴. Alternatively, transduced cells can release antiangiogenic factors to suppress the growth of the entire tumor or, in the process of their own death, prime the immune system against tumor antigens and activate T cells specific for untransduced cells^{35,36,37,38,39,40,41}.

We have observed that mixing untreated Pan02 cells with those preinfected with Ad-CaSm resulted in a significant inhibition of sc tumor establishment and growth, further implying bystander effect activity. To define this phenomenon further, we performed additional experiments in vitro and have determined that culture medium from Panc02 cells treated with Ad-CaSm is capable of substantially reducing the viability of untreated Panc02 cells, predominantly through induction of apoptosis. Thus, it appears that at least a part of the bystander effect is mediated by a soluble factor and is therefore independent of such in vivo mechanisms as anti-angiogenesis and induction of a T-cell-mediated immune response. Ongoing studies are focused on elucidating the molecular mechanisms underlying this bystander effect and on a more thorough evaluation of Ad-CaSm safety in a murine model.

In summary, our studies in both subcutaneous and portal circulation tumor challenge models of PC demonstrate that downregulation of CaSm results in the inhibition of tumor growth and metastasis. Of particular note, systemic administration of Ad-CaSm has a significant therapeutic impact in a preclinical model of advanced PC in the absence of any obvious toxicity. As most human PC overexpress CaSm, this promising therapy is potentially widely applicable. Of course, the effectiveness with which adenoviral vectors can be delivered to metastatic PC in human patients is unknown and may differ substantially from what we have observed in this preclinical model. Therefore, it would be valuable to evaluate Ad vector delivery to primary PC isolates, including hepatic and omental metastases, and compare it to Panc02 isolates extracted from tumor-bearing mice. Overall, the results of this study suggest that molecular targeting of the oncogene CaSm is likely to lead to promising new treatment modalities for pancreatic cancer.

Materials and methods

Cell culture

The Panc02 murine PC cell line was originally established by Corbett et al.⁴². This cell line was provided by Dr. James A. Nelson (University of Texas M. D. Anderson Cancer Center, Houston, TX, USA) and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin (Gibco BRL, Rockville, MD, USA) at 37°C in 5% CO₂. Cultured cells were tested and found to be negative for mycoplasma and viral contamination.

Adenoviral vectors

All experiments using recombinant adenoviral vectors were approved by the Institutional Biosafety Committee at the Medical University of South Carolina. Recombinant adenoviral vectors expressing an antisense sequence specific for human CaSm (Ad-CaSm) or LacZ (Ad-LacZ) have been previously described¹⁷. Two types of reporter Ad vectors expressing the -galactosidase gene (Ad-LacZ) and green fluorescent protein (Ad-GFP) from a backbone similar to that of Ad-CaSm were purchased from Quantum (Montreal, QC, Canada).

Subcutaneous tumor challenge

All experiments involving mice were approved by the Animal Care and Use Committee of the Medical University of South Carolina. Mice were housed under specific-pathogen-free conditions. For subcutaneous tumor challenge experiments, 5 10⁵ Panc02 cells were injected subcutaneously into the flanks of female C57BL/6 mice. When tumors reached approximately 100 mm³ in size, all of the mice were randomly divided into groups and treated with intratumoral injection of 1 10⁹ pfu of the desired Ad vector in 100 l of PBS on days 0, 3, and 6. To examine Ad-CaSm-induced bystander effect on the growth of PC, Panc02 cells were pretreated for 16 h with Ad-CaSm, Ad-LacZ, or sham treatment at an m.o.i. of 100. Treated cells (or a mixture of treated and untreated cells) were injected subcutaneously into the flanks of female C57BL/6 mice as indicated. Animals were monitored for tumor growth, with tumor size measured by digital calipers twice a week. Tumor volume was calculated as V = L W W (/6), where L is the rostral/caudal measurement and W is the dorsal/ventral measurement.

Portal circulation tumor challenge

Panc02 cells (5 10⁶) were injected into the spleens of female C57BL/6 mice to establish a tumor model as described previously²⁰. As indicated, mice were treated systemically with 100 l of PBS containing 1 10⁹ pfu of the indicated Ad vector on days 3, 6, and 9 (or 10, 13, and 16) after Panc02 injection. The animals were monitored for health and survival as described previously²⁰. To assess the formation of hepatic metastases, animals were euthanized by CO₂ exposure. After visual inspection of the abdominal organs, the liver, spleen, omentum, lung, and pancreas were removed and fixed in 10% formalin overnight. The tissues were photographed using a Kodak digital camera (Kodak DC290, Japan) under a dissecting microscope. The tissues were embedded in paraffin, sectioned, and stained with H&E.

Histology

Tissues were snap frozen in Tissue-Tek OCT compound (Sakura Finetek UAA, Inc., Torrance, CA, USA) and then sectioned in 10-m sections using a cryostat. The sections were fixed for 10 min in a 2% formaldehyde solution. After being rinsed in PBS, cryosections were incubated overnight at 37°C in a PBS solution containing 5 mM potassium ferrocyanide, 2 mM magnesium chloride, and 1 mg/ml X-gal (Sigma, St. Louis, MO, USA). The slides were washed three times in PBS and counterstained with eosin. The stained slides were dehydrated and mounted in Permount and visualized on a light microscope using an attached Pixera Professional digital camera (Pixera Corp., Los Gatos, CA, USA). Images were captured with an attached camera linked to a computer, and the numbers of X-gal-positive cells (staining blue) and negative cells (staining red) were counted from five fields (magnification 400).

Quantitative determination of GFP fluorescence in mouse tissues

The tumor-bearing animals were given three intravenous tail vein injections of 1 10⁹ pfu Ad-GFP and sacrificed on day 3 after final vector administration. Liver, kidneys, pancreas, lungs, and heart were removed, as well as the large omental metastases. The tissues were weighed and mechanically disrupted with a tissue homogenizer in the presence of two volumes (w/v) of 1% Triton X-100 in PBS. Mixtures were centrifuged at 15,000g and cleared supernatants were decanted. GFP fluorescence in the supernatants was analyzed using a FLUOstar dual fluorescence/absorbance plate reader (BMG Labtechnologies, Durham, NC, USA). Background was calculated by determining fluorescence of the same organs from control mice injected with PBS. Background was subtracted from experimental values, which were reported as relative fluorescence units per gram of organ tissue.

In vitro proliferation/cell viability assay

Panc02 cells were seeded in flat-bottom, 96-well plates (2000 cells/well) in RPMI 1640 plus 10% FBS. The next day, CM was mixed with serum-free medium as indicated and added to Panc02 cells, and the final concentration of FBS was adjusted to 2%. Following 96 h incubation, cell viability was determined by MTT assay according to the manufacturer's instructions (Roche). Percentage viability was calculated using the formula (corrected OD of CM treated cells/corrected OD of mock control) 100%.

Preparation of conditioned medium

Panc02 cells were plated in triplicate in 175-cm² flasks (1 10⁷ cells/flask) and infected with either Ad-CaSm or Ad-LacZ at an m.o.i. of 100 in a total volume of 4 ml of FBS-free RPMI 1640 medium. After 90 min at 37°C, the virus-containing medium was removed, the cells were washed three times with PBS, and 18 ml of fresh RPMI containing 2% FBS was added. Uninfected cells were used as a control. The culture medium (which contained floating debris) was harvested at different time points (24, 48, and 72 h) and centrifuged at 2000g for 10 min. Following centrifugation, the supernatant was filtered through a 0.22-m filter and stored at -80°C. Before use, the CM was pretreated as indicated with 26 ng/l of the anti-fiber knob neutralizing antibody 1D6.14 (gift from Dr. James S. Norris, MUSC) for 1 h at 25°C⁴³.

Detection of apoptosis

Panc02 cells were plated in six-well plates at a concentration of 2 10⁵ cells/well. CM prepared from Ad-CaSm- or Ad-LacZ-transduced Panc02 cells was then added at the concentrations indicated, and the final concentration of FBS was adjusted to 2%. The cells were then cultured for 48 to 72 h. Floating and adherent cells were collected in ice-cold PBS and fixed in 70% ethanol overnight at -20°C, stained with propidium iodide at a concentration of 50 g/ml (Sigma), and analyzed using a FACSCalibur flow cytometer (Becton–Dickinson, San Diego, CA, USA). Cells with a sub-G1 DNA content were considered to have undergone apoptosis and were reported as a percentage of the total cell population.

Statistics

One-way ANOVA was used to determine significant differences among groups. The T statistic was used to determine significant differences between two groups. Kaplan–Meier survival analysis was used to determine survival differences between treatment and control groups using SigmaStat software. A P value less than or equal to 0.05 was considered to be statistically significant.

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Acknowledgements

We thank Dr. David N. Lewin (Department of Pathology, Medical University of South Carolina) for technical support and Dr. Demetri Spyropolous and James Nicholson (Department of Pathology and Laboratory Medicine, Medical University of South Carolina) for photographic assistance. We also thank Dr. James A. Nelson (The University of Texas M. D. Anderson Cancer Center, Houston, TX) for kindly providing the Panc02 cells and Dr. James S. Norris (Department of Microbiology and Immunology, Medical University of South Carolina) for his gift of the anti-fiber knob neutralizing antibody 1D6.14. This work was funded by a grant from the Department of Defense, GC-3532-03-42153CM (Y. Yan, D. J. Cole); ACSIRG Sub-award 2270301 85244 5921 01 (Y. Yan); and Department of Defense N6311601MD10004 (D. K. Watson).