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10.1245/ASO.2003.10.006
Annals of Surgical Oncology 10:810-820 (2003)
© 2003 Society of Surgical Oncology
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ORIGINAL ARTICLES

Synergistic Effect of a Granulocyte-Macrophage Colony-Stimulating Factor–Transduced Tumor Vaccine and Systemic Interleukin-2 in the Treatment of Murine Colorectal Cancer Hepatic Metastases

Ajay Jain, MD, Jill E. Slansky, PhD, Laurel C. Matey, BA, Heather E. Allen, BS, Drew M. Pardoll, MD, PhD and Richard D. Schulick, MD

From the Departments of Surgery (AJ, HEA, RDS), Oncology (LCM, DMP), and Medicine (DMP), Division of Immunology and Hematapoiesis, Johns Hopkins University School of Medicine, Baltimore, Maryland; and the Department of Immunology (JES), University of Colorado Health Sciences Center, Denver, Colorado.

Correspondence: Address correspondence and reprint requests to: Richard D. Schulick, MD, the Johns Hopkins Hospital, 600 North Wolfe Street, Blalock 657, Baltimore, MD 21287; Fax: 410-614-9880; E-mail: rschulick{at}jhmi.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Granulocyte-macrophage colony-stimulating factor–transduced tumor cell vaccines are less effective against cancer as the interval between metastasis and the initial vaccination increases.

Methods: Hepatic metastases were generated in BALB/c mice by using a syngeneic colorectal cancer line (CT26) with a splenic injection model. Irradiated CT26 cells transduced to secrete granulocyte-macrophage colony-stimulating factor were used as vaccine. Treatment groups received vaccine, systemic interleukin (IL-2), or both. Livers were examined for gross metastases 21 days after tumor challenge. Splenocytes were analyzed for in vitro activity against CT26 by using an enzyme-linked immunospot assay and a cytotoxic T lymphocyte assay.

Results: Eighty-eight percent of mice treated with vaccines and IL-2 were tumor free on day 21 (P <= .001 vs. control). Treatment with vaccines or IL-2 alone did not result in a significant treatment effect. Splenocytes from mice treated with both vaccines and IL-2 showed greater CT26 lysis than splenocytes from mice treated with vaccines alone at effector:target ratios of 100, 30, and 10 (P < .05 for all). More splenocytes from these mice released interferon-{gamma} in response to stimulation with the CT26 tumor antigen AH1 compared with mice treated with vaccines alone (P = .05).

Conclusions: Systemic IL-2 augments tumor vaccine efficacy in the treatment of microscopic murine colorectal hepatic metastases.

Key Words: GM-CSF • IL-2 • Tumor vaccine • Colorectal • Metastases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal cancer is the second leading cause of cancer death in the United States. There were an estimated 148,300 new cases and 56,600 colorectal cancer–related deaths in the United States in 2002 alone. In patients with advanced disease, colorectal cancer is often metastatic, and the most common site of metastasis is the liver. Current treatment options for colorectal cancer hepatic metastases include surgical resection, ablation, and chemotherapy. New therapies are needed, because even with successful resection followed by chemotherapy, 5-year survival ranges only from 30% to 40%. Recurrence of disease after surgical resection is typically attributed to incomplete removal of residual microscopic disease.1,2 One potential approach to improve 5-year survival after surgical resection of colorectal cancer hepatic metastases is to use cytokine-transduced tumor cell vaccines capable of inducing tumor-specific immune responses.

Immunotherapy with granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting tumor cell vaccines has been shown to induce significant protection against the formation of tumors in laboratory animals when administered before a cancer-cell challenge. The protective effects have been attributed to the activation of antitumor responses via CD4 and CD8 T-cell pathways. Some experiments have even shown tumor cell vaccines to cause regression of preexisting, small-volume tumors, but results have not been consistent. Often a reduction in tumor volume is seen, but complete eradication of preexisting tumors does not always occur.3–11 Preexisting tumor burden may be less amenable to immunotherapeutic treatment strategies because growing tumors have the ability to tolerize the host immune system.12–13 In clinical practice, treatment of colon cancer is often initiated long after the cancer has metastasized from its primary location. For the use of cytokine-secreting tumor cell vaccines to become clinically feasible, their efficacy must be improved when administered after cancer dissemination has already occurred.

It may be possible to improve the efficacy of GM-CSF–secreting tumor cell vaccines by administering adjuvant doses of systemic interleukin (IL-2). IL-2 is a potent cytokine capable of activating multiple immune cell types. Activated T cells upregulate receptors for IL-2.14,15 IL-2 has a broad range of immune regulatory effects, including the expansion of lymphocytes after activation by tumor antigens. The antitumor effects of IL-2 in vivo may be derived from its ability to expand and activate lymphocytes with antitumor activity.16–19 Because GM-CSF–secreting tumor cell vaccines can work through the activation of T cells, it is hypothesized that systemic IL-2 given after vaccination might result in improved treatment success. Use of adjuvant IL-2 has shown promising results in mice and in human melanoma patients when used with peptide pulsed vaccines.18,20 GM-CSF vaccines might increase local recruitment of antigen-presenting dendritic cells at the site of tumor cell vaccination, resulting in better immune cell priming and activation, whereas systemic IL-2 might stimulate the vaccine-activated immune cells.19

To study the effectiveness of combined GM-CSF tumor cell vaccination and systemic IL-2 in treating microscopic colon-liver metastasis, we established a mouse model that mimicked the progression of colorectal cancer metastases in the liver. In other mouse models, colorectal cancer tumors have been generated in the liver by using primary hepatic injection, portal vein injection, or whole-spleen injection of tumor cells, with or without splenic excision. Others have attempted to test immunotherapeutic treatment strategies by using subcutaneous tumor models outside of the liver.11,21–23

Although other tumor models provide very valuable information regarding immunotherapy efficacy in the treatment of many cancers, special considerations might limit their ability to discern treatment effects in the event of isolated colorectal cancer hepatic metastases. Portal vein injection is technically difficult in mice and can result in tumor spillage and peritoneal carcinomatosis in addition to hepatic metastases. Whole-spleen injection of tumor cells results in a large primary splenic tumor burden. This may compromise immune function in the animal by inducing immunosuppressive cytokine secretion by tumor-encountering splenic macrophages.23 Removal of the spleen, an important site of antigen presentation, after tumor injection may also diminish the host animal’s immune responses. The primary hepatic injection and subcutaneous tumor models are not models of metastasis, and treatment strategies that are effective on solitary tumors might not translate to disseminated metastatic processes. It is necessary to test therapeutic vaccines in a model of isolated hepatic metastases so the immune system can encounter cancer cells in a more clinically relevant context. Such a model of isolated hepatic metastases in the absence of a primary tumor may accurately reflect a subset of patients who undergo hepatic resection and could potentially benefit from adjuvant systemic immunotherapy.

Here we introduce a unique colorectal cancer hepatic metastasis model: the hemispleen model. This model uses surgical division of a whole spleen into two halves, followed by tumor cell injection into one of the "hemispleens." The tumor-injected hemispleen is excised after allowing seeding of the liver with tumor cells entering through the portal circulation. The remaining hemispleen is immunologically intact, and splenocytes can be harvested from it after the animal’s death and analyzed for effector properties and activation state. The hepatic metastases generated in the hemispleen model simulate colorectal cancer recurrence in the liver after surgical resection of a primary colonic tumor.

Using this model, we evaluated the use of adjuvant IL-2 in conjunction with GM-CSF–secreting tumor cell vaccines. Results indicate that although GM-CSF–secreting vaccines alone provide some therapeutic benefit for established hepatic metastases, the addition of systemic IL-2 to the vaccine therapy regimen synergizes to provide significant antitumor immunity in this system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Murine Tumor Cell Lines
CT26 BALB/c colorectal carcinoma was generated by injection of methylcholanthrenes as described by Corbett et al.24 The vaccine cell line was derived from CT26 cells transduced to secrete murine GM-CSF by using a replication-defective MFG retroviral vector as described by Dranoff et al.25 For target controls in the cytotoxic T lymphocyte (CTL) experiments, we used an MC57G fibrosarcoma line transduced to express Ld.26 All tumor cell lines were grown in RPMI 1640 media containing 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), streptomycin (100 U/mL), 1x minimum essential medium nonessential amino acids (Sigma, St. Louis, MO), 10 mM of HEPES buffer (pH 7.5), 1 mM of sodium pyruvate, and 2 mM of L-glutamine.

Treatment of Animal Subjects
All experimental subjects were treated ethically in accordance with an animal-use protocol approved by the Johns Hopkins Animal Care and Use Committee and in compliance with the Animal Welfare Act.

Establishment of Hepatic Tumors by Hemispleen Injection of Tumor Cells
BALB/c mice were anesthetized with pentobarbital (50 mg/kg intraperitoneally). A left flank incision was made to expose the spleen (Fig. 1A). The spleen was divided into two hemispleens by using medium-size Horizon surgical titanium clips (Weck Closure Systems, Research Triangle Park, NC), leaving the vascular pedicles intact (Fig. 1B). With a 27-gauge needle, 1 x 105 viable CT26 cells in 400 µL of Hanks’ balanced salt solution (HBSS) buffer were injected into the spleen. Cells then flowed into the splenic and portal veins and were deposited in the liver (Fig. 1C). The vascular pedicle draining the cancer-contaminated hemispleen was ligated with a small surgical clip. The CT26-contaminated hemispleen was then excised, leaving a functional hemispleen free of tumor cells (Fig. 1D).



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  		FIG. 1. Establishment of hepatic metastases by hemispleen injection. (A) Anatomical relationship of the colon, spleen, and liver. Both the colon and the spleen are drained by the portal venous system, which empties into the liver. (B) The spleen is divided into two hemispleens by using medium surgical clips. (C) One hemispleen is injected with 1 x 105 CT26 colon cancer cells suspended in 400 µL of Hanks’ balanced salt solution buffer. The injected tumor cells flow into the liver via the portal vein and establish microscopic hepatic metastases. The uninjected hemispleen remains free of tumor cells. (D) A small surgical clip is used to ligate the vasculature of the injected hemispleen, and the injected hemispleen is then excised. A viable, uncontaminated hemispleen remains.

 
Immunotherapy With GM-CSF–Secreting Tumor Cell Vaccines
Treated mice received 1 x 106 irradiated (50 Gy) CT26 cells from a line that has been transduced to express GM-CSF (GM/CT26). Enzyme-linked immunosorbent assay confirmed that 1 x 106 GM-CSF–transduced CT26 cells secreted 140 to 200 ng of GM-CSF per 24 hours after irradiation. Vaccination treatment consisted of three 100-µL subcutaneous injections in three separate limbs. A mouse received a total dose of 1 x 106 GM/CT26 cells in 300 µL of HBSS buffer. Mice were vaccinated at various time points in relation to the establishment of hepatic metastases. Mice were vaccinated at day -7, 0, +3, or +7 in relation the date of tumor metastasis (day 0). After the initial vaccination, each mouse was give booster vaccines twice weekly with the same dose until day +21.

Administration of Adjuvant Systemic IL-2
Proleukin IL-2 (Chiron, Emeryville, CA) was administered systemically as adjuvant therapy. Twenty-two million international units of IL-2 was reconstituted in 1.2 mL of distilled water. Then, 1.44 mL of D5W was added, resulting in a final concentration of 8.3 x 106 IU/mL. Each mouse received 200 µL of IL-2 (1.6 x 106 IU) via intraperitoneal injection for 5 days, beginning on day 6 after the hemispleen tumor challenge.

Stimulation of Splenocytes for CTL Assay
Splenocytes were isolated from the remaining hemispleens by using a standard protocol with ACK lysing buffer.27 Before the CTL assay was conducted, splenocytes had to be stimulated in vitro for 5 days with irradiated CT26 cells transfected to express the co-stimulatory molecule B7-1 (B7-CT26). This cell line has been previously described by Huang et al.28 Four million splenocytes from each group were added to 1 x 105 irradiated (7500 rad) B7-CT26 cells (40:1 ratio). The cells were incubated in a 37°C incubator for 5 days.

Functional Assessment: Methods of Conducting CTL Assays
To evaluate in vitro tumor lysis by the splenocytes of the experimental mice, CTL assays were conducted on the stimulated splenocytes. CT26 target cells were labeled with 200 µCi of chromium-51 per million cells at 37°C for 1 hour. Target cells were washed three times to remove excess chromium. Three thousand chromium-labeled CT26 cells were added to varying concentrations of the stimulated splenocytes (effector:target ratios of 100:1, 30:1, 10:1, and 3:1). MC57G cells pulsed with either 5 µM of the tumor peptide AH1 (SPSYVYHQF) or 5 µM of the nontumor peptide ß-gal (TPHPARIG) were also used as control targets. The chromium-release assays were performed in a total volume of 200 µL in a V-bottom 96-well plate for 4 hours at 37°C. After the incubation, the plates were centrifuged at 115 x g. One hundred microliters of supernatant was aspirated from each well without disturbing the pelleted cells. The supernatant was transferred to 1.2-mL microtubes and loaded into a Wizard 1470 automated gamma counter (PerkinElmer Wallac, Gaithersberg, MD).

Functional Assessment of Splenocytes: Enzyme-Linked Immunospot Assay
The splenocytes were also tested for interferon (IFN)-{gamma} production on stimulation with the AH1 peptide. AH1 is a nine–amino acid (SPSYVYHQF), H-2Ld-restricted peptide that is part of the GP70 surface protein on the CT26 colorectal cancer cell line. It is the immunodominant antigen of CT26.26 The enzyme-linked immunospot (ELISPOT) assay described by Miyahira et al.29 and Murali-Krishna et al.30 was modified to detect AH1-specific T cells. The assay was performed in 96-well filtration plates coated with 10 µg/mL of rat anti-mouse IFN-{gamma} antibody (PharMingen, San Diego, CA) in 50 µL of phosphate-buffered saline. After overnight incubation at 4°C, the wells were washed and blocked with culture medium containing 10% fetal bovine serum. Freshly isolated splenocytes from mice in the different treatment groups were plated (5 x 105 cells per well). Splenocytes were incubated at 37°C for 24 hours with no stimulating antigen in the ELISPOT well, 5 µg/mL of AH1 tumor antigen in the well, or 5 µg/mL of murine cytomegalovirus (MCMV) peptide (YPHFMPTNL) in the well. Additionally, 10 U/mL of IL-2 was added to each well. After culture, the plate was washed and incubated with 5 µg/mL of biotinylated IFN-{gamma} antibody (PharMingen) in 50 µL of phosphate-buffered saline at 4°C overnight. After washing, 1.25 µg/mL of avidin-alkaline phophatase (Sigma) in 50 µL of phosphate-buffered saline was added and incubated for 2 hours at room temperature. After washing once more, the assay was developed with 50 µL of BCIP/NBT solution (Boehringer Mannheim, Indianapolis, IN). Spots were counted automatically with a KS ELISPOT reader (Zeiss, Halbermoos, Germany) or manually by two trained individuals.

Footpad Vaccination With GM-CSF–Secreting Tumor Cell Vaccines
To confirm where tumor-specific T cells were generated, one group of mice was given footpad vaccinations with GM-CSF–transduced tumor cell vaccines followed by systemic IL-2. Mice received vaccines on days 0, 3, 7, and 10, and a 5-day course of systemic IL-2 (1.6 x 106 IU per dose) was given on days 9 to 13 after initiation of the first vaccination. On day 14, lymphocytes were isolated from the vaccine site–draining popliteal nodes and the non–vaccine site–draining axillary nodes. No tumor challenge was given. T cells were isolated from vaccine site–draining popliteal nodes and nondraining axillary nodes and analyzed with the ELISPOT and CTL assays. Two 50-µL injections of 5 x 105 GM/CT26 cells were given to each mouse, one per each lower-extremity footpad.

Harvesting of Lymph Nodes and Isolation of T Cells From Lymph Nodes
Mice were killed with CO2 inhalation and then degloved of their skins. Popliteal and axillary lymph nodes were harvested from vaccinated mice. Lymph nodes were also harvested from naïve mice as controls. Nodes were suspended in 2 mL of medium, crushed with the plunger of a sterile 10-mL syringe, strained into separate 50-mL conical tubes, and diluted to a total volume of 20 mL. The tubes were centrifuged at 500 x g for 10 minutes. The pelleted cells were resuspended in 2 mL of medium, transferred to new tubes, and diluted to a total volume of 10 mL. The cells were once again spun at 500 x g for 5 minutes to remove all fat from the T cells. T cells from each node basin were then analyzed by using CTL and ELISPOT assays.

Statistical Analysis and Interpretation of Data
Analysis of the statistical significance of disease-free survival at 21 days between mice in the different groups was conducted with a standard {chi}2 algorithm with two rows and two columns (1 df). For ELISPOT and CTL data, a Student’s t-test was used to compare the numerical data (number of IFN-{gamma}–secreting cells or percentage lysis) of the different treatment groups. Survival data were evaluated with GraphPad Prism (GraphPad, San Diego, CA) software and Kaplan-Meier analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To establish hepatic metastases, we injected tumor cells into mice after dividing their spleens into two separate hemispleens with the approach outlined in Fig. 1. After injection of the tumor cells, the injected hemispleens were removed, leaving non–tumor-bearing, functional hemispleens in the mice. To determine the optimal number of tumor cells required to establish isolated hepatic metastases, we injected three separate tumor concentrations into mice. We then sectioned the livers 1 or 2 weeks after tumor injection and stained with hematoxylin and eosin to assess for metastatic tumor burden. Fig. 2 demonstrates the efficacy of hemispleen injection of tumor cells. Injection of 1 x 104 CT26 cells did not result in the establishment of hepatic metastases. If 1 x 105 cells were injected, small micrometastatic tumor foci were visible 1 week after tumor injection. These foci were larger at 2 weeks, and gross tumor metastases were visible 3 to 4 weeks after tumor injection. These metastases were discrete and confined solely to the liver. If the injected tumor load was increased to 1 x 106 cells, large-volume micrometastatic tumor burden was seen at 1 and 2 weeks after injection. At this dose, the injected tumor cells did not form solitary metastases, but instead they formed large, confluent areas of gross tumor 2 to 3 weeks after injection.



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  		FIG. 2. Hepatic tumor burden at 1 and 2 weeks after hemispleen injection of tumor cells. Mice were given hemispleen injection with saline or 1 x 104, 1 x 105, or 1 x 106 CT26 tumor cells. Mice were killed 1 or 2 weeks after the injection of tumor cells. Livers were removed from the mice, sectioned, and stained with hematoxylin and eosin to evaluate microscopic hepatic tumor burden.

 
Once the proper CT26 tumor load resulting in discrete hepatic metastases was determined (1 x 105 cells), the efficacy of GM-CSF–secreting CT26 tumor cell vaccines in protecting against or treating CT26 hepatic metastases was tested. Fig. 3 demonstrates that vaccine efficacy diminished as the interval between vaccination and the establishment of tumor metastases increased. All mice prevaccinated 7 days before tumor metastasis were protected from developing hepatic metastases at 21 days (P <= .001). If the first vaccination and tumor challenge were given on the same day (day 0), only 5 of 10 mice were tumor free on day 21—a significant reduction in vaccine efficacy compared with vaccination before tumor challenge and a significant improvement compared with no treatment (P <= .01). If vaccination was delayed until 3 days after metastasis (GM+3), only 3 of 10 remained tumor free. This treatment effect was not statistically significant compared with control. Finally, if vaccination was delayed until 7 days after tumor metastasis, only 1 in 10 mice remained tumor free. This treatment effect was also not statistically significant compared with control.



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  		FIG. 3. Vaccine efficacy 21 days after metastasis in relation to the initiation of vaccine timing. Mice were vaccinated with granulocyte-macrophage colony-stimulating factor–secreting tumor cell vaccines (1 x 106 cells per vaccination) initiated at varying time points in relation to the establishment of tumor metastases on day 0 (GM0). Booster vaccines were given twice weekly until completion of the experiment on day 21 after metastasis. The percentage of mice within each treatment group that were disease free on day 21 is shown. GM-7, mice were prevaccinated 7 days before tumor metastasis; GM+3, vaccination was delayed until 3 days after metastasis; GM+7, vaccination was delayed until 7 days after tumor metastasis; N.S., not significant.

 
Using systemic IL-2 as an adjuvant improved the efficacy of GM-CSF–secreting tumor cell vaccination initiated 3 days after tumor metastasis. As Fig. 4 illustrates, all mice in the control group (14 of 14) developed gross hepatic tumors 21 days after the injection of tumor cells. In contrast, all (15 of 15) mice vaccinated with GM-CSF–secreting tumor cell vaccines 4 days before tumor metastasis (GM-4) remained free of tumor (P <= .001 compared with control). Only 2 of 17 mice first vaccinated 3 days after metastasis (GM+3) and 2 of 11 mice treated with systemic IL-2 alone on days 6 to 10 after metastasis remained tumor free. These treatment effects were not significant when compared with control. Finally, of mice that received both vaccination 3 days after metastasis and systemic IL-2 (GM+3/IL-2), 15 of 18 were free of tumor on day 21. This treatment effect was significant compared with control, treatment with vaccination alone initiated 3 days after metastasis (GM+3), and treatment with IL-2 alone on days 6 to 10 after metastasis (P <= .001 for all). Thus, adjuvant IL-2 significantly increased the efficacy of vaccination initiated 3 days after tumor metastasis.



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  		FIG. 4. The effect of adjuvant interleukin (IL)-2 on vaccination initiated 3 days after metastasis. Mice were vaccinated with granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting tumor cell vaccines (1 x 106 cells per vaccination) initiated either 4 days before metastasis on day 0 (GM-4) or 3 days after metastasis (GM+3). One group of mice received adjuvant doses of systemic IL-2 (1.6 x 106 IU per dose) for 5 consecutive days (days 6–10 after metastasis) in addition to GM-CSF tumor cell vaccination initiated 3 days after metastasis (GM+3/IL-2). One group received systemic IL-2 therapy alone without vaccination. Booster vaccines were given twice weekly until completion of the experiment on day 21 after metastasis. The percentage of mice within each treatment group that were disease free on day 21 is shown. N.S., not significant.

 
In addition to determining the disease status of mice in the different treatment groups 21 days after tumor challenge, we determined whether splenocytes from the mice were specific for the CT26 tumor antigen AH1. As previously described, AH1 is the immunodominant antigen of the CT26 colorectal cancer cell line.26 Fig. 5 is a histogram of an ELISPOT assay conducted with splenocytes isolated from the mice 21 days after tumor metastasis. Table 1 shows the average number of IFN-{gamma}–secreting splenocytes in mice after in vitro stimulation with tumor antigen AH1 and non–tumor antigen MCMV. Mice prevaccinated with GM-CSF–secreting tumor cell vaccines 4 days before metastasis (GM-4) had 66 of 500,000 IFN-{gamma}–releasing splenocytes after stimulation with tumor antigen AH1 and 12 of 500,000 IFN-{gamma}–releasing splenocytes after simulation with non–tumor antigen MCMV. The increase in the fraction of AH1 tumor antigen–specific splenocytes relative to non–AH1-specific splenocytes was significant (P = .01). GM-4 mice also had significantly more AH1 tumor antigen–specific splenocytes than mice first vaccinated 3 days after metastasis (GM+3; P = .009) and untreated control mice (P = .004). GM+3 mice had 5 of 500,000 IFN-{gamma}–releasing splenocytes after stimulation with tumor antigen AH1 and 10 of 500,000 IFN-{gamma}–releasing splenocytes after stimulation with non–tumor antigen MCMV. The fraction of AH1 tumor antigen–specific splenocytes relative to non–AH1-specific splenocytes was not statistically different in GM+3 mice. Additionally, GM+3 mice did not have more AH1 tumor antigen–specific splenocytes than untreated control mice (P = not significant). In contrast, mice treated with both vaccination and systemic IL-2 (GM+3/IL-2) had 17 of 500,000 IFN-{gamma}–releasing splenocytes after stimulation with tumor antigen AH1 and 0 in 500,000 IFN-{gamma}–releasing splenocytes after stimulation with non–tumor antigen MCMV. The increase in the fraction of AH1 tumor antigen–specific compared with non–AH1-specific splenocytes in GM+3/IL-2 mice was significant (P = .007). GM+3/IL-2 mice also had more tumor antigen AH1–specific splenocytes than GM+3 mice (P = .05) and control mice (P = .04).



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  		FIG. 5. Enzyme-linked immunospot (ELISPOT) assay for interferon (IFN)-{gamma} release by splenocytes of mice in different treatment groups in response to antigen stimulation. ELISPOT assay flat projection: the graph depicts the number of peptide-specific IFN-{gamma}–releasing cells per 500,000 splenocytes isolated from individual mice 21 days after tumor challenge. Five hundred thousand splenocytes from each mouse were incubated with 10 µg/mL of rat antimouse IFN-{gamma} antibody (PharMingen, San Diego, CA) at 37°C with either IL-2 alone (10 U/mL), CT26 tumor antigen AH1 (5 µg/mL) and IL-2, or non–tumor antigen murine cytomegalovirus peptide (MCMV; 5 µg/mL) and IL-2 for 24 hours. Each incubation was performed once. IFN-{gamma}–releasing cells were represented as spots on the bottom of the plate after antibody counterstaining and counted twice using an automated Zeiss ELISPOT counter.

 

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  		TABLE 1. IFN-{gamma} release by splenocytes from mice in different treatment groups in response to antigen stimulationa
 
Figure 6 shows that vaccination with GM-CSF–secreting CT26 tumor cell vaccines can induce mice to generate splenocytes capable of lysing target cells loaded with AH1, the immunodominant antigen of the CT26 cancer cell line. Splenocytes from vaccinated mice cause the greatest lysis when incubated with CT26 tumor cell targets. Significant lysis was also seen when these splenocytes were incubated with AH1 tumor peptide pulsed Ld–expressing MC57G fibrosarcoma cells. Lysis was reduced dramatically when the vaccinated mice splenocytes were incubated with MC57G targets pulsed with the negative control peptide ß-gal. Splenocytes from vaccinated mice caused statistically greater lysis of AH1 pulsed MC57G targets compared with ß-gal pulsed targets at effector:target ratios of 100:1, 30:1, 10:1 (P <= .0001 for all), and 3:1 (P <= .025).



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  		FIG. 6. Lysis of CT26 cancer cells, AH1 tumor antigen pulsed MC57G cells, and ß-gal pulsed MC57G cells by splenocytes from mice vaccinated with GM/CT26. Cytotoxic T lymphocyte (CTL) assay: splenocytes from mice vaccinated twice weekly with 1 x 106 irradiated GM/CT26 cells were harvested 24 days after the first vaccination and stimulated in vitro for 5 days with irradiated CT26 cells expressing the co-stimulatory molecule B7-1 (B7-CT26; described by Huang et al.26). A CTL assay was conducted in triplicate using chromium-labeled CT26 cells, AH1 tumor antigen pulsed MC57G cells, and ß-gal pulsed MC57G cells as targets at effector:target ratios of 100, 30, 10, and 3 in a volume of 200 µL for 4 hours. One hundred microliters of supernatant was analyzed in a Wizard 1470 automated gamma counter (PerkinElmer Wallac).

 
A second CTL assay was performed to determine whether adjuvant systemic IL-2 could augment vaccine-mediated lysis of CT26 tumor cells in vitro. Figure 7 demonstrates that splenocytes from prevaccinated (GM-4) mice caused the greatest lysis of CT26 colorectal cancer cells in vitro at all effector:target ratios (100:1, 30:1, 10:1, and 3:1). Splenocytes from mice treated with both vaccines and systemic IL-2 initiated after metastasis (GM+3/IL-2) also caused CT26 lysis in vitro, although the lysis was attenuated compared with that caused by GM-4 mice splenocytes. GM+3/IL-2 mice splenocytes did, however, cause significantly greater lysis of CT26 than splenocytes from mice treated with vaccines alone (GM+3) at effector:target ratios of 100:1, 30:1, and 10:1 (P = .008, .04, and .03, respectively).



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  		FIG. 7. Lysis of CT26 cancer cells in vitro by splenocytes from mice in the different treatment groups. Cytotoxic T lymphocyte (CTL) assay: splenocytes were harvested 21 days after tumor challenge from mice treated with either prevaccination (GM-4), vaccination initiated 3 days after metastasis (GM+3), systemic interleukin (IL)-2 on days 6 to 10 after metastasis, or both vaccines and IL-2 (GM+3/IL-2). The splenocytes were stimulated in vitro for 5 days with irradiated CT26 cells expressing the co-stimulatory molecule B7-1 (B7-CT26; described by Huang et al.26). A CTL assay was done in triplicate using chromium-labeled CT26 cells as targets at effector:target [E:T] ratios of 100, 30, 10, and 3 in a volume of 200 µL for 4 hours. One hundred microliters of supernatant was analyzed in a Wizard 1470 automated gamma counter (PerkinElmer Wallac).

 
After determining that IL-2 augments the efficacy of vaccination in the treatment of microscopic CT26 metastases, we attempted to elucidate whether the adjuvant IL-2 effects were AH1 tumor antigen specific. Figure 8 depicts the results of an ELISPOT assay conducted on lymphocytes isolated from the vaccine site–draining popliteal nodes and nondraining axillary nodes of mice that received footpad vaccinations followed by a course of systemic IL-2. T cells isolated from the lymph nodes of naïve mice were used as a negative control. The axillary nodes of footpad-vaccinated, systemic IL-2–treated mice contained 1 in 500,000 lymphocytes that released IFN-{gamma} after stimulation with tumor antigen AH1. This was not statistically different from the number of lymphocytes that released IFN-{gamma} in response to stimulation with non–tumor antigen MCMV (0 in 500,000; P = not significant). In contrast, vaccine site–draining popliteal nodes contained many lymphocytes that released IFN-{gamma} specifically in response to stimulation with tumor antigen AH1 (228 of 500,000). These popliteal node lymphocytes did not release IFN-{gamma} in response to stimulation with non–tumor antigen MCMV (0 of 500,000). Thus, vaccine site–draining popliteal lymph nodes contained more AH1 tumor antigen–specific lymphocytes than non–vaccine site–draining axillary lymph nodes of the same animal (P <= .02).



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  		FIG. 8. Enzyme-linked immunospot (ELISPOT) assay for interferon (IFN)-{gamma} release by CD8 T cells from vaccine site–draining popliteal versus nondraining axillary lymph nodes of mice given vaccination and systemic interleukin (IL)-2. ELISPOT assay: mice were vaccinated in their hind-limb footpads on days 0, 3, 7, and 10 and given a 5-day course of systemic IL-2 on days 9 to 13. On day 14, lymphocytes were isolated from the vaccine site–draining popliteal nodes and the non–vaccine site–draining axillary nodes. The graph depicts the number of peptide-specific IFN-{gamma}–releasing cells per 500,000 lymphocytes. Five hundred thousand pooled lymphocytes from each node basin were incubated with 10 µg/mL of rat anti-mouse IFN-{gamma} antibody (PharMingen, San Diego, CA) and either IL-2 alone (10 U/mL), CT26 tumor antigen AH1 (5 µg/mL) and IL-2, or non–tumor antigen murine cytomegalovirus peptide (MCMV; 5 µg/mL) and IL-2 for 24 hours. IFN-{gamma}–releasing cells were represented as spots on the bottom of the plate after antibody counterstaining. Two separate manual counts were performed for each plate, and these were averaged after subtracting background.

 
Because vaccine site–draining popliteal nodes contained more AH1 tumor antigen–specific T cells than nondraining axillary nodes, we wished to determine whether these vaccine site–draining T cells could cause greater lysis of CT26 in vitro than T cells from non–vaccine site–draining axillary nodes. Figure 9 depicts the results of the CTL assay by using CT26 tumor cells as targets. At all effector:target ratios, lymphocytes from vaccine site–draining popliteal nodes induced the greatest lysis of CT26 cells in vitro. Nondraining axillary node lymphocytes also induced lysis of CT26 tumor cells in vitro, but the lysis was not as great as that induced by popliteal node lymphocytes. The lysis induced by popliteal node lymphocytes was significant compared with the lysis induced by axillary node lymphocytes at effector:target ratios of 100:1, 30:1, 10:1, and 3:1 (P <= .01, .002, .004, and .0001, respectively). Naïve mouse lymphocytes caused negligible lysis at all effector:target ratios.



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  		FIG. 9. Lysis of CT26 cells in vitro by CD8 T cells from vaccine site–draining popliteal versus nondraining axillary lymph nodes of mice given vaccination and systemic interleukin (IL)-2. Mice were vaccinated in their hind-limb footpads on days 0, 3, 7, and 10 and given a 5-day course of systemic IL-2 on days 9 to 13. On day 14, lymphocytes were isolated from the vaccine site–draining popliteal nodes and the non–vaccine site–draining axillary nodes. Pooled lymphocytes from the different node basins were stimulated in vitro for 5 days with irradiated CT26 cells expressing the co-stimulatory molecule B7-1 (B7-CT26; described by Huang et al.26). A CTL assay was conducted in triplicate using chromium-labeled CT26 cells incubated with the stimulated lymphocytes at effector:target [E:T] ratios of 100, 30, 10, and 3 in a volume of 200 µL for 4 hours. One hundred microliters of supernatant was analyzed in a Wizard 1470 automated gamma counter (PerkinElmer Wallac).

 
Finally, as Fig. 10 shows, therapy with both vaccines and IL-2 prolonged survival in tumor-bearing mice compared with therapy with vaccines alone (GM+3; P = .02) and no therapy (control; P = .02). Therapy with vaccines alone (GM+3) did not result in a significant treatment effect compared with control. Although survival was prolonged in mice treated with both vaccines and IL-2, most of the mice ultimately died from metastatic cancer.



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  		FIG. 10. Survival of mice with CT26 hepatic metastases in different treatment groups. Mice were vaccinated with granulocyte-macrophage colony-stimulating factor–secreting tumor cell vaccines (1 x 106 cells per vaccination) initiated at varying time points in relation to tumor metastasis on day 0. Booster vaccines were given twice weekly until day 21 after metastasis (the same day on which mice were killed in previous experiments to check for hepatic metastases). Long-term survival was then followed up as an experimental end point. GM-4, mice prevaccinated 4 days before metastasis; GM+3, vaccination initiated 3 days after metastasis; GM+3/IL2, mice treated with vaccine and interleukin-2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study introduces a novel mouse model of colorectal cancer hepatic metastasis that generates isolated metastases in the liver without a residual primary tumor in the spleen. These gross metastases form from microscopic tumor burden in much the same way that resected hepatic metastases recur in humans after incomplete surgical resection. In addition, this technique leaves splenic tissue from which lymphocytes can be isolated and assayed at a later time. Treatment with tumor vaccines alone is insufficient to delay or eradicate the formation of gross tumors in this model. Thus, the system is well suited for the study of adjuvant strategies to improve vaccine efficacy in the treatment of clinically significant micrometastatic disease.

Using the hemispleen model, we demonstrated that the efficacy of GM-CSF–secreting tumor cell vaccines diminishes as the time interval between tumor dissemination and initiation of vaccine therapy increases. Indeed, treatment of CT26 colorectal cancer liver metastases with vaccines initiated 3 days after tumor challenge is ineffective. Addition of systemic IL-2 to a vaccine treatment regimen initiated 3 days after tumor metastasis, however, significantly increases the number of disease-free mice 21 days after tumor challenge.

Others have shown that GM-CSF–secreting CT26 tumor cell vaccines induce immune responses that are directed primarily against AH1, the major histocompatibility complex (MHC-I)–restricted immunodominant antigen of the CT26 colorectal cancer cell line.26 Our data supported those findings. As Fig. 6 shows, splenocytes from mice injected with GM-CSF–secreting tumor vaccines alone caused the greatest lysis when incubated with CT26 tumor cell targets. Splenocytes from vaccinated mice also caused significant lysis of Ld-expressing MC57G fibrosarcoma targets pulsed with the CT26 tumor peptide AH1. Splenocyte-mediated lysis of MC57G targets pulsed with a negative control peptide ß-gal was greatly attenuated in comparison. Although CT26 tumor cells may contain other antigens that are targets for vaccine-induced antitumor responses, AH1 seemed to be a highly significant target antigen in our experiments.

Our data also suggest that adjuvant IL-2 may augment vaccine efficacy 21 days after tumor metastasis by increasing the number of splenocytes capable of responding to the CT26 tumor antigen AH1. As the ELISPOT assay in Fig. 5 demonstrates, mice treated with both vaccines and IL-2 had more splenocytes that released IFN-{gamma} in response to stimulation with AH1 tumor antigen than mice treated with vaccines alone. In the ELISPOT assay, AH1 peptide was added directly to harvested splenocytes during the incubation period. These splenocytes represented a mixed population of immune cells (CD4 T cells, CD8 T cells, B cells, and antigen-presenting cells). Thus, the harvested splenocytes served as both effector and target cells in the assay. The increase in the number of AH1 tumor antigen–specific, IFN-{gamma}–secreting splenocytes may have reflected an increase in the number of tumor antigen–primed effector cells (e.g., increased numbers of tumor antigen–primed CD8 effector T cells), or it may have reflected an increase in the number of antigen-presenting cells (dendritic cells or monocytes) that could prime effector cells more efficiently.

We have shown that although adjuvant IL-2 given after vaccination could increase the number of AH1 tumor antigen–specific, IFN-{gamma}–secreting splenocytes, IL-2 alone did not induce AH1 antigen–specific antitumor effects. Tumor-challenged mice treated with IL-2 alone did not have a significant number of AH1 tumor antigen–specific, IFN-{gamma}–releasing splenocytes, and splenocytes from IL-2–treated mice did not cause significant lysis of CT26 in vitro. It is interesting to note that the non-AH1 tumor antigen–specific lymphocytes from the axillary nodes of footpad-vaccinated, systemic IL-2–treated mice caused significant lysis of CT26 tumor cells in vitro. The lysis caused by axillary node lymphocytes was not as great as the CT26 lysis induced by popliteal node lymphocytes, but it was far greater than that induced by naïve lymphocyte controls. As mentioned previously, AH1 tumor antigen is an MHC-I–restricted peptide. It is possible that adjuvant systemic IL-2 given after vaccination may have augmented the antitumor immune responses of immune cells that lack MHC-I restriction (e.g., natural killer cells). It is also likely that systemic IL-2 enhanced the antitumor effects of AH1 antigen–specific T cells generated by vaccines in the vaccine site–draining nodes.

The data from the footpad vaccination experiment also raise some interesting issues about lymphocyte trafficking. We have shown that the AH1 tumor antigen–specific T cells were generated primarily in vaccine site–draining lymph nodes. Presumably, these cells would have to migrate to the liver to halt the progression of hepatic metastases. One potential strategy to augment the efficacy of tumor cell vaccines may be to alter the microenvironment of the target organ of metastasis to increase trafficking of vaccine-activated immune cells from the periphery into the organ itself.

In our model, therapy with both vaccines and systemic IL-2 significantly prolonged survival in hepatic tumor-bearing mice compared with therapy with vaccines alone. Ultimately, however, most vaccination- and IL-2–treated mice ultimately developed metastases and died. It remains to be tested whether booster courses of IL-2 may result in more significant disease-free survival. Different doses of IL-2 should also be tested. In this study, the dose of IL-2 administered to the mice as a vaccine adjuvant was higher than that usually administered to humans. Human patients being treated for metastatic melanoma have received multiple courses of high-dose IL-2 consisting of 600,000 to 720,000 IU/kg administered by intravenous injection every 8 hours for 5 days, with an average cumulative dose of 1.2 x 107 IU/kg per course. This dose of IL-2 can result in serious systemic toxicities in humans, including hypotension, thrombocytopenia, renal insufficiency, vascular leak, and systemic manifestations similar to septic shock. High-dose IL-2 is therefore typically administered in an intensive care setting.31 In this study, we used a dose of systemic IL-2 that caused mice minimal distress and no adverse outcomes. We determined this dose to be 8 x 107 IU/kg/day (for 5 days), for a total cumulative dose of 40 x 107 IU/kg in a 20-g mouse. Although this dose is much higher than the highest dose of IL-2 normally administered to humans, it is possible that IL-2 dosing in humans and mice cannot be directly compared. As mentioned previously, mice experienced no systemic toxicities from the doses of IL-2 we administered. It is possible that IL-2 could augment vaccine efficacy in humans at lower doses that do not cause systemic toxicities. Although further investigation regarding the dose titration of adjuvant IL-2 is necessary, the data suggest that systemic IL-2 has good potential to augment the efficacy of tumor vaccine–based immunotherapy approaches.


   ACKNOWLEDGMENTS
 
The acknowledgments are available online at www.annalssurgical.oncology.org.

Supported by the National Institutes of Health Special Projects in Oncology Research (SPORE) Program 5P50 CA 62924 and the Richard Starr Ross Clinician Scientist Award of the Johns Hopkins University. The authors thank Robert W. and Jacquelyn M. Alvord and Mareen D. and R. Bruce Hughes of the Charles Delmar Foundation, who made this work possible.


   FOOTNOTES
 
Cytokine-secreting tumor cell vaccine efficacy diminishes as the interval between cancer metastasis and vaccination increases. Adjuvant systemic interleukin-2 significantly improves the efficacy of granulocyte-macrophage colony-stimulating factor–secreting tumor cell vaccines in a murine colorectal cancer hepatic metastasis model.

Received for publication October 3, 2002. Accepted for publication May 5, 2003.


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