Gene Ther Mol Biol Vol 12, 1-6, 2008

Advantages of intracerebral versus systemic administration of a DNA-based vaccine in treatment of an intracerebral tumor

Research Article

Terry Lichtor^1,*, Roberta P Glick¹, Goro Osawa, Julian Hardman, Lisa A Feldman²

Department of Neurological Surgery, Rush University Medical Center

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*Correspondence: Terry Lichtor, MD, PhD, Department of Neurosurgery, Rush University Medical Center, 1725 West Harrison Street, Suite 1115, Chicago, Illinois 60612, USA; Tel: 312-942-6628; Fax: 312-942-2176; E-Mail: Terry_Lichtor@rush.edu

Key words: Brain Tumors, cDNA, Gene Therapy, Interleukin-2, Local Delivery, Tumor Vaccine

Abbreviations: complementary DNA, (cDNA); enzyme-linked immunosorbent assay, (ELISA); fetal bovine serum, (FBS); interleukin-2, (IL-2); intracerebral, (i.c.); subcutaneously, (s.c.); tumor associated antigens, (TAA)

¹Supported in part by a grant from The CINN Foundation

²Supported in part by a fellowship award from the American Brain Tumor Association

Received: 17 December 2007; Revised: 17 March 2008

Accepted: 21 March 2008; electronically published: April 2008

Summary

Structural differences between malignant and nonmalignant cells of the same individual form the basis of clinical immunotherapeutic strategies. Previously, we reported the therapeutic properties of a vaccine prepared by transfer of a cDNA-expression library from breast cancer cells into a highly immunogenic allogeneic fibroblast cell line where genes specifying an array of breast cancer antigens were expressed. In addition we have demonstrated the application of this cell-based vaccination strategy for breast cancer metastatic to the brain. In this study we explored the efficacy of the vaccine upon intracerebral versus subcutaneous injection in the treatment of an intracerebral tumor. Although the vaccine was efficacious in prolonging survival upon subcutaneous or intracerebral injection, there did not seem to be any synergy in either the development of systemic antitumor immunity or in prolonging survival when the vaccine was administered both intracerebrally and subcutaneously. However the intracerebral lymphocytic response was more intense following injection of the vaccine into the brain in the region of the tumor cells. Furthermore regulatory T cells (CD4⁺CD25⁺Foxp3⁺-positive) which inhibit antitumor immunity were not increased in the spleen cells from tumor-bearing mice injected intracerebrally but were increased in those injected subcutaneously with the enriched vaccine. This data suggests that local delivery of the cytokine-secreting DNA vaccine in treatment of an intracerebral tumor has certain advantages.

I. Introduction

Under ordinary circumstances, proliferating tumor cells fail to provoke anti-tumor immune responses that are capable of controlling tumor growth. The neoplastic cells escape recognition by the immune system in spite of the fact that they form weakly immunogenic tumor associated antigens (TAA). The successful induction of immunity to unique TAA expressed by the malignant cells could result in tumor cell destruction and prolongation of survival of cancer patients.

Antigenic differences between neoplastic and non-neoplastic cells in the tumor-bearing host form the rationale for clinical immunotherapy protocols. Because the antigenic phenotype varies widely among different cells within the same tumor-cell population, immunization with a vaccine that stimulates immunity to multiple TAA expressed by the population of malignant cells is likely to be more effective than immunization with a vaccine that expresses only a single epitope. Variants that fail to express the antigen chosen for therapy can avoid destruction. A vaccine strategy has been developed by transfer of a cDNA-expression library from breast cancer cells into a highly immunogenic fibroblast cell line, where genes specifying an array of breast cancer antigens were expressed (Cohen, 2001). As the transferred DNA integrates spontaneously into the genome of the recipient cells and replicates as the cells divide, sufficient quantities of DNA to prepare the vaccine could be obtained from quite small amounts of tumor tissue, enabling treatment of early metastatic neoplasms.

However, as only a small proportion of the tumor cDNA-transfected cell population was expected to have incorporated genes responsible for inducing immunity to the tumor, a novel strategy was devised to enrich the transfected cell-population for immunotherapeutic cells (Kim et al, 2006). We have tested the immunotherapeutic properties of the enriched IL-2 secreting DNA vaccine in mice with intracerebral (i.c.) breast cancer, and have found that mice with intracerebral breast cancer treated solely with the enriched vaccine survived significantly longer than mice in control groups (Lichtor et al, 2008). In addition the introduction of cells from the enriched vaccine into an intracerebral tumor resulted in a relative deficiency of T-regulatory cells.

It has been demonstrated that local immunotherapy with interleukin-2 (IL-2) is more effective against systemic tumors than systemic IL-2 therapy (Jacobs et al, 2005). The mechanism for this appears to be related to vascular leakage and/or local tissue destruction inside the tumor secondary to the direct intratumoral IL-2 injection. The goal of the current study is to determine the efficacy of the enriched IL-2 secreting DNA vaccine upon intracerebral versus systemic administration in treatment of an intracerebral tumor.

II. Materials and Methods

A. Cell lines and experimental animals

Pathogen-free 6-8 week old female C3H/He (H-2^k) mice from Charles River Breeding Laboratories (Portage, MI) were used in the experiments. The animals were maintained in the animal care facilities of the University of Illinois (Chicago, IL), according to National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. SB5b cells were a breast cancer cell line derived from an adenocarcinoma that arose spontaneously in the mammary gland of a C3H/He mouse in our animal colony. LM cells, a fibroblast cell line of C3H/He mouse origin, were from the American Type Culture Collection (Manassas, VA). Each of the cell cultures was maintained at 37⁰C in a humidified 7% CO₂/air atmosphere in Dulbecco modified Eagle medium (DMEM; GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO) and antibiotics (growth medium; Life Technologies, Grand Island, NY).

B. Modification of fibroblasts to secrete IL-2

To augment their non-specific immunogenic properties, the fibroblasts were modified before transfection to secrete IL-2 (LM-IL-2 cells), as described previously (Lichtor et al, 2002). An IL-2-specific enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Diego, CA) was used to determine the quantity of IL-2 secreted by the modified cells.

C. Modification of cytokine-secreting fibroblasts to express H-2Kb class I-determinants

LM cells, of C3H/He mouse origin, express H-2^k determinants constitutively. To further augment their non-specific immunogenic properties, the fibroblasts were modified to express H-2K^b class-I determinants allogeneic in C3H/He mice (H-2^k), as described previously (de Zoeten et al, 1999). Quantitative immunofluorescence measurements were used to detect the expression of H-2K^b-determinants by the modified fibroblasts.

D. Preparation of a vaccine for the treatment of intracerebral breast cancer

The vaccine was prepared by transfection of the modified fibroblasts with a cDNA expression library derived from SB-5b cells, as described previously (Kim et al, 2006).

E. Strategy for the enrichment of the vaccine for immunotherapeutic cells: Identification of highly immunogenic cell pools (immuno^high)

The strategy used to enrich the vaccine for immunotherapeutic cells was described previously (Kim et al, 2006). The LM-IL-2K^b/SB5b cells were prepared by transfer of unfractionated complementary DNA (cDNA) derived from a malignant breast neoplasm (SB5b) that arose spontaneously in a C3H/He mouse into a highly immunogenic cell line (LM-K^b). In brief, 1 X 10⁵ transfected cells (master pool; LM-IL-2K^b/cSB5b cells) were added to each of 15 T-75 flasks. As the cell numbers increased, cells from individual flasks were transferred to progressively larger cell culture flasks. Afterward, one-half of the expanded populations from the individual pools was maintained frozen/viable. The remaining portion was used to immunize C3H/He mice. Pools after four rounds of enrichment that stimulated spleen cell–mediated immunity toward SB5b cells to the greatest extent (immuno^high) were identified by both ELISPOT IFN-g and ⁵¹Cr-release cytotoxicity assays. Aliquots of the cell suspensions from the immuno^high pool were recovered and subjected to two additional rounds of positive or negative immune selection.

F. Intracerebral injection of C3H/He mice with SB-5b breast cancer cells

C3H/He mice were injected intracerebrally with a mixture of SB5b breast cancer cells as a model of intracerebral metastatic breast cancer in patients, as described previously (Lichtor et al, 2005). The treatment cells (immuno^high pool cells) were administered either intracerebrally mixed with the tumor cells or subcutaneously. The total intracerebal injection volume was 10 ml.

G. Spleen cell-mediated cytotoxicity by ⁵¹Cr-release assay

Mononuclear cells from the spleens of C3H/He mice immunized with the various cell constructs were used as sources of effector cells for the cytotoxicty studies using a standard 4 hour chromium release assay as previously described (Lichtor et al, 2003).

H. Analysis for CD3⁺, CD4⁺, CD8⁺ and CD25⁺ cells

To determine the nature of the immune response elicited by the immuno^high pool vaccine, brain and spleen tissue from animals in the various treatment groups were removed from two animals and single cell homogenates were prepared. The spleen cells were prepared as described previously (Lichtor et al, 2008), and the brain tissue was processed according to the procedure described by Hellums and colleagues in 2005. The following fluorochrome-conjugated antibodies (1.0 μl) were added to 500,000 viable cells (trypan blue) in 200 ml of FACS buffer containing 5% bovine serum albumin: CD3, CD4, CD8 and CD25/CD4/CD3 (E Bioscience, California) followed by incubation for 30 minutes at 4⁰C. After washing, the labeled cells were re-suspended in 200 μl of PBS containing 2% formalin. FACS analysis was performed at the Flow Cytometry Service, at the CORE facility of University of Illinois at Chicago. Data was acquired on a Cyan machine, and analyzed with Summit software (DakoCytomation, v4.2). FACS results are shown as the percentage of gated cells for each cell type.

I. Statistical Analysis

Student’s t test was used to determine the statistical differences between the survival of mice in various experimental and control groups. A p value less than 0.05 was considered significant.

III. Results

A. Secretion of IL-2 by mouse fibroblasts transfected with pZipNeoSVIL-2, a plasmid vector specifying IL-2

LM mouse fibroblasts were modified to secrete IL-2, as a means of augmenting their inherent immunogenic properties. A plasmid vector, pZipNeoSVIL-2, specifying human IL-2 (human IL-2 and mouse IL-2 are functionally indistinguishable in mice) was used for this purpose. The vector, zZipNeoSVIL-2, is a replication-defective retroviral vector specifying human IL-2 provided by MKL Collins, University College, London. An IL-2 ELISA was used to determine the quantity of IL-2 secreted by the transduced cells. Cells from the immuno^high pools formed 357.8 pg IL-2/10⁶ cells/ 48 hours (Table 1). This level was significantly higher than the IL-2 level detected in the SB5b breast cancer cells and well in excess of the level required to elicit an immune response. Equivalent quantities of IL-2 were secreted by cells from the the immuno^high pool after three months of continuous culture.

B. T-cell-mediated toxicity toward breast cancer in mice bearing an i.c. breast cancer injected with cells from the immuno^high pool of transfected cells

A spleen cell assay was used to determine the presence of systemic immunity against breast cancer in the animals treated either i.c. or s.c. with the immuno^high pool cells. Using a ⁵¹Cr-release cytotoxicity assay on spleen cells taken from these animals (Figure 1), antitumor immunity against breast cancer was detected in the animals treated either i.c. or with both i.c. and s.c. immuno^high pool transfected cells relative to ⁵¹Cr-release for spleen cells injected i.c. with SB5b tumor cells alone (p < 0.025). Antitumor immunity was also detected using the ⁵¹Cr-release cytotoxicity assay in spleen cells taken from those animals treated s.c. with the immuno^high pool transfected cells, although due to the large standard deviation statistical significance was not obtained.

FACS analysis of the brain tissue from the animals (Figure 2) revealed some increase in the total T cells, CD4⁺, and CD8⁺ T cells in those animals that received the immuno^high pool cells i.c.. No such findings were seen upon FACS analysis of the spleen cells from those animals that received the immuno^high pool cells s.c. alone. Regulatory T cells (CD4⁺/CD25⁺) are potent inhibitors of natural antitumor immunity (Fecci et al, 2006). The success of immunotherapeutic protocols may depend upon the relative numbers of T-reg cells and cytotoxic T lymphocytes in tumor-bearing animals and patients. FACS analysis of the spleen cells from the same animals (Figure 3) revealed a corresponding decrease in CD4⁺/CD25⁺ regulatory T cells in those animals treated with the immuno^high pool transfected cells injected i.c. alone, but injection of the immuno^high pool transfected cells s.c. either with or without an additional i.c. injection of the immuno^high pool cells actually resulted in a stimulation of the regulatory T cells.

C. Prolonged survival of mice with i.c. breast cancer treated by injection into the tumor bed of cells from the immuno^high pool

The enhanced immunotherapeutic properties of cells from the immuno^high pool were indicated by the results of ⁵¹Cr-release cytotoxicity assay in tumor-bearing mice. T-reg cells were relatively deficient in the spleens of mice injected i.c. with cells from the immuno^high pool. In previous studies, a modest prolongation of survival was demonstrated in animals with intracerebral breast cancer that were treated solely using cells from the non-enriched master pool injected intracerebrally (Lichtor et al, 2005). We have also done a study (unpublished data) that demonstrated no prolongation of survival when mice with an intracerebral breast cancer were treated by injection into the tumor bed with cells from the immuno^low pool. To determine if the immunotherapeutic properties of the immuno^high pool cells affected the survival of mice with i.c. breast cancer, C3H/He mice were injected i.c. with 5.0 X 10⁴ SB5b cells and 1.0 X 10⁶ cells from the immuno^high pool. As controls, the mice were injected i.c. with SB5b cells alone. The results (Figure 4) indicated that mice injected i.c. with breast cancer and cells from the immuno^high pool survived significantly longer than untreated mice injected with SB5b cells alone (p < 0.05) [also reported in Figure 7 by Lichtor et al, 2008]. Analogous findings were obtained if the mice were injected i.c. with the breast cancer cells and subcutaneously (s.c.) with cells from the immuno^high pool.

Table 1. Interleukin-2 Secretion

Cell Type IL-2	(pg/10^6 cells/48 hrs ± St Dev)
Media	13.8 ± 0.4
SB5b	65.0 ± 1.0
LM-IL-2K^b/cSB5b Immuno^high cells	357.8 ± 17.3

Figure 1. ⁵¹Cr-release cytotoxicity assay on spleen cells taken from two animals with an intracerebral tumor treated either subcutaneously or intracerebrally with the high pool vaccine. C3H/He mice received an injection of 5.0 X 10⁴ SB5b cells and 1.0 X 10⁶ immuno^high pool cells s.c, i.c. or both i.c. and s.c.. Untreated controls received only an injection of 5.0 X 10⁴ SB5b cells i.c.. The groups s.c., i.c. or s.c. + i.c. received 1.0 X 10⁶ immuno^high pool cells s.c., i.c. or both i.c. and s.c. respectively. Probability values are as follows: p < 0.025 for those animals treated either i.c. or with both i.c. and s.c. immuno^high cells relative to ⁵¹Cr-release for spleen cells injected i.c. with SB5b tumor cells alone in assays done at spleen cell/SB5b cell ratio of 30:1; p < 0.05 for those animals treated with i.c. immuno^high pool cells in comparison to untreated animals at a spleen cell/SB5b cell ratio of 60:1. The error bars represent one standard deviation of triplicate determinations.

Figure 2. FACS analysis of brain tissue for CD4⁺, CD8⁺ and total T cells (CD3⁺). C3H/He mice received an injection of 5.0 X 10⁴ SB5b cells and 1.0 X 10⁶ LMIL-2K^b/SB5b immuno^high pool cells s.c, i.c. or both i.c. and s.c. respectively. Ten days following the initial injection, mononuclear cells from the brains from two of the immunized mice were obtained for analysis by FACS. Data is expressed as percentage of gated cells.

Figure 3. FACS analysis of spleen cells for CD4⁺/CD25⁺ regulatory T cells. C3H/He mice received an injection of 5.0 X 10⁴ SB5b cells and 1.0 X 10⁶ LMIL-2K^b/SB5b immuno^high pool cells s.c, i.c. or both i.c. and s.c. respectively. Ten days following the initial injection, mononuclear cells from the spleens from two of the immunized mice were obtained for analysis by FACS. Data is expressed as percentage of gated cells.

Figure 4. Survival of mice with i.c. breast cancer treated by immunization with cells from the immuno^high pool of transfected cells. C3H/He mice (eight animals/group) were injected with 5.0 X 10⁴ SB5b cells and 1.0 X 10⁶ cells from the immuno^high pool through a small burr hole. At the same time the animals were injected s.c. with an equivalent number of cells from the immuno^high pool alone. As controls, the mice were injected i.c. with 5.0 X 10⁴ SB5b cells and 1.0 X 10⁶ cells from the immuno^high pool or with SB5b cells i.c. and s.c. with cells from the immuno^high pool. Mean survival time (MST) in days: Injected with SB5b alone, 12.7 ± 1.0; injected with SB5b and cells from immuno^high pool s.c., 15.6 ± 3.9; injected with SB5b cells and cells from immuno^high pool i.c., 15.4 ± 3.3; injected with SB5b cells and cells from the immuno^high pool i.c. and s.c., 17.4 ± 5.9. Probability values were as follows: p < 0.05 for mice injected with SB5b cells and cells from the immuno^high pool s.c., i.c. or i.c. and s.c. versus untreated mice.

IV. Discussion

The immuno^high pool vaccine whether injected i.c. or s.c. was effective in prolonging survival in animals with an i.c. breast carcinoma, and there did not seem to be any synergy in either the development of systemic antitumor immune responses or in prolonging survival when the vaccine was administered both i.c. and s.c.. The intracerebral lymphocytic response was more intense following administration of the IL-2 secreting DNA vaccine into the brain although this did not translate into any survival advantages. However it should be noted that administration of the vaccine s.c. resulted in some increase in CD4⁺CD25⁺ regulatory T cells, and this may be secondary to the fact that the high pool vaccine also secretes IL-2 which plays a critical role in the maintenance of regulatory T cells that inhibit the development of antitumor immunity (Wan and Flavell, 2006; Zorn et al, 2006).

It is likely that little IL-2 reaches outside the central nervous system when the vaccine is injected i.c., and therefore administration of this vaccine directly into the tumor bed of an i.c. tumor may have certain advantages. In particular i.c. administration of IL-2 likely avoids the systemic toxicity commonly seen when IL-2 is introduced systemically. In addition smaller doses of IL-2 are required in general when this agent is administered directly into the tumor region in comparison to the injections required when IL-2 is given systemically (Maas et al, 1991; Moiseeva et al, 2003). The mechanism for this is likely in part due to the fact that some local tissue destruction inside the tumor secondary to the presence of IL-2 stimulates the development of potent anti-tumor immunity.

The ultimate goal of cancer therapy is the elimination of every remaining tumor cell from the patient. It is unlikely that a single form of therapy is capable of achieving this goal. However immunotherapy in combination with surgery, radiation therapy and chemotherapy will likely find a place as a new and important means of treatment for patients with brain tumors. A major advantage of DNA-based vaccines is that they do not require protein purification or its production and yet they are able to elicit robust and long-lasting activation of the immune response, which results in tumor rejection. From a practical point of view, these vaccines are easy to prepare and they are relatively inexpensive. Only a limited quantity of tumor-derived DNA is required, which can be obtained from small surgical specimens. Overall, the disadvantages of DNA-based vaccines are few and are certainly no more difficult to overcome than those associated with other types of vaccines. Thus, DNA-based vaccines offer a number of advantages, which greatly encourage their further development for cancer immunotherapy. The enrichment strategy enables the generation of highly immunogenic pools of transfected cells with enhanced immunotherapeutic properties. Finally intracerebral delivery of the cytokine-secreting DNA vaccine has certain advantages in treating a brain tumor in comparison to systemic administration.

Acknowledgements

This work was supported in part by a grant from the CINN foundation awarded to Drs. Lichtor and Glick. Lisa Anne Feldman received a fellowship award from the American Brain Tumor Association in support of this work.

References

Cohen EP (2001) DNA-based vaccines for the treatment of cancer-an experimental model. Trends Mol Med 7, 175-179.

de Zoeten E, Carr-Brendel V, Markovic D, Taylor-Papadimitriou J, Cohen EP (1999) Treatment of breast cancer with fibroblasts transfected with DNA from breast cancer cells. J Immunol 162, 6934-6941.

Fecci PE, Mitchell DA, Whitesides JF, Xie W, Friedman AH, Archer GE, Herndon JE, Bigner DD, Dranoff G, Sampson JH (2006) Increased regulatory T-cell fraction amidst a diminished CD4 compartment explains cellular immune defects in patients with malignant glioma. Cancer Res 66, 3294-3302.

Hellums EK, Markert JM, Parker JN, He B, Perbal B, Roizman B, Whitley RJ, Langford CP, Bharara S, Gillespie GY (2005) Increased efficacy of an interluekin-12-secreting herpes simplex virus in a syngeneic intracranial murine glioma model. Neuro-Oncology 7, 213-224.

Jacobs JJ, Sparendam D, Den Otter W (2005) Local interleukin 2 therapy is more effective against cancer when injected intratumourally Cancer Immunol Immunother 54, 647-654.

Kim TS, Chopra A, O-Sullivan I, Cohen EP (2006) Enhanced immunity to breast cancer in mice immunized with fibroblasts transfected with a complementary DNA expression library from breast cancer cells. Enrichment of the vaccine for immunotherapeutic cells. J Immunother 29, 261-273.

Lichtor T, Glick RP, Cohen EP (2003) Approaches to the treatment of brain tumors using cytokine-secreting allogeneic fibroblasts. Cancer Therapy 1, 109-120.

Lichtor T, Glick RP, Lin H, O-Sullivan I, Cohen EP (2005) Intratumoral injection of IL-secreting syngeneic/allogeneic fibroblasts transfected with DNA from breast cancer cells prolongs the survival of mice with intracerebral breast cancer. Cancer Gene Therapy 12, 708-714.

Lichtor T, Glick RP, Lisa A Feldman, Goro Osawa, Julian Hardman, InSug O-Sullivan, Edward P Cohen (2008) Enhanced Immunity to Intracerebral Breast Cancer in Mice Immunized with a cDNA-based Vaccine Enriched for Immunotherapeutic Cells. J Immunother 31, 18-27.

Lichtor T, Glick RP, Tarlock K, Moffett S, Mouw E, Cohen EP (2002) Application of interleukin-2-secreting syngeneic/allogeneic fibroblasts in the treatment of primary and metastatic brain tumors. Cancer Gene Ther 9, 464-469.

Maas RA, Van Weering DH, Dullens HF, Den Otter W (1991) Intratumoral low-dose interleukin-2 induces rejection of distant solid tumour. Cancer Immunology Immunotherapy 33, 389-394.

Moiseeva EV, Merkulova IB, Bijleveld C, Koten JW, Miroshnikov AI, Den Otter W (2003) Therapeutic effect of a single peritumoural dose of IL-2 on transplanted murine breast cancer. Cancer Immunology Immunotherapy 52, 487-496.

Wan YY, Flavell RA (2006) The roles for cytokines in the generation and maintenance of regulatory T cells. Immunol Rev 212, 114-130.

Zorn E, Nelson EA, Mohseni M, Porcheray F, Kim H, Litsa D, Bellucci R, Raderschall E, Canning C, Soiffer RJ, Frank DA, Ritz J (2006) IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood 108, 1571-1579.