Cancer Therapy Vol 4, 163-170, 2006

Advantages of a unique DNA-based vaccine in comparison to paclitaxel in treatment of an established intracerebral breast cancer in mice

Research Article

Terry Lichtor^1,*, Roberta P Glick¹, Henry Lin¹, Amla Chopra², InSug O-Sullivan² and Edward P Cohen²

¹Department of Neurological Surgery, Rush University Medical Center and John H Stroger Hospital of Cook County

²Department of Microbiology and Immunology, University of Illinois at Chicago; Chicago, Illinois

__________________________________________________________________________________

*Correspondence: Terry Lichtor, MD, PhD, Department of Neurosurgery, Rush University Medical Center, 1900 West Polk Street, Chicago, Illinois 60612, Telephone: 312-864-5120, Fax: 312-864-9606, E-Mail: Terry_Lichtor@rush.edu

Key words: DNA-Based Vaccine, IL-2, breast cancer, intracerebral cannula

Abbreviations: Intracerebrally, (i.c.); intraperitoneal, (i.p.); tumor associated antigens, (TAA)

This work was supported by a grant from the CINN Foundation awarded to Drs Lichtor and Glick, and by NIDCR grant number 1 RO1 DEO13970-01A2 awarded to Dr. Cohen.

Received: 19 April 2006; Accepted: 03 May 2006; electronically published: May 2006

Summary

In this study we compared the benefits of treating C3H/He mice with an established intracerebral breast carcinoma by immunization with a unique DNA-based vaccine to chemotherapy with paclitaxel. Prior studies revealed the immunotherapeutic properties of a vaccine prepared by transfer of genomic DNA from breast cancer cells into a highly immunogenic cell line. Here, C3H/He mice with an established intracerebral breast cancer were treated either by injection into the tumor bed through a unique cannula system with the cell based vaccine or with paclitaxel administered intraperitoneally. Both treatment strategies were effective in prolonging survival and stimulating a systemic anti-tumor immune response (p< 0.025). However, unlike mice treated with the vaccine, the animals that received paclitaxel alone displayed significant toxic side effects. No additional therapeutic advantage was detected when these two treatment strategies were combined. The vaccine tended to provide a somewhat better therapeutic and clearly better systemic immunologic effect based on two independent spleen cell assays in comparison to paclitaxel.

I. Introduction

We recently reported on the immunotherapeutic properties of a unique DNA-based cell vaccine for treatment of intracerebral (i.c.) breast cancer in C3H/He mice (Lichtor et al, 2005). In particular we showed that C3H/He mice injected with a cell mixture containing a breast carcinoma (SB5b) along with a vaccine prepared by transfection of mouse fibroblasts with DNA from the breast carcinoma (SB5b) survived longer than mice in various control groups. Systemic cellular tumor immunity was generated in the mice injected intracerebrally with the transfected cells, which was mediated predominantly by CD8⁺ T cells (Lichtor et al, 2005). The vaccine was prepared by transfer of sheared genomic DNA-fragments from an adenocarcinoma of the breast into a highly immunogenic mouse fibroblast cell line. The cells used as DNA-recipients were modified in advance of DNA-transfer to secrete various immune-augmenting cytokines. The rationale was that genes specifying tumor associated antigens (TAA) would be expressed in a highly immunogenic form by the transfected cells. As the transferred DNA is integrated into the genome of the recipient cells, and replicated as the cells divide, the vaccine could be prepared with DNA derived from microgram amounts of tumor tissue. It is likely that the multiple mutant and/or dysregulated genes in the breast cancer cells specifying an array of unidentified weakly immunogenic tumor associated antigens were expressed in a highly immunogenic form by the transfected cells. Since the tumor cell population is known to be heterogeneous and includes cells that are resistant to cellular immune mechanisms, the tumor cell population must include a subpopulation of breast cancer cells that are resistant to host immune mechanisms.

In an attempt to more closely simulate the clinical situation, mice bearing an established i.c. malignant breast carcinoma were treated with the DNA-based vaccine or with paclitaxel, a chemotherapeutic agent commonly used in breast cancer therapy (Conte et al, 2004; Eralp et al, 2004). Paclitaxel has been shown to be active against gliomas and various brain metastases, although its use in treatment of brain tumors is limited due to low blood-brain barrier permeability (Koziara et al, 2004). There is synergy between radiation therapy and paclitaxel in treatment of mice astrocytoma, yet clinical trials involving patients bearing a supratentorial high-grade glioma undergoing combination therapy of external beam radiation along with paclitaxel have not established any additional benefit of paclitaxel (Langer et al, 2001).

This study was designed both to compare and determine the possible benefits of combining paclitaxel with immunotherapy in the treatment of C3H/He mice bearing an established, highly aggressive intracerebral breast cancer. The mice were treated by injection into the tumor bed with the DNA-based vaccine, with paclitaxel administered intraperitoneally or by paclitaxel followed by immunization with the DNA-based vaccine. The results indicated that the survival of mice with an established intracerebral breast cancer was prolonged by treatment with either paclitaxel or the DNA-transfected fibroblasts (p < 0.025), but survival of mice receiving the combined therapy did not exceed that of tumor-bearing mice receiving either form of treatment alone.

II. Materials and Methods

A. Cell lines and experimental animals

Four to six-week-old pathogen-free C3H/He (H-2^k) mice were obtained from Charles River Breeding Laboratories (Portage, MI). The mice were maintained in the animal care facilities of the University of Illinois, according to National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. They were 6-8 weeks old when used in the experiments. SB-5b cells were derived from an adenocarcinoma of the breast, which formed spontaneously in a C3H/He mouse in our animal colony. The SB-5b cells were grown by in vitro passage. LM cells, a fibroblast cell line of C3H/He mouse origin, were obtained from the American Type Culture Collection (Manassas, VA). All the cells were maintained at 37⁰C in a humidified 7% CO₂/air atmosphere in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Sigma, St Louis, MO) and antibiotics (Life Technologies) (growth medium).

B. Modification of fibroblasts to secrete IL-2

To augment their non-specific immunogenic properties, before transfection, the fibroblasts modified to secrete IL-2 (LM-IL-2 cells) were prepared as described previously (Kim et al, 1992). In brief, a gene specifying human IL-2 [the biological properties of human IL-2 in mice are equivalent to that of mouse IL-2 (Kim et al, 1992)] was transduced into LM fibroblasts with the retroviral vector pZipNeoSVIL-2 (obtained originally from T. Taniguchi, Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan) (Yamada et al, 1987). The vector specified the human IL-2 gene and the neo^r gene (confers resistance to the neomycin analog, G418) (Cobere-Garapin et al, 1981). Every third passage, the cells were placed in growth medium containing 300 mg/ml G418. Under these circumstances, the quantity of IL-2 secreted by the cells after three months of continuous culture was equivalent to that of cells from primary cultures. Like unmodified cells, LM fibroblasts transduced with pZipNeoSVIL-2 divided approximately every 24 hours.

C. Modification of cytokine-secreting fibroblasts to express H-2K^b class I-determinants

LM cells, of C3H/He mouse origin, express H-2^k determinants. Allogeneic class I-determinants are strong immune adjuvants. To further augment their non-specific immunogenic properties, the LM fibroblasts were further modified to express H-2K^b class-I determinants as described previously (Lichtor et al, 2005). Confirmation of the expression of H-2K^b-determinants by the fibroblasts was confirmed by quantitative immunofluorescence measurements; more than 99 percent of the transduced fibroblasts stained positively for H-2K^b-determinants. Under similar conditions, nontransduced fibroblasts or fibroblasts stained with FITC-conjugated isotype serum failed to stain. The expression of H-2K^b-determinants was a stable property of the cells, and the intensity of staining for H-2K^b determinants was essentially unchanged after three months of continuous culture. Therefore the LM fibroblasts (LMK^b/IL-2) possess both syngeneic and allogeneic determinants when injected into C57Bl/6 mice (H-2^b).

D. Transfection of modified fibroblasts with sheared genomic DNA from a breast carcinoma that arose spontaneously in a C3H/He mouse (SB-5b)

Genomic DNA was isolated (Qiagen, Chatsworth, CA) from a mammary adenocarcinoma (SB-5b) that arose spontaneously in a C3H/He mouse. The genomic DNA was used to transfect the modified fibroblasts, using the method described by Wigler et al, (1979), as modified. Briefly, high molecular weight DNA was sheared by three passages through a 25-gauge needle. The approximate size of the DNA at the time it was used in the experiments was 25 kb, as determined by agarose gel electrophoresis. Afterward, 100 mg of sheared DNA was mixed with 10 mg pCDNA6/V5-HisA, a plasmid which gives resistance to the antibiotic Blasticidin. The sheared tumor-DNA and plasmid DNA (the DNA : plasmid ratio was 10 : 1 to ensure that cells that were converted to Basticidin-resistance took up DNA from the breast carcinoma cells as well) were then mixed with Lipofectamine 2000, according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA). The DNA/Lipofectamine mixture was added to a population of 1 X 10⁷ actively proliferating modified fibroblasts cells divided into ten dishes containing 1 X 10⁶ cells each. Eighteen hours afterward, the medium was replaced with fresh growth medium. The fibroblasts were maintained for 14 days in growth medium containing 2-5 mg/ml Blasticidin HCl (Invitrogen, Carlsbad, CA). One hundred percent of the cells transfected with tumor-DNA alone maintained in the Basticidin growth medium died within this period. The surviving colonies (at least 2.5 X 10⁴) were pooled and maintained as a cell line for use in the experiments.

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

As a model of intracerebral metastatic breast cancer in patients, C3H/He mice were injected intracerebrally with the breast cancer cells through a small cannula (Griffitt et al, 1999) that was modified as follows for injection of the tumor cells and the modified fibroblasts. Small screws (0-80 X 1/16; 1.6 mm in length) were obtained from Plastics One (Roanoke, VA) and a .022 diameter hole was subsequently drilled through the center of the screw. Anesthetized mice and were placed into a stereotactic frame and a small burr hole was placed with a D#60 drill bit (Plastics One, Roanoke, VA) over the right frontal lobe in the region of the coronal suture. The screws bearing a central hole were subsequently secured into the small burr hole using Elmer’s Super Glue Gel. The mice were allowed to recover and on specified days injections were made using a Hamilton syringe containing a 26 gauge needle with a small piece of solder placed 5 mm from the tip of the needle to maintain a uniform depth of injection. The total injection volume was 5-10 ml.

F. T cell mediated cytotoxicity toward breast cancer cells in mice immunized with the transfected fibroblasts

A CellTiter 96 aqueous non-radioactive cell proliferation assay kit (Promega, Madison WI) was used to measure T cell mediated cytotoxicity toward the breast cancer cells in mice immunized with the transfected fibroblasts. Effector T cell [recovered from the spleens of immunized mice by Histopaque (Sigma) density gradient (Kim and Cohen, 1994)] and mitomycin C-treated (50 μg/ml for 45 min) SB-5b target cells were co-cultured at 37⁰ C for 18 hrs at a 30:1 effector:target cell ratio. Afterward, the non-adherent cells were removed, washed and viable SB-5b cells were added at various E:T ratios for 4 hrs at 37⁰ C in a 7% CO₂/air atmosphere. The number of remaining viable cells was measured by methylthiazolyl tetrazolium salt (MTS), which is bioreduced by viable cells into a formazan product that can be detected at 490 nm. Negative control wells were treated with 2% Triton-100 to cause total lysis of the cells. Positive control wells did not receive effector cells. Next 20 μl of MTS and 1 μl of phenazine methosulfate (PMS), an electron coupling reagent, were mixed and added to each well, followed by incubation at 37⁰C for 1-4 hrs in a 7% CO₂/air atmosphere after which the absorbance was read. The percent specific lysis was calculated from the absorbance using the formula as follows:

G. ELISPOT IFN-g assays

Spleen cells from C3H/He mice injected i.c. with the various cell constructs were analyzed for T cells reactive with SB-5b cells in ELISPOT IFN-g assays. T cells recovered by Histopaque density gradient from the spleens of mice injected i.c. with the transfected cells were co-incubated with SB-5b tumor cells (the ratio of spleen cells : SB-5b cells = 10:1) for 16 hours at 37⁰C in wells precoated with a high-affinity monoclonal antibody for IFN-g according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA). After incubation, the cells were washed before the addition of biotinylated anti-IFN-g detection antibody and horse radish peroxidase labeled streptavidin (Streptavidin-HRP). The spots were counted using computer-assisted image analysis (ImmunoSpot Series 2 analyzer: Cellular Technology Limited, Cleveland, OH).

H. 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. Treatment of intracerebral breast cancer in C3H/He mice with Paclitaxel

Paclitaxel is a potent inhibitor of cell division (Gaitanos et al, 2004; Nettles et al, 2004; Ross et al, 2004). It blocks cells in the G2/M phase of replication through its effect on the formation and function of microtubules in the cell. To determine the effect of paclitaxel on an intracerebral breast cancer, naïve C3H/He mice were injected intracerebrally into the right frontal lobe with the malignant cells (SB5b). One day afterward, the mice received a single intraperitoneal injection of varying amounts of paclitaxel (range = 1.75 to 2.75 mg/kg). The results (Figure 1) indicated that the two higher doses of

paclitaxel (2.25 and 2.75 mg/kg) resulted in a modest but not statistically significant effect in prolonging the survival of mice with an intracerebral breast cancer. In the experiments to follow, mice with intracerebral breast cancer receiving the combined therapy were treated with a single intraperitoneal injection of 2.25 mg/kg of paclitaxel before the first immunization.

B. The effect of paclitaxel on the white blood cell count in C3H/He mice

Paclitaxel is highly toxic. Since the development of an effective immune response is dependent on white cell proliferation following antigen administration, peripheral white blood counts were measured at varying times after an injection of paclitaxel. The results (Figure 2) indicate that four days after injection of 2.25 mg/kg paclitaxel, the white blood count had returned to pre-injection levels consistent with a recovery from the toxic effects of the drug.

C. Cytokine-secretion by LM mouse fibroblasts transduced with a plasmid vector specifying IL-2

To augment their nonspecific immunogenic properties, the fibroblasts used as recipients of DNA from the breast cancer cells were modified before DNA-transfer to secrete IL-2. A gene specifying IL-2 was introduced into the cells by transduction with a plasmid vector. The vector also specified a gene conferring resistance to neomycin, an antibiotic used for selection. An ELISA assay was used to measure IL-2 secretion by the transduced fibroblasts. The results indicated that antibiotic-resistant cells transduced with pZipNeoSVIL-2 (specifies IL-2) formed 2214 pg IL-2/ml/10⁶ cells/72 hrs. Every third passage, the transduced fibroblasts were passaged in medium containing antibiotic. Under these circumstances, equivalent quantities of IL-2 were present after three months of continuous culture. The generation time of transduced and non-transduced fibroblasts, approximately every 24 hours, was equivalent.

D. Expression of H-2K^b, MHC class 1-determinants, by LM fibroblasts transduced with the vector, pBR327H-2K^b

H-2K^b-determinants are allogeneic in C3H/He mice (H-2^k). Allogeneic MHC-determinants are strong immune adjuvants (deZoeten et al, 2002). To further augment their immunogenic properties, the cytokine-secreting fibroblasts used as DNA recipients were subsequently modified to express H-2K^b-determinants. A plasmid, pBR327H-2K^b, was used for this purpose. The results indicated that more than 99 percent of the transduced fibroblasts stained positively for H-2K^b-determinants. Under similar conditions, nontransduced fibroblasts or fibroblasts stained with FITC-conjugated isotype serum failed to stain. The expression of H-2K^b-determinants was a stable property of the cells, and the intensity of staining for H-2K^b determinants was essentially unchanged after three months of continuous culture.

E. Treatment of mice bearing an established intracerebral breast cancer with transfected fibroblasts modified to secrete IL-2 and/or paclitaxel

The therapeutic properties of intratumoral injections of the DNA-based vaccine were compared with those of paclitaxel in the treatment of mice with an established intracerebral breast cancer. A cannula was placed into the right frontal lobe of C3H/He mice. One day afterward the animals received an injection intracerebrally (i.c.) through the cannula with 1.0 X 10⁴ SB-5b breast carcinoma cells. On the following day those animals treated with paclitaxel received a single intraperitoneal (i.p.) injection of 2.25 mg/kg paclitaxel. On days two and nine following tumor injection, the animals treated with the vaccine received 1.0 X 10⁶ transfected fibroblasts introduced through the cannula into the tumor region. The results (Figure 3) indicated that mice with an established breast cancer injected either i.c. with fibroblasts transfected with tumor DNA and modified to secrete IL-2 or i.p. with pacitaxel survived significantly longer than untreated mice (p < 0.025). In addition mice with an established breast cancer that received a combination of i.p. paclitaxel followed by immunization with the transfected fibroblasts survived significantly longer (p < 0.05) than untreated mice. However there was no difference in survival in the mice treated by a combination of paclitaxel and transfected fibroblasts in comparison to those animals treated with either therapy alone. Finally it should be noted that those animals treated with paclitaxel exhibited significant lethargy and cachexia that was not observed in either the controls or those animals treated only with DNA-transfected fibroblasts modified to secrete IL-2.

F. T cell mediated toxicity toward breast cancer in mice immunized with transfected fibroblasts modified to secrete IL-2 and/or paclitaxel

To determine if the immunity in mice injected i.c. with the transfected fibroblasts was systemic, spleen cells from mice injected i.c. with the transfected cells were analyzed for cytotoxic effects toward the breast cancer cells. An MTS based cytotoxicity assay was used for this purpose. The analysis was performed two weeks after the i.c. injection of breast cancer cells. The results (Figure 4) indicated that the spleen cell-mediated cytotoxic responses of greatest magnitude were in mice injected i.c. with breast cancer cells and transfected fibroblasts modified to secrete IL-2. Somewhat lesser cytotoxic effects were present in mice with an established i.c. breast cancer treated with paclitaxel with or without transfected fibroblasts modified to secrete IL-2. Thus, systemic immune responses directed toward the breast cancer cells were induced in mice injected i.c. with either IL-2 secreting transfected cells or paclitaxel.

IV. Discussion

This study has demonstrated that anti-tumor immune responses are generated in C3H/He mice with an established i.c. breast cancer injected i.c. with cytokine-secreting mouse fibroblasts transfected with unfractionated genomic DNA from the breast cancer cells. The immunity was sufficient to prolong survival, although the mice eventually died of the disease. A major advantage of this type of vaccine is that the fibroblasts could be genetically modified in advance of DNA-transfer to augment their immunogenic properties. In this instance, the cells were modified to express allogeneic class I MHC-determinants (allogeneic MHC-determinants are strong immune adjuvants and ensure that the cells will be rejected) and to secrete IL-2. The prolonged survival of mice with i.c. breast cancer treated solely by immunization with the cytokine-secreting cells points toward the potential of this form of therapy in patients with breast cancer metastatic to the brain.

Because the tumor cell population is known to be heterogeneous and includes cells that are resistant to cellular immune mechanisms, a subpopulation of malignant breast cancer cells, resistant to host immune mechanisms must have survived. For further control, a combination of therapeutic strategies will be required. In this study we compared the benefits of combining immunotherapy with paclitaxel, a standard chemotherapeutic agent. The dose and dosing schedule of paclitaxel used in this study is identical to that used in similar studies with this animal model (Chopra et al, 2006). Although paclitaxel suppresses the peripheral white cell population for several days, an anti-tumor immune response was found in the spleen cells taken from those animals treated with both paclitaxel and the cellular vaccine. However the best systemic immunologic effect was detected in those animals treated with vaccine alone. A statistically significant prolongation of survival (p< 0.025) was found in mice receiving either form of treatment alone. Combination therapy did not appear to provide synergistic potential.

The results of this study are consistent with other reports that paclitaxel is effective in the treatment of metastatic brain tumors (Cortes et al, 2003; Koziara et al, 2004). It is also evident that paclitaxel is toxic since animals treated with paclitaxel were cachectic and lethargic. The toxic side effects largely attributed to the paclitaxel solvent, Cremophor EL, have limited the use of paclitaxel in patients (Cortes et al, 2003). Furthermore the suppression of the peripheral white blood cell count attributed to paclitaxel, although relatively brief, makes paclitaxel along with most chemotherapeutic agents somewhat antagonistic when administered with immunotherapeutic treatment strategies. Nevertheless the combination of systemic chemotherapy along with immunotherapy has been used to treat patients with advanced-stage carcinoma (Yin et al, 2005). It has been proposed that dying tumor cells, particularly those killed by chemotherapy, engage with anti-tumor immune responses (Lake and Robinson, 2005). Although immunization at an appropriate interval following chemotherapy may result in an enhanced tumor immune response and therapeutic synergy, it is difficult to find such an interval in this animal model in which the life expectancy is approximately three weeks following i.c. injection of this highly aggressive tumor. It remains possible that other chemotherapeutic agents may have synergistic effects when administered in combination with immunotherapeutic treatments including the DNA-based vaccine used in this study. Finally given that the vaccine and paclitaxel have distinct mechanisms which may not complement each other, it is conceivable that some increase in the therapeutic benefits of these two treatments might exist using a different dosing schedule perhaps for example if the vaccine is given prior to administration of paclitaxel.

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Terry Lichtor