Matrix metalloproteinase-2 (MMP-2) has been used as a target for cancer immunotherapy. The activation of immunization by breaking immune tolerance to self-MMP-2 may be one of the promising approaches for the treatment of MMP-2-positive tumors. In this study, we constructed the xenogeneic tumor cell vaccine c-MMP-2 by transfecting CT26 and LLC cells with chicken MMP-2 cDNA constructs. MMP-2-specific autoantibodies in sera and tumor cells were found in mice immunized with c-MMP-2. Protection against tumor growth was evaluated in respect of the relative contributions of autoantibodies, CD4+, and CD8+ T cells. Treatment with this vaccine (c-MMP-2) also prolonged the survival time of mice bearing cancer. The specific cytotoxic T-cell responses suggested that the treatment increased CD8+ T-cell activity. The antitumor activity of c-MMP-2 was abrogated by in vivo depletion of CD4+ and CD8+ T-lymphocytes and improved by adoptive transfer of CD4+ and CD8+ T-lymphocytes from the mice treated with c-MMP-2. An alternative DNA vaccination strategy for cancer therapy was identified in this study by eliciting humoral and cellular immunoresponse with a crossreacting transfectant.
Matrix metalloproteinase-2 (MMP-2), a type IV collagenase,1 degrades a broad range of substrates including type IV, V, VII and X collagens as well as elastin and fibronectin. Thus, it plays an important role in the degradation of the extracellular matrix, such as inflammation, tissue repair, tumor invasion, metastasis, etc.2 MMP-2 is primarily expressed in mesenchymal cells during proliferation and tissue regeneration.3 It is also shown to be expressed in many connective tissue cells as well as in neutrophils, macrophages and monocytes. According to previous pathological and clinical studies, MMP-2 is expressed in various tumor cells especially in the metastasizing tumor cells to traverse basement membranes at tissue boundaries and in blood vessels.4, 5, 6 The synthesis and secretion of MMP-2 is controlled by transcriptional regulation, zymogen activation and specific tissue inhibitors of metalloproteinases (TIMPs).7, 8 Because of its important role in tumor growth and tumor invasion, MMP-2 has been a target for the development of antitumor therapeutic approaches that inhibit the motility of malignant cells, a prerequisite for tumor invasion and metastasis formation.3 Several approaches have been considered to inhibit the activity of MMP-2, including using TIMPs as the natural inhibitors of metalloproteinases, or using synthetic compounds to inhibit the enzyme activity.9 Several inhibitors, including Marimatat and Batimasta, have been tested for clinical trial in patients with cancer.10, 11 However, the results have been disappointing in a Phase III clinical trial.12 The recent induction of an autoimmune response against MMP-2 by DNA immunization with a xenogeneic homologous MMP-2 cDNA has shed light in clinical application.13
Although the identification of tumor antigens has propelled the development of many different approaches to cancer immunotherapy, whole-tumor cell vaccines have the advantage of immunization with diverse antigens that are present on the tumor surface without knowing the exact antigen(s). A major disadvantage of whole-cell tumor vaccines is that tumor cells are generally not immunogenic, but they may be injected and genetically altered to express cytokines, human leukocyte antigen (HLA) or costimulatory molecules.14, 15 In addition, the cell-associated antigen of the whole-tumor cells is associated with better presentation of antigen to CD8+ T cells than the soluble antigen. Several studies have demonstrated that breaking immune tolerance against tumor growth may be a useful approach for cancer therapy.16, 17 Sequence analysis indicates that mouse MMP-2 is 82% identical with that of chicken and 89% identical with that of humans at the amino-acid level. Thus, we constructed chicken MMP-2 cDNA transfected cells as a vaccine to break immunity tolerance to MMP-2. In the present study, we tested the possibility of inducing antitumor immunity in mouse models with whole-tumor cells transfected with chicken MMP-2 cDNA (c-MMP-2) as the tumor vaccine.
Materials and methods
Tumor cells and vaccine preparation
The plasmid pcDNA3.1 (+) containing the full-length cDNA of chicken and mouse MMP-2 was constructed in our laboratory before the study.13 According to the manufacturer's instruction, 2 μg of constructed plasmid or empty vector pcDNA3.1 (+) with Lipofectamine reagent (Life Technologies, Grand Island, NY) was transfected into CT26 colon adenocarcinoma (BALB/C origin) or Lewis lung carcinoma (C57BL/6 origin) cells, respectively, in six-well Petri dishes. Forty-eight hours later, the cells were placed into selective medium containing G418 (400 μg/ml). G418-resistant clones were isolated and maintained in culture medium containing 200 μg/ml of G418.18
Reverse transcription-polymerase chain reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was used to choose MMP-2 highly expressed cell lines. The total cellular RNA isolation and RT-PCR were carried out according to the manufacturer's instructions. The primers for amplification of mouse MMP-2 were 5′-IndexTermTAGAATTCATGGAGGCACGAGTGGCTTGG-3′ and 5′-IndexTermTATAAGCTTCGCAGCCCAGCCAGTCTG-3′, and the primers for chicken MMP-2 were 5′-IndexTermGCGAATTCATGAAGACTCACAGTGTTTTTG-3′ and 5′-IndexTermTATAAGCTTCGCAACCCAACCAGTCAG-3′. The PCR products were checked and purified by agarose gel electrophoresis. Cell clones which showed high transfection efficiency were chosen as tumor cell vaccines of chicken MMP-2 (c-MMP-2), mouse MMP-2 (m-MMP-2) and vector control (tumor cells transfected with empty vector (e-p)).
CT26 colon adenocarcinoma was established in BALB/C mice, and Lewis lung carcinoma was established in C57BL/6 mice. Mice at 6–8 weeks of age (n=10) were injected subcutaneously (s.c.) with freshly prepared cell suspensions (total 2 × 105 cells) with c-MMP-2, m-MMP-2 or e-p in normal saline. The same amount of parental cells was injected for controls.
To test the protective efficacy of xenogeneic transgenic cells as a vaccine, 1 × 106 parental CT26 or LLC cells were rechallenged in the mice in the opposite s.c. tissue 4 weeks after the inoculation of c-MMP-2. The same amount of parental cells was injected into the normal mice for controls. The tumor volume was determined by the following formula: tumor volume (mm3)=π/6 × length (mm) × width (mm) × width (mm). All studies involving mice were approved by the Institute's Animal Care and Use Committee.
Western blot assay
Western blot analysis was performed as described.16 The membrane blots were probed with experimental mouse sera or anti-MMP-2 antibody. Blots were then washed and incubated with a biotinylated secondary antibody (biotinylated horse anti-mouse IgG or IgM), which was then transferred to Vectastain ABC buffer (Vector Laboratories, Burlingame, CA). Meanwhile, commercially available mouse monoclonal anti-MMP-2 antibodies were used as a positive control.
Immunohistochemistry was used to determine the antibody deposited in tumor tissues. Sample slides were collected and stained by a standard protocol as described previously.19 Frozen sections were fixed in acetone, washed with phosphate-buffered saline and incubated with goat fluorescein isothiocyanate-conjugated antibody against mouse IgA, IgM or IgG (Sigma-Aldrich, St Louis, MO). The slides were examined by fluorescence microscopy.
To determine the antiangiogenesis activity of the vaccine, the tumors with around 1 cm diameter were chosen. After dewaxing and rehydration, paraffin sections were treated with a microwave oven twice for 5 min. Then the sections were stained with a rat anti mouse CD31 antibody as described previously.20 The vessel density was determined by counting the microvessels per high power field in the sections staining with the antibody reactive to CD31 as described.20
Adoptive transfer in vivo
Purification of immunoglobulins and adoptive transfer in vivo were performed as described previously.13 Briefly, immunoglobulins were purified from the pooled sera derived from the immunized mice or from the control mice by affinity chromatography (CM Affi-gel Blue Gel Kit; Bio-Rad, Richmond, CA). To assess the efficacy of Ig in antitumor in vivo, the purified Ig (10–300 mg/kg) was adoptively i.v. transferred 1 day before the mice were challenged with 1 × 106 tumor cells and then treated every 4 days for 4 weeks. As a control, Ig was adsorbed four times by the incubation with the fixed MMP-2-positive or -negative tumor cells at 4°C for 1 h with rocking.
Cytotoxic T-lymphocyte analysis
For the determination of the possible tumor-specific cytotoxicity mediated by cytotoxic T-lymphocyte (CTL), a 4 h 51Cr release assay was performed as described previously.21 Splenocytes were isolated from effectively immunized mice as well as the control mice. One hundred micro liter of effector cells and 51Cr-labeled target cells were assigned at different E:T (effector-to-target) ratios to each well of the microtiter plates and incubated at 37°C for 4 h. Samples were then harvested, and the activity was calculated by the formula: %cytotoxicity=((experimental release–spontaneous release)/(maximum release–spontaneous release))100. In inhibition experiments using anti-MHC monoclonal antibodies (mAb), target cells were initially incubated at 37°C for 30 min with anti-mouse H-2Kb/H-2Db or anti-mouse I-A/I-E mAb (as negative control)(BD Pharmingen, San Diego, CA) before they were added in the cytotoxicity assay. The final mAb concentration was 50 μg/ml.22
Depletion of immune cell subsets
The anti-CD4 (clone GK1.5, rat IgG), anti-CD8 (clone 2.43, rat IgG), anti-natural killer (NK) (clone PK136) hybridomas were obtained from the American Type Culture Collection (Manassas, VA). All hybridomas were grown as ascites in nude mice, and the mAb was purified from ascites by using ion exchange chromatography (Harlan Bioproducts for Science, San Diego).23 The mice were injected i.p. with 500 μg of either anti-CD4+, anti-CD8+ or anti-NK mAb 1 day before each transfectant s.c. injection and were then observed every 4 days for 4 weeks. The rate of depletion of CD4+, CD8+ and NK cells was consistently greater than 90%, as determined by a Coulter Elite ESP flow cytometry.
Adoptive transfer of purified CD4+ and CD8+ T cells
CD4+ and CD8+ T cells were purified by negative selection as previously reported.24 Single splenic cell suspensions were prepared, and red blood cells were lysed. Spleen cells were incubated at 4°C for 45 min with rat anti-CD8+ or anti-CD4+ antibody (BD Pharmingen). After being washed, cells were incubated at 4°C for 30 min with magnetic beads coated with sheep anti-rat (Dynal, Oslo, Norway), and antibody-coated cells were removed by magnetic separation to isolate CD4+ and CD8+ T cells. Then 1 day after the adoptive transfer of 2 × 106 cells, the mice were challenged with 1 × 105–106 tumor cells.
Expression of MMP-2 in transfectants
Fourteen independent G418-resistant clones of CT26 cells and nine independent clones of LLC cells were isolated and expanded. Highly expressed clones were chosen to designate as c-MMP-2, m-MMP-2 and e-p cell vaccines. In addition, the plasmid with chicken MMP-2, or with mouse MMP-2, or the plasmid alone had no direct effect on the proliferation of CT26, LLC tumor cells in vitro (data not show).
Inhibition of tumor growth in vivo
Mice were inoculated with transfectants (c-MMP-2, m-MMP-2 or e-p), or untransfected cells (2 × 105 per mouse). The mean tumor volume of mice injected with c-MMP-2 was significantly smaller than that of others which grew progressively until the animals died (Figure 1a and b). The survival rate of the tumor-bearing mice immunized with c-MMP-2 was also significantly greater than that of controls (P<0.05) (Figure 1c and d). To examine the protective immunity, mice were injected with 1 × 106 parental cells in the left s.c. tissue 4 weeks after c-MMP-2 was implanted. Tumors were rejected in the inoculated mice whereas others grew progressively in the control mice (Figure 2a). The survival rate of the tumor-bearing mice immunized with c-MMP-2 was also significantly greater than that of controls (P<0.05) (Figure 2b).
Characterization of autoantibodies
In an attempt to explore the possible mechanism underlying the induction of antitumor activity by c-MMP-2, we identified the autoantibodies against MMP-2 in the inoculated mice. Sera from c-MMP-2 inoculated mice recognized not only the protein of chicken MMP-2, but also the protein of mouse MMP-2 in Western blot analysis (Figure 3a). In contrast, the sera from the control mice showed negative staining (Figure 3b). As a control, commercially available mouse monoclonal anti-MMP-2 antibodies also recognized a single Mr 72 000 band, with the same molecular size as the sera from mice inoculated with c-MMP-2 (Figure 3c).
To identify the possible deposition of autoantibodies in the tumor and its surrounding tissues, we investigated the tissues by immunofluorescence staining. The deposition of IgG within the tumor tissues in c-MMP-2 inoculated mice was observed (Figure 3d), but this was not present in controls (Figure 3e–g). In addition, no IgM- or IgA-specific fluorescence was found. The results suggest that the adoptive transfer of purified immunoglobulins isolated from c-MMP-2 inoculated mice provides effective protection against tumor growth, compared with those from controls (Figure 4).
Inhibition of angiogenesis
Since MMP-2 is required for the angiogenesis during the tumor development. To degrade the basement membrane in microvessel walls, we inoculated c-MMP-2 against the MMP-2 activity. By counting the number of microvessels, the vaccination with c-MMP-2 resulted in apparent inhibition of angiogenesis in tumors, compared with control groups (Figure 5).
Function of T subsets in the antitumor activity
The T-cell response to tumor cells was measured in a 4 h 51Cr release assay. T cells derived from the spleens of c-MMP-2 inoculated mice showed higher cytotoxicity against LLC and CT26 cells than did T cells from m-MMP-2, e-p and parental cells inoculated mice (Figure 6a and b). Moreover, the anti-MMP-2 tumor cytolytic activity of these effector T cells was blocked by the addition of anti-H-2Kb/H-2Db, but not anti-I-A/I-E mAb, indicating that these CTL were MHC class I-restricted (Figure 6c).
To further explore the roles of immune cell subsets in antitumor efficacy elicited by c-MMP-2, CD4+, CD8+ T-lymphocytes or NK cells were depleted in mice by the injection of the corresponding mAb. With the inoculation of c-MMP-2, both normal mice and the mice depleted of NK cells demonstrated a significant reduction in tumor growth (P<0.05), suggesting that NK cells were not required for the response (Figure 7). Mice depleted of CD4+ T cells could partially abrogate the antitumor activity with the inoculation of c-MMP-2, and mice depleted of CD8+ T cells also showed partial abrogation. This pattern of response was also observed in the adoptive transfer assay. The adoptive transfer of CD8+, or CD4+ T-lymphocytes isolated from mice inoculated with c-MMP-2 exhibited the antitumor activity against MMP-2 positive tumor cells (Figure 8).
Using whole-tumor cells transfected with xenogeneic MMP-2 as an alternative DNA vaccine, we found that an autoreactive immune response against the MMP-2-positive tumor cells may be provoked in a crossreaction with the transfectants expressing xenogeneic MMP-2, and that the antitumor activity following xenogeneic MMP-2 vaccination may be involved in both humoral and cellular immune responses. Autoantibodies against MMP-2 in the sera and the tumor tissues were examined. The antitumor activity was also introduced by the adoptive transfer of the purified immunoglobulins. These findings suggest that humoral immunity might be responsible for the antitumor activity by the vaccination of xenogeneic MMP-2 vaccine. Moreover, the antitumor activity and the production of autoantibodies against MMP-2 could be abrogated by the depletion of CD4+ or CD8+ T-lymphocytes, whereas the depletion of NK cells has no significant effect on immune activity. MHC I-dependent CD8+ CTL activity was found in in vitro cytotoxicity assay. More importantly, the antitumor activity can be acquired by the adoptive transfer of CD8+ and CD4+ T-lymphocytes isolated from c-MMP-2 immunized mice. These findings suggest that a cellular immune response might be required for the antitumor immune response against MMP-2-positive tumors with xenogeneic MMP-2 transfectants.
It is presumably difficult to elicit the immunity to MMP-2 with vaccines based on self- or syngeneic MMP-2 molecules because of the immune tolerance acquired during the development of the immune system. However, several experiments demonstrated that the breaking immune tolerance against tumor growth should be a useful approach for cancer therapy.16, 17, 18 Sequence analysis indicates mouse MMP-2 has 82% identical residues with chicken MMP-2. Thus, chicken MMP-2 was used as a vaccine to raise antibodies which crossreacted with mouse MMP-2. In mice immunized with c-MMP-2, these antibodies acted as autoantibodies against mouse MMP-2 which successfully broke the mouse immunity tolerance to MMP-2. In this study, these antibodies appeared to be the important effector for clearing or preventing tumor cells replication.
In addition to their role in B-cell activation and switching, activated CD4+ T cells also produce several cytokines, such as IFN-γ, TNF-α, and they provide costimulatory molecules for activating antigen presenting cells (APCs). Th1 cells secreting IFN-γ play an integral role in providing help for the development and maintenance of both CTL and neutralizing antibody response.25 For the antibody-dependent immunity, CD4+ T-lymphocytes can be required at the immunization phase as well as at the effector phase.26 Through the interaction with B cells and the secretion of cytokines to initiate and amplify the CD8+ T-cell response, CD4+ T cells link the humoral and cellular immune response. The present study indicated that in vivo depletion of CD4+ T-lymphocytes could abrogate the antitumor activity through the immunization of c-MMP-2, suggesting that the abrogation of the antitumor activity by the depletion of CD4+ lymphocytes may be attributable to the failure in the induction of both CTL and autoantibodies.
In this study, activated CD8+ T cells were able to efficiently cause cell death in vitro. The cytolytic activity of T cells was blocked by the addition of anti-H-2Kb/H-2Db, but not anti-I-A/I-E mAb, indicating that CTL activity is mediated by CD8+ T cells. Our results also showed that the depletion of CD8+ lymphocytes lead to the partial abrogation of the antitumor activity in vivo. As the quality and quantity of CD8+ CTL determines the completeness of immunosurveillance and is controlled by CD4+ T cells,27 the depletion of CD8+ lymphocytes may only result in the loss of CD8+ CTL. The autoantibodies may still be present and play a role in antitumor activity. To date, several studies have demonstrated that the gelatinases, MMP-2 and MMP-9, are involved in vascular cell migration and invasion assays.28, 29 It has been documented that MMP-2 mediates the tumor-associated angiogenesis.30, 31 Microvessel density has been found to correlate with the capacity of the tumor to metastasize, and thus is considered to be one of the most important predictors of tumor progression.32 In this study, angiogenesis was apparently inhibited within the tumors. In conclusion, the cell vaccine based on whole-tumor cells transfected with homologous chicken MMP-2 as a cancer antigen may provide an alternative strategy for DNA vaccination therapy for cancer through the induction of autoimmune response against self-molecules associated with tumor growth through a crossreaction. This may be important to the further exploration of the role of the breaking immune tolerance.
tumor cells transfected with plasmid DNA encoding chicken MMP-2
tumor cells transfected with plasmid DNA encoding mouse MMP-2
tumor cells transfected with empty vector
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This work was supported by National Key Basic Research Program of China (2004CB518800 and 2001CB510001), Project of National Natural Sciences Foundation of China, national 863 project.
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