Human Prostate Stem Cell Antigen and HSP70 Fusion Protein Vaccine Inhibits Prostate Stem Cell Antigen-Expressing Tumor Growth in Mice

Abstract

Prostate stem cell antigen (PSCA) has been considered a potentially worthwhile target for prostate cancer therapy with its overexpression in both androgen-dependent and androgen-independent prostate cancers. However, PSCA is an autoantigen that can evoke immunological tolerance and hardly incite effective immunologic response. In this study, we sought to construct the fusion protein vaccines based on PSCA and heat shock protein 70 (HSP70) and to evaluate their immune responses and therapeutic efficacy. A series of recombinant proteins were prepared, and then, the male C57BL/6 mice were immunized subcutaneously by inoculation with RM-PSCA/Luc cells. The PSCA-specific cellular immune responses were monitored with ELISPOT and intracellular cytokines staining assay, and ELISA assay was used to detect humoral immune responses. The tumor growth was observed by in vivo bioluminescence imaging. The results showed that the mice vaccinated with PSCA-HSP could induce the PSCA-specific cellular and humoral immune responses. Tumor progression could be quantitatively monitored by in vivo bioluminescence imaging. Animal experiments showed that PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the survival time of vaccinated mice. This study supported and confirmed the potential of HSP70 as a chaperone for protein vaccines, and PSCA-HSP could be of potential value for prostate cancer treatment.

Introduction

Prostate cancer represents the most common noncutaneous cancer with high mortality rate in American men.¹ Although great progress has been made in treatment, the therapy of prostate cancer is still confronting challenges.^2,3 In recent years, the researches on novel anticancer strategies have shed light on prostate cancer immunotherapy, which represents a highly promising therapeutic strategy for cancer treatment. Immunotherapeutic strategies based on T cells and antibodies represent interesting approaches that inhibit prostate tumor growth for improving survival of patients. CD8⁺ cytotoxic T lymphocytes (CTLs) efficiently recognize and destroy tumor cells. Antibodies mediate their antitumor effects via antibody-dependent cellular cytotoxicity and activation of the complement system to destroy tumor cells. Moreover, systemic immunity induced by vaccines for specific antigens expressed in cancer cells probably reduces tumor load and produces an immunological memory that will prevent disease recurrence. To date, several prostate-confined targeted antigens, including prostate-specific antigen (PSA),⁴ six-transmembrane epithelial antigen of the prostate,⁵ prostate-specific membrane antigen,⁶ and prostate acid phosphatase,⁷ have been described as the targets of prostate cancer immunotherapy. However, the therapeutic efficacy of vaccines based on these antigens or antigen derived peptides used in clinical trials is far from satisfactory. Therefore, the development of novel tumor markers and therapeutic strategies for prostate cancer immunotherapy are extremely urgent.

Prostate stem cell antigen (PSCA), first described by Reiter et al.,⁸ is a surface glycoprotein that has 30% of homology with stem cell antigen-2 (SCA-2). The PSCA gene encodes a 123-amino-acid protein and is expressed in 85% of prostate cancer specimens with high tissue specificity. In addition, there is a direct correlation between the expression level of PSCA and the tumor stage, grade, and the bone metastases.⁹ So far, large amounts of scientific literature have confirmed that vaccination based on PSCA enhances the cellular and humoral immune responses and inhibits PSCA-expressing tumors growth in mice.^10
–12 Therefore, PSCA could be considered a potentially worthwhile target for prostate cancer immunotherapy.

When faced with thermal stress and other stressors, cells from bacteria to humans respond to express heat shock proteins (HSPs).¹³ HSPs are a group of phylogenetically conserved proteins with the function of molecular chaperones. HSP70 is a major molecular chaperone of HSPs family, which assists in transport, transmembrane, folding, and assembly of proteins in the cytoplasm, mitochondria, endoplasmic reticulum, and nucleus. In recent years, vaccination with HSP70-peptide complexes has been confirmed to elicit specific antitumor responses.^14
–16 These findings suggest that HSP70 is involved in the process of antigen presentation and has potential worth as chaperone for specific antigens in vaccines.

In this present study, we evaluated the immune effectiveness and antitumor activity of recombinant fusion protein vaccines based on PSCA and HSP70 following immunization in a mouse model. Meanwhile, the tumor growth was monitored with noninvasive, sensitive, and quantitative localization by in vivo bioluminescence imaging.

Materials and Methods

Preparation of recombinant proteins

For the generation of the pQE30-PSCA, the primers 5′-CCG GGA TCC TGC TAT AGC TGC AAA GCC-3′ and 5′-CCG AAG CTT CAG GGC ATG GGC CCC GCT-3′ were used to amplify PSCA gene, and the fragment was cloned into the BamHI and HindIII sites of pQE30 vector. The recombinant pQE30-PSCA was transformed into the Escherichia coli expression strain M15. After induction with 1 mM IPTG at 30°C for 5 hours, the cells were disrupted by sonication in 20 mM phosphate buffer, pH 7.4. The solution was centrifuged at 12,000 g, and the supernatant was purified with a His-binding Ni-NTA resin (Qiagen GmbH). For the generation of the pET21-HSP expression vector, the primers 5′-CCG GAA TTC ATG GCC AAA GCC GCG GCG-3′ and 5′-CCG CTC GAG CTA ATC TAC CTC CTC AAT GG-3′ were used to amplify the HSP gene, and the fragment was cloned into the EcoRI and XhoI sites of pET21a(+) vector. The recombinant pET21-HSP plasmid was transformed into the E. coli expression strain BL-21 (DE3). After induction with 1 mM IPTG at 30°C for 5 hours, the cells were disrupted by sonication in 20 mM phosphate buffer, pH 7.4. The solution was centrifuged at 12,000 g, and the supernatant was purified with DEAE (Amersham) chromatography followed by phenyl sepharose HP (Amersham). For the generation of the pET21-PSCA-HSP expression vector, the PSCA gene was amplified with the primers 5′-CCG CAT ATG TGC TAT AGC TGC AAA GCC-3′ and 5′-CCG GAA TTC CAG GGC ATG GGC CCC GCT-3′, and was cloned into the NdeI and EcoRI sites of pET21-HSP. The recombinant pET21-PSCA-HSP plasmid was transformed into the E. coli expression strain BL-21 (DE3). After induction with 1 mM IPTG at 30°C for 5 hours, the cells were disrupted by sonication in 20 mM phosphate buffer, pH 7.4. The solution was centrifuged at 12,000 g, and the supernatant was purified with Ni-NTA resin, phenyl sepharose HP, and Superdex 75 (Amersham). The purity of all recombinant proteins was about 95%, and the identity of the purified proteins was verified by western blotting with anti-PSCA antibody (sc-28820) and HSP mAb (sc-24), respectively.

Mice and cell lines

Male C57BL/6 mice at the age of 4–6 weeks old were purchased from the Center for Laboratory Animals (Beijing, China). All the study protocol about experimental animals was approved by the Animal Care and Use Committee of Academy of Military Medical Sciences. The mouse prostate tumor cell line RM-1, syngeneic to C57BL/6, was purchased from Shanghai Cell Institute. To generate a luciferase (Luc)-expressing and human PSCA-expressing cell population RM-PSCA/Luc, RM-1 was transfected with pcDNA-PSCA and pcDNA-Luc plasmids followed by using Geneticin (G418) selection. Expression of Luc was tested by luminometer detection, and expression of PSCA was examined by RT-PCR analysis and flow cytometric analysis.

Vaccination

Recombinant proteins were diluted to 1 mg/mL with phosphate-buffered saline (PBS) and stored at −20°C on standby. Mice were vaccinated subcutaneously twice with a 2-week interval with 100 μg recombinant proteins PSCA, HSP, PSCA+HSP, and PSCA-HSP in 100 μL PBS at both hind limbs, respectively. Mice in the control group were mocked with an injection of 100 μL PBS. Mice were randomly divided into 5 groups, and 5 mice were assigned for each group.

Preparation of splenocytes

Splenocytes were isolated as previously described.¹⁷ Splenocytes from vaccinated and control mice were harvested. Under aseptic condition, spleens were grinded and pushed through a 70-μm cell strainer in complete RPMI-1640 medium to prepare a single cell suspension. Splenocytes were centrifuged at 500 g for 5 minutes, and then, the supernatant was discarded. Red blood cells were lysed with ACK lysing buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, and pH 7.2–7.4), and the debris was removed, leaving a fine suspension of splenocytes. Cells were washed twice in complete RPMI-1640 medium and were resuspended at a concentration of 4×10⁶/mL in complete RPMI-1640 medium.

ELISPOT assay

Two weeks after the second vaccination, splenocytes were isolated from vaccinated mice. The ELISPOT assay kit purchased from U-CyTech Biosciences was used to detect effector T cells releasing interferon (IFN)-γ. Being coated with anti-mouse IFN-γ monoclonal antibody, nitrocellulose bottomed 96-well plates were incubated at 4°C for 24 hours followed by washing and blocking with RPMI-1640 medium containing 5% fetal bovine serum. Fresh isolated splenocytes (1×10⁵) were added to the wells and incubated with 5 μg/mL PSCA-specific H-2D^b epitope (PSCA aa 28–36) for 20 hours. After washing, the plates were added with biotinylated anti-IFN-γ antibody and incubated for 1 hour at 37°C. The spots were counted using a Bioreader 4000 (Bio-Sys GmbH).

Intracytoplasmic cytokine staining and flow cytometric analysis

Splenocytes from vaccinated mice were pooled and incubated with 5 μg/mL PSCA- specific H-2D^b epitope (PSCA aa 28–36) for 20 hours. Being added with golgistop (BD Biosciences) for 6 hours, the cells were stained with allophycocyanin-conjugated CD3e antibody (BD Biosciences) and R-phycoerythrin-conjugated CD8a antibody. Subsequently, the intracellular cytokines were stained with FITC-conjugated IFN-γ antibody (BD Biosciences) after fixation and permeabilization of cells with the Cytofix/Cytoperm kit (BD Biosciences). Flow cytometric analysis was performed by using BD FACSCalibur. The percentage data was subjected to arcsine square root transformation and was statistically analyzed with the Student–Newman–Keuls test and LSD test.

ELISA for serum PSCA antibodies

For analysis of the humoral responses, anti-PSCA antibodies in the serum of vaccinated mice were detected by enzyme-linked immunosorbent assay (ELISA). The 96-well microplates were coated with 100 μL of 10 μg/mL recombinant PSCA protein, and then, were incubated at 4°C overnight. After incubation, the wells were blocked with 3% bovine serum albumin contained in PBS. Serum collected from the mice after the second immunization were serially diluted in PBS and incubated on the plates at 37°C for 2 hours. After PBS washing, the plates were incubated at room temperature for 1 hour with 1:2000 dilution of a horseradish peroxidase-conjugated rabbit antimouse IgG antibody (Sigma-Aldrich), followed by stopping the reaction with H₂SO₄. Then, the plates were read at 450 nm.

In vivo tumor treatment experiments and bioluminescent reporter imaging

For detecting the ability of recombinant protein vaccines to treat tumors in the mouse model, 4–6 week-old male C57BL/6 mice were first challenged with a subcutaneous injection of 1×10⁶ RM-PSCA/Luc cells in the right flank. Three days after the challenge with the tumor cells, mice were vaccinated subcutaneously with 100 μg recombinant proteins PSCA, HSP, PSCA+HSP, and PSCA-HSP in 100 μL PBS in both hind limbs, respectively. Boost was performed 1 week later. Mice in the control group were mocked with an injection of 100 μL PBS at the same time point. After inoculation with RM-PSCA/Luc cells, the mice were monitored twice a week when the tumor could be touched. Tumor size was measured by vernier caliper, and the tumor volume was calculated according to the formula: V=0.5a×b ², where a and b were the long and short diameters of the tumor, respectively. Meanwhile, the tumor growth was monitored by in vivo bioluminescence imaging (IVIS Imaging System 50; Xenogen Corp.). An aqueous solution of luciferin was injected intraperitoneally after the mice had been anesthetized. Then, being placed in the light-tight chamber, the amount of photon emitted by RM-PSCA/Luc cells was quantified to monitor the tumor growth. The survival time of the mice was recorded.

Results

PSCA-specific T-cell-mediated immune response

ELISPOT assay and intracellular cytokine staining were utilized to detect the IFN-γ-secreting cells at the individual cell level for determination of PSCA-specific CD8⁺ T-cell responses. Both assays were utilized to detect the IFN-γ-secreting cells at the individual cell level. Mice with immunization of recombinant protein PSCA-HSP generated the highest number of IFN-γ spot-forming cells (24/5×10⁵ splenocytes, p<0.05, compared with other groups) (Fig. 1). However, mice vaccinated with other recombinant proteins and PBS had no such effect, and there was no significant difference in spots formed among these groups. The number of spot-forming cells from splenocytes incubated without peptides was not significantly different among these groups (p>0.05).

FIG. 1.

ELISPOT assay of IFN-γ-secreting PSCA-specific T-cells. Male C57BL/6 mice were vaccinated subcutaneously twice at a 2-week interval with recombinant proteins PSCA, HSP, PSCA+HSP, PSCA-HSP, and PBS. Five mice were assigned for each group. Splenocytes isolated from vaccinated mice 2 weeks after the final vaccination were stimulated with MHC-I-restricted PSCA peptide (aa 28–36) in vitro. (A) Pictures captured by Bioreader 4000. Each individual spot represents an individual IFN-γ–secreting cell. (B) The number of spots (mean±SD) per 5×10⁵ splenocytes for each group with or without stimulation with the MHC-I-restricted PSCA peptide (aa 28–36) is shown. The data were statistically analyzed with the Student–Newman–Keuls test and LSD test. Mice with immunization of recombinant protein PSCA-HSP generated the highest number of spot-forming cells (p<0.05, compared with other groups). The data of ELISPOT assay shown here are from one of three repeated independent experiments. PSCA, prostate stem cell antigen; HSP, heat shock protein; PBS, phosphate-buffered saline; IFN, interferon.

The results of flow cytometric analysis were consistent with the ELISPOT assay (Fig. 2). Mice with immunization of recombinant protein PSCA-HSP generated the highest number of IFN-γ-secreting PSCA-specific CD8⁺ T cells (p<0.05, compared with other groups). The data showed that the numbers of IFN-γ-secreting PSCA-specific CD8⁺ T cells generated by mice vaccinated with other recombinant proteins and PBS were not significantly different (p>0.05).

FIG. 2.

Flow cytometric analysis of IFN-γ-secreting PSCA-specific CD8⁺ T-cells. Male C57BL/6 mice were vaccinated subcutaneously twice at a 2-week interval with different recombinant proteins and PBS. Splenocytes isolated from vaccinated mice were stimulated with MHC-I-restricted PSCA peptide (aa 28–36) in vitro and stained for both CD8 and intracellular IFN-γ. CD8⁺ T-cells were confirmed by costaining with CD3. The number of CD3⁺ /CD8⁺/IFN-γ ⁺ cells per 5×10⁵ splenocytes after stimulation with PSCA-specific peptide (aa 28–36) is shown as a percentage of CD3⁺ T-cells (mean±SD). The data subtracting the number of CD3⁺ /CD8⁺/IFN-γ ⁺ cells without stimulation of PSCA peptide was statistically analyzed. Mice with immunization of recombinant protein PSCA-HSP generated the highest number of IFN-γ-secreting PSCA-specific CD8⁺ T-cells (p<0.05, compared with other groups). The flow cytometric data shown here are from one of three repeated independent experiments.

The results from both the ELISPOT assay and the intracellular cytokine staining assay showed that human HSP70 could enhance the PSCA-specific cellular immune response in mice.

PSCA-specific humoral immune response

ELISA was performed for detection of anti-PSCA antibodies to determine the humoral immune response (Fig. 3). The results showed that mice with immunization of recombinant protein PSCA-HSP generated the strongest anti-PSCA antibody response compared with other groups (p<0.05). The anti-PSCA antibody titers in the groups vaccinated with PSCA and PSCA+HSP were higher than those in the groups vaccinated with HSP and PBS (p<0.05). However, there was no significant difference between the anti-PSCA antibody titers in the groups vaccinated with PSCA and PSCA+HSP, or vaccinated with HSP and PBS, respectively (p>0.05).

FIG. 3.

ELISA was performed to test the humoral immune responses in mice vaccinated with various recombinant proteins and PBS. Serum samples were obtained from immunized mice 14 days after the second vaccination. The results from the 1:100 dilution are shown as the mean absorbance (A450nm)±SD and were statistically analyzed using the Student–Newman–Keuls test. Mice with immunization of recombinant protein PSCA-HSP generated a significantly higher level of anti-PSCA antibodies compared with PSCA and PSCA+HSP vaccinated groups. The ELISA results shown here are from a representative experiment of three repeated independent experiments. ELISA, enzyme-linked immunosorbent assay.

In vivo bioluminescence imaging of prostate tumor growth

In vivo bioluminescence imaging was performed to detect the potency of recombinant protein vaccines to treat tumors in the mouse model. Meanwhile, the tumor volume and the survival time of the mice were recorded. After being challenged with RM-PSCA/Luc cells, all of the mice exhibited a detectable tumor within 2 weeks. The tumor growth was significantly retarded in the groups vaccinated with recombinant proteins PSCA, PSCA+HSP, and PSCA-HSP, compared with those in the groups treated with HSP and PBS (Fig. 4A). The mean size of tumors in the group vaccinated with PSCA-HSP at day 30 was significantly smaller than those in the groups vaccinated with PSCA and PSCA+HSP (p<0.05), and there was no significant difference between the groups vaccinated with PSCA and PSCA+HSP, or vaccinated with HSP and PBS, respectively (p>0.05). The bioluminescent emission increased substantially from first appearance until the day 28 (Fig. 4D). Quantification of Luc signal was used for in vivo monitoring of tumor growth (Fig. 4B), and the bioluminescent signal was related with the tumor burden. The results demonstrated that an in vivo imaging system could be used to monitor tumor growth as a quantitative tool. The tumor progressed quickly, and vaccination with recombinant protein PSCA-HSP significantly prolonged the survival time of mice (ranging from 36 to 49 days) as compared with other groups (Fig. 4C, p<0.05). These data showed that PSCA-specific immune responses inhibited the PSCA-expressing tumors growth in mice and prolonged the life span of tumor bearing mice.

FIG. 4.

In vivo tumor treatment experiment. Male C57BL/6 mice were injected subcutaneously with 1×10⁶ RM-PSCA/Luc cells and immunized with 100 μg recombinant proteins PSCA, HSP, PSCA+HSP, and PSCA-HSP, or injected with PBS on days 3 and 10 after tumor cells challenge. (A) Tumor growth in vaccinated mice. The data are presented as mean tumor volume (mm³)±SD. (B) Quantification of Luc signal in vaccinated mice. The bioluminescent emission registered at day 14 increased substantially from first appearance until the day 28, and the photon counts were correlated with tumor load. (C) Survival of vaccinated mice. The percentage of survival is shown for each group at the indicated time points. Vaccination with PSCS-HSP was most effective at inhibiting tumor growth and could significantly prolong the mouse survival (p<0.05, compared with other groups). The results shown here are from three repeated independent experiments. (D) Monitoring of tumor growth by in vivo bioluminescence imaging. Visualizing images were obtained extracorporeally 1 week (a), 2 weeks (b), 3 weeks (c) and 4 weeks (d) after an injection of 1×10⁶ RM-PSCA/Luc tumor cells. The respective photon counts of each mouse are represented by the scales beside the mouse pictures.

Discussion

As a therapeutic target for tumor treatment, PSCA has several advantages compared with other prostate-confined tumor antigens. Studies suggested that PSCA-specific T-cell immune response in human lymphocyte from patients with prostate cancer could be induced by MHCI-restricted PSCA-derived peptides.^18,19 PSCA was overexpressed in prostate tumor tissues, and there was a direct correlation between the intensity of PSCA expression and the tumor stage, grade, and the bone metastases. These findings suggest the potential of PSCA as a therapeutic target for prostate cancer immunotherapy, as it is not shed from the surface of the prostate cancer cells.

The ideal tumor vaccines not only induce strong CTL responses, but also generate robust tumor specific antibody responses, which elicit drastic and effective antitumor immunological effects. PSCA, as an autoantigen, may cause immune tolerance that hardly incites strong immune responses. Therefore, if assisted with suitable adjuvants, the immunogenicity and the solubility of PSCA can be enhanced, which may improve the potency of protein vaccines based on PSCA. In the past few years, the function of HSP70 with immunoadjuvants has been identified, and large amounts of scientific literature have confirmed that vaccination with both HSP70-peptide complexes and HSP70-antigen fusion proteins reconstituted in vitro with genetic recombination can elicit antitumor immune responses.^20

–23 Zhang et al.²⁴ reported that HSP70 could increase the potency of DNA vaccine based on PSCA with strong cellular and humoral immune responses, which inhibited the growth of PSCA-expressing tumors and prolonged the survival time of vaccinated mice. Moreover, coupling antigens to the amino-terminus of HSP70 could induce stronger immune responses than to the carboxyl-terminus.²⁴ Zhang et al.²⁵ recently prepared the DNA/peptide combined vaccine based on PSCA, and the vaccination of mice with this complex induced a potent antitumor immunity to prostate carcinomas in a xenograft tumor model in nude mice. Karan et al.²⁶ showed that adenovirus vaccine with PSCA and PSA coexpressing could induce strong PSCA-specific immune responses, and inhibit the growth of PSCA-expressing and PSA-expressing tumors. These findings, once again, proved the significance of PSCA as a potentially worthwhile target for prostate cancer immunotherapy.

In this study, we used a prostate tumor animal model with an injection of Luc-reporter-positive transfected cancer cells (RM-PSCA/Luc cells), and tumor progression could be quantitatively monitored by in vivo bioluminescence imaging. However, compared with the results of Zhang et al.,²⁵ the tumor cells established in our study grew faster in vivo. Both Zhang et al.²⁴ and Zhang et al.²⁵ suggested that immunization with pcDNA-PSCA alone hardly incited strong PSCA-specific immune responses and inhibited the growth of PSCA-expressing tumors, while we found that immunization with PSCA protein alone could incite effective immunologic response. In addition, human HSP70 could enhance the potency of the fusion protein vaccine based on PSCA in vivo, which enhanced PSCA-specific cellular and humoral immune responses and inhibited the growth of PSCA-expressing tumors of vaccinated mice. The tumor growth was retarded, and the life span of vaccinated mice was prolonged. Our data showed that mice with immunization of recombinant protein PSCA-HSP generated higher level IFN-γ-secreting CD8⁺ T-cells compared with other groups. Moreover, mice with a vaccination of recombinant protein PSCA-HSP induced the strongest anti-PSCA antibody response. However, the number of IFN-γ-secreting CD8⁺ T-cells induced by recombinant protein PSCA-HSP was significantly lower than that induced by DNA vaccine. Due to the better immune effectiveness and antitumor activity induced by DNA and protein vaccines, the immune strategy of “prime-boost” should be adopted to improve the effect of vaccines in further studies. In addition, the research of Karan et al.²⁶ suggested that dual antigen target-based vaccine could be a better choice for prostate cancer immunotherapy. Animal experiments demonstrated that the antitumor effect in mice with vaccination of recombinant protein PSCA-HSP was better than those in the groups vaccinated with PSCA and PSCA+HSP. The reason may be related to the fact that the cellular and humoral immune levels induced by PSCA-HSP are relatively high and the conformation of recombination protein PSCA purified through inclusion renaturation may be different from nature PSCA protein. In a word, anti-PSCA antibody was confirmed to be effective for inhibiting prostate tumor growth in vivo.

In this present study, the group of mice only immunized with HSP70 had a similar tumor volume as the group of mice immunized with PBS. Although it has been reported that HSPs can evoke inhibition of some tumor growth,^27,28 our results showed that the innate immune responses induced by HSP70 cannot protect mice against the prostate tumor growth.

Conclusions

The present study demonstrated the efficacy of the PSCA-HSP vaccine against the PSCA-expressing tumors in mice and the HSP70 as a potent adjuvant for the vaccine, which lays the foundation for the development of prostate cancer vaccines and its future clinical application.

Footnotes

Acknowledgment

The authors thank Dr. Gao B for providing the statistical analysis.

Disclosure Statement

All authors declare that they have no proprietary, financial, professional, or other personal interest in any product, service, and/or company that could be construed as influencing the position presented in this article.

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