Abstract
To develop effective anti-lung cancer vaccines, we directly mixed mycobacterial heat shock protein 65 (MHSP65) and tumor cell lysate (TCL) from Lewis lung cancer cells in vitro and tested its efficacy on stimulating anti-tumor immunity. Our results showed that MHSP65–TCL immunization significantly inhibited the growth of lung cancer in mice and prolonged the survival of lung cancer bearing mice. In vivo and in vitro data suggest that MHSP65–TCL could induce specific CTL responses and non-specific immunity, both of which could contribute to the tumor inhibition. Thus, this report provides an easy approach to prepare an efficient TCL based tumor vaccine.
Keywords: Lung cancer, Tumor cell lysate, Heat shock protein, Anti-tumor immunity
Introduction
Tumor cell lysates (TCLs), containing multiple tumor antigens, have been tried to use as tumor vaccines [1, 2]. However, TCLs themselves are poorly immunogenic, and fail to stimulate efficient anti-tumor immune responses [3]. To increase the immunogenicity of the TCLs, varied approaches have been tested. In a Phase I/II clinical trial, DETOX was included in the TCL mechanically prepared from melanoma cell line cells, and the DETOX–TCL was evaluated for the treatment of melanoma. DETOX is an immunological adjuvant containing detoxified endotoxin from Salmonella Minnesota, cell wall skeletons of mycobacterium pitici, squalane, and emulsifier. The immunization with DETOX–TCL induced cytotoxic T lymphocytes (CTLs) specific to melanoma cells [4]. Alternatively, fusogenic liposomes (FLs) were included in the TCL from melanoma cells. The FLs–TCL loaded dendritic cells (DCs) induced anti-melanoma immunity in mice [5].
In a decade, heat shock proteins (HSPs) have been found to facilitate the exogenously applied tumor antigens to generate tumor cell specific CTLs and to simulate nonspecific antitumor immune responses. Intrigued by the properties, HSPs were also tested to improve the immunogenicity of TCLs [6]. Tumor-derived chaperone-rich cell lysate (CRCL) prepared via a free solution-isoelectric focusing (FS-IEF) technique was demonstrated therapeutic and prophylactic in mice against B-16 melonoma, leukemia and lymphoma [7]. Tumor cell lysate-pulsed human DCs heat-shocked were found efficient to generate CTL responses against medullary thyroid carcinoma tumor cells in vitro [8]. It was noteworthy that HSPPC-96, a heat shock protein gp96 peptide complex isolated from autologous tumors, was evaluated for the treatment of renal cell carcinoma in phase III clinical trials. The preparations failed to show clinical benefit in the recurrence-free survival of the patients with the tumor [9]. In another trial, HSPPC-96 did not show improvement in overall survival of the patients with IV melanoma [10].
In this study, we tried an approach to prepare a tumor vaccine (MHSP65–TCL) by directly mixing TCL of mouse Lewis lung cancer cells with recombinant mycobacterial heat shock protein (MHSP65). The MHSP65–TCL was tested for its efficacy to induce anti-tumor immunity in a lung cancer model of mice.
Materials and methods
Mice and cell lines
Female C57BL/6 mice were purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd and maintained in microisolator cages under pathogen-free conditions. All mice were used at 6–8 weeks of age. The experimental manipulation of mice was undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Science and Technology of Jilin Province. Mouse Lewis lung cancer cell is a cell line derived from C57BL/6 strain. YAC-1 cell is a mouse lymphoma cell line. The cells were cultured in IMDM before use. The Lewis cells cultured in vitro were used to prepare TCL.
TCL, MHSP65 and MHSP65 plus TCL (MHSP65–TCL)
To prepare TCL, cultured tumor cells were lysed by freezing–thawing in 0.85% NaCl solution for five times in rapid succession between −70 and 37°C, and then refrozen and stored in −70°C refrigerator until use. Each of the TCL was detected under microscope by trypan blue staining after the final thawing. pET28a-MHSP65 recombinant plasmid for expressing MHSP65 in E. coli was constructed by cloning the gene encoding BCG HSP65 into pET28a plasmid vector (Novagen) [11]. To obtain MHSP65, one BL21 colony transformed with pET28a-MHSP65 was inoculated and fermented in 10 L LB medium. Following the induction with isopropyl b-d-thiogalactopyranoside (IPTG) (0.4 mmol/l) for 3 h, the bacteria were harvested by centrifugation and broken by sonication at 4°C. MHSP65 was purified by successive applications of Ni+ column affinity chromatography. LPS was removed by passing the protein through a polymyxin B-agarose column (Detoxigel column; Pierce, Rockford, IL, USA). MHSP65–TCL was prepared by directly mixing MHSP65 with TCL in vitro. Then the mixture was placed in refrigerator overnight for immunization.
Immunization and tumor challenge
Female C57BL/6 mice were immunized with TCL, MHSP65, or TCL plus MHSP65 (MHSP65–TCL) or injected with 0.85% NaCl s.c. at right inguinal lymph node area on day −7, 1, 8 and 15. In therapeutic scheme, mice were immunized on day 1, 8 and 15. One dose of the TCL was derived from 1 × 106 Lewis lung cancer cells. The MHSP65–TCL was prepared by directly mixing the TCL with various amount of MHSP65 in total 150 μl. For tumor inoculation, the mice were challenged with 0.033 g Lewis lung cancer gruel in a volume of 0.2 ml serum-free medium s.c. at back near right hind leg. Lewis lung cancer gruel was prepared as the following. The tumor lump generated by inoculating Lewis lung cancer cells into C57BL/6 mice was removed, weighed and triturated with vitric grinder into gruel. The gruel was suspended in serum-free medium and used for tumor challenge. Tumor volume was measured every 2 days with a caliper and tumors greater than 2 mm in diameter with progressive growth were recorded as positive. Survival of mice was monitored for 50–60 days.
Histological analysis
On day 16 after tumor challenge, three mice in each group were killed and the tumor lumps were removed from the mice, dissected and fixed in 4% (wt/vol) paraformaldehyde. The fixed tumor tissues were embedded into paraffin, cut into sections and stained by hematoxylin/eosin (H–E).
Specific cytotoxicity assay
C57BL/6 mice were immunized with TCL, MHSP65 or MHSP65–TCL or injected with 0.85% NaCl s.c. at right inguinal lymph node area on day 0, 7 and 14 and killed on day 21 for isolating spleens. 1 × 106/ml spleen cells were prepared and cultured with TCL for 4 days in a humidified atmosphere of 5% CO2 at 37°C. Mouse IL-2 (20 U/ml) was added to the culture 48 h later. The spleen cells were centrifuged, counted and used as effector cells. Lewis lung cancer cells were used as target cells. Briefly, 4.5 × 105 spleen cells were serially diluted and cultured with Lewis lung cancer cells per well in a 96-well plate at effector/target ratios of 5:1, 15:1 and 45:1 at 37°C. After 4 h, the supernatant in the culture was discarded and remaining adherent cells were collected, stained with trypan blue and counted. The amount of the remaining live cells was calculated followed by the formula: cells number/ml = total cell number in four big squares/4 × dilute multiple × 104. The cell number in four big squares must be between 200 and 500, and concentration of cells must be higher than 104/ml. The cytotoxicity of the spleen cells was calculated based on the formula: [1 − experiment cell number/cell number in medium control] × 100%.
Nonspecific cytotoxicity assay
1 × 106/ml spleen cells from naive mice were cultured in medium added with 0.85% NaCl, TCL at a ratio of 5:1, 10 μg/ml MHSP65, or MHSP65–TCL for 48 h. Mouse IL-2 (20 U/ml) was added into the culture at the same time. The spleen cells were used as effector cells The effector cells were cultured with 1 × 104 YAC-1 cells or Lewis lung cancer cells per well in a 96-well plate at effector/target ratios of 25:1, 50:1 and 100:1 (for YAC-1 cells) or 5:1, 15:1 and 45:1 (for Lewis lung cancer cells) at 37°C. 24 h later, the cells in each well were stained with 20 μl MTT for 4 h at 37°C and decolored by adding 150 μl DMSO per well for 20 min with shaking. Then, OD value of each well was detected using spectrophotometer at 578 nm. The cytotoxicity was calculated based on the formula: [1 − (OD value of experimental well − OD value of splenocyte control)/OD value of medium control] × 100%.
Flow cytometry analysis
Mouse spleen cells from either immunized mice or naïve mice were stained with FITC-labeled anti-CD8, CD19 or NK1.1 mAbs and/or PE-labeled anti-CD69 mAb for analyzing activation of lymphocytes. Briefly, the 1 × 105 spleen cells were stained with fluorescence conjugated monoclonal antibodies for 30 min on ice in dark, washed with FACS buffer (1 × PBS) and analyzed by flow cytometry.
Statistical analysis
Tumor growth curves were plotted based on tumor size until the first mouse was died. One-way analysis of variance (ANOVA) was used to analyze experimental data. A two-sided Student’s t test was adopted to compare the mean values of individual treatments when the primary outcome was statistically significant. Survival was estimated by the Kaplan–Meier method and evaluated with a log-rank test. A P value of <0.05 was considered statistically significant. All statistical analyses were performed with the SPSS 13.0 software.
Results
MHSP65 assists TCL to display anti-lung cancer effect in mice
To explore a new approach for developing an efficient vaccine against lung cancer, MHSP65 was directly mixed with the TCL prepared from Lewis cells, lung cancer cell line cells derived from C57BL/6 mouse, and the resultant preparation (MHSP65–TCL) was tested for its effect for evoking anti-tumor immunity in mice. As shown in Fig. 1a, C57BL/6 mice were s.c. injected with TCL or MHSP65 or MHSP65–TCL or 0.85% NaCl at inguinal lymph node area on right side on day −7, 1, 8 and 15 and challenged with Lewis lung cancer gruel on day 0. Tumor volumes were measured every other day. The result showed that TCL or MHSP65, when used alone, failed to induce tumor growth inhibition (P > 0.05), and that MHSP65–TCL induced significant growth inhibition of the tumors (P < 0.05) (Fig. 1b). The tumor incidence in mice immunized with MHSP65–TCL was significantly lower than that in other groups (P < 0.05) (Fig. 1c). On day 11, tumor incidence of mice injected with NaCl or TCL or MHSP65 reached 66.67, 50 and 83.33%, respectively, whereas the incidence in MHSP65–TCL group was only 16.67%. 100% of mice vaccinated with MHSP65–TCL was still alive but 83.33% of mice in NaCl group succumbed to the tumors on day 50 (Fig. 1d) (P < 0.005). To conduct histological examination, three of mice in each group were killed on day 16 post-tumor inoculation. The tumors were removed and sectioned. The sections were stained by H–E and observed under microscope. As shown in Fig. 1e, massive death of tumor cells was induced by MHSP65–TCL. The dead tumor cells were surrounded by large number of infiltrated lymphocytes.
Fig. 1.
The growth inhibition of lewis lung cancer induced by MHSP65–TCL. C57BL/6 mice were immunized on day −7 with TCL or MHSP65 or MHSP65–TCL and transplanted with Lewis lung cancer cells on day 0, followed by three immunizations from day 1 in 7-day interval. Tumor volume was measured every 2 days, survival of mice was calculated and histological section of tumors was analyzed. *, versus NaCl; #, versus TCL; ∆, versus MHSP65. a The experimental procedure. b Tumor growth curves. Each line represents tumor growth kinetics in each mouse. c Tumor incidence. Each line represents tumor incidence in each group. d Survival curves of mice in each group. e Histological sections of tumors
To find an optimal dose of MHSP65, MHSP65 at 100 or 10 or 1 μg was used to prepare MHSP65–TCL from the TCL of 1 × 106 Lewis lung cancer cells. The dose analysis was conducted in a prophylactic/therapeutic scheme. As shown in Fig. 2a, the MHSP65–TCL prepared with MHSP65 at three dosages all induced significant inhibition of tumor growth, compared with NaCl (P < 0.005) and TCL (P < 0.05). The MHSP65–TCL prepared with 100 μg MHSP65 was less effective than MHSP65–TCL prepared with 10 μg MHSP65 (P = 0.014) or 1 μg MHSP65 (P = 0.006). As shown in Fig. 2b, on day 29 after tumor inoculation, 60% of the mice immunized with MHSP65–TCL prepared with MHSP65 at three dosages still survived, and all of the mice in the control groups succumbed to the tumors (P < 0.05). On day 50 after tumor inoculation, when all of the mice received MHSP65–TCL prepared with 100 μg MHSP65 succumbed to advancing tumors, 37.5 or 62.5% of the mice still survived in the mice immunized with MHSP65–TCL prepared with 10 μg MHSP65 or with 1 μg MHSP65, respectively. To find the lower optimal dose of MHSP65, another experiment was conducted. The mice were immunized with MHSP65–TCL prepared with 0.1 or 0.01 μg MHSP65. As shown in Fig. 2c, d, MHSP65–TCL prepared with 0.1 or 0.01 μg MHSP65 could prolong the survival of the mice (P < 0.05), and failed to induce significant inhibition of tumor growth.
Fig. 2.
Dose-effect of MHSP65 on tumor inhibition induced by MHSP65–TCL. C57BL/6 mice were immunized with TCL containing 100, 10, 1 or 0.1 μg, 0.01 μg MHSP65 or TCL or MHSP65 on day −7, 1, 8 and 15, respectively. NaCl-injected mice were as negative controls. On day 0, the mice were transplanted with Lewis lung cancer. Tumor volume was measured every 2 days and survival of mice was calculated. *, versus NaCl; #, versus TCL; ∆, versus MHSP65; a, versus 100 μg MHSP65–TCL. a Tumor growth curves. Each line represents tumor growth kinetics in each mouse. b Survival curves. c Tumor growth curves. Each line represents tumor growth kinetics in each mouse. d Survival curves
To study whether MHSP65–TCL is therapeutic to already established tumors, mice were therapeutically immunized three times after tumor challenge on day 1, 8 and 15. As a control, the mice were prophylactically/therapeutically immunized both before and after the tumor challenge on day −7, 1, 8 and 15 (Fig. 3a). The tested mice were observed after tumor inoculation, and the tumor growth was measured and recorded as tumor volumes. As shown in Fig. 3b, the therapeutic immunization with MHSP65–TCL could induce significant inhibition of the tumor growth, compared with that in NaCl-injected mice (P = 0.004). In contrast, the prophylactic/therapeutic immunization with MHSP65–TCL displayed more effective role in tumor inhibition in mice than the therapeutic immunization (P = 0.014).
Fig. 3.
Tumor inhibition induced by therapeutic or prophylactic/therapeutic immunization of MHSP65–TCL. C57BL/6 mice were immunized either with MHSP65–TCL on day 1, 8 and 15 or with MHSP65–TCL on day −7, 1, 8 and 15. NaCl-injected mice were used as negative controls. On day 0, the mice were transplanted with Lewis lung cancer. Tumor volume was measured every 2 days. *, versus NaCl; a, versus MHSP65–TCL therapeutically immunized. a The experimental procedure. b Tumor growth curves. Each line represents average tumor growth kinetics of the mice in different groups
MHSP65 assists TCL to initiate specific anti-lung cancer immunity
To detect the specific anti-tumor immunity induced by MHSP65–TCL, mice were injected with TCL or MHSP65 or MHSP65–TCL or 0.85% NaCl for three times in a 7-day interval. On day 21 after the first immunization, the mice were killed, and their spleens were isolated (Fig. 4a). The spleen cells were cultured with TCL for 4 days, and then co-cultured with the adherent Lewis lung cancer cells at effector/target ratio of 5:1, 15:1, 45:1, respectively, for another 4 h. After discarding the suspended spleen cells, the remaining adherent cells were stained by trypan blue and counted. The result showed that the spleen cells from the mice immunized with MHSP65–TCL could significantly kill the Lewis lung cancer cells (P = 0.02) (Fig. 4b). To observe the activation of CD8+ T cells, the spleen cells from the immunized mice were cultured with TCL for 8 h, and then stained with FITC-labeled anti-CD8 mAb and PE-labeled anti-CD69 mAb, followed by flow cytometry analysis. The result showed that the average percentage of CD8 and CD69 double-positive cells in spleen cells from mice immunized with MHSP65–TCL was 2.7% that was significantly higher than 1.7% in the spleen cells from the mice injected with NaCl (P = 0.001) (Fig. 4c).
Fig. 4.
Specific anti-tumor immunity induced by MHSP65–TCL. C57BL/6 mice were immunized with TCL or MHSP65 or MHSP65–TCL on day 0, 7 and 14. On day 21, mice were killed for isolating spleens. a The experiment procedure. b Specific CTL killing assay. The splenocytes were stimulated with TCL for 4 days and used as effector cells. Lewis lung cancer cells were used as target cells. c Flow cytometry analysis of CD8+ T cell activation. After stimulation with TCL for 8 h, the splenocytes were stained with FITC-labeled anti-CD8 mAb and PE-labeled anti-CD69 mAb and then analyzed by flow cytometry. *, versus NaCl
MHSP65–TCL activates non-specific immune response in vitro and in mice
To investigate whether MHSP65–TCL can activate non-specific anti-tumor immunity, spleen cells from naive mice were cultured with TCL, MHSP65 or MHSP65–TCL for 48 h (Fig. 5a), and then co-cultured with NK sensitive cell YAC-1 cells or Lewis lung cancer cells at different ratio for testing their cytotoxic activities. The results showed that spleen cells stimulated by TCL, MHSP65 or MHSP65–TCL displayed significant cytotoxicity (P < 0.01) at effector/target ratio of 100:1 to YAC-1 cells or 45:1 to Lewis lung cancer cells. MHSP65–TCL was more effective on activating spleen cells than TCL (P = 0.02) or MHSP65 (P = 0.02) (Fig. 5b, c). To observe the nonspecific activation, spleen cells from naive mice were cultured with TCL, MHSP65 or MHSP65–TCL for 48 h and then stained with PE-labeled anti-CD69 mAb, followed by flow cytometry analysis. The result revealed (Fig. 5d) that the CD69 on spleen cells was significantly up-regulated by TCL or MHSP65 (P < 0.001) or MHSP65–TCL (P < 0.001).
Fig. 5.
Non-specific immune responses induced by MHSP65–TCL in vitro. a The experimental procedure. b YAC-1 cell killing assay. Splenocytes from naive mice were treated with different stimulators for 48 h and used as effector cells. YAC cells were used as targets. c Lewis lung cancer cell killing assay. The splenocytes were treated with different stimulators as above and used as effectors. Lewis lung cancer cells were used as targets. d Flow cytometry analysis. The splenocytes were treated with different stimulators for 48 h, stained with PE-labeled anti-CD69 mAb and then analyzed by flow cytometry. Representative data from one of three experiments are shown. *, versus NaCl; #, versus TCL; ∆, versus MHSP65
Furthermore, we tested the activation status of lymphocytes including NK, CD8+ T and B cells in the spleens of vaccinated mice. Mice were immunized with TCL, MHSP65 or MHSP65–TCL for four times and killed at different time points for isolating spleens (Fig. 6a). The spleen cells were directly stained with FITC-labeled anti-NK1.1 mAb or FITC-labeled anti-CD8 mAb or FITC-labeled anti-CD19 mAb and PE-labeled anti-CD69 mAb, followed by flow cytometry analysis. The results showed that the activated NK cells were observed in the spleens isolated from MHSP65–TCL immunized mice on day 6 post-primary immunization and in 24 h post-third immunization, and could not be observed in the spleens from the mice received NaCl, TCL or MHSP65 (Fig. 6b). Interestingly, CD8/CD69 double-positive cells were obviously decreased in the spleen from MHSP65–TCL immunized mice on day 19 after the first immunization (P < 0.01) (Fig. 7a), compared with those in the spleens of NaCl injected mice (Fig. 7b). In contrast, the number of CD19/CD69 double-positive cells in the spleens did not changed by the immunization with TCL or MHSP65 or MHSP65–TCL (Fig. 7c).
Fig. 6.
Ex vivo activation of NK cells in splenocytes from immunized mice. C57BL/6 mice were immunized with TCL or MHSP65 or MHSP65–TCL for four times and killed for isolating spleens. The splenocytes were stained with FITC-labeled anti-NK1.1 mAb and PE-labeled anti-CD69 mAb and then analyzed by flow cytometry. Representative data from one of three mice in each group are shown. *, versus NaCl; #, versus TCL. a The experimental procedure. b Percentage of NK1.1+CD69+ cells
Fig. 7.
Ex vivo activation of T cells and B cells in splenocytes from immunized mice. C57BL/6 mice were immunized with TCL or MHSP65 or MHSP65–TCL for four times and killed for isolating spleens. The splenocytes were stained with FITC-labeled anti-CD8 or CD19 mAb and PE-labeled anti-CD69 mAb and then analyzed by flow cytometry. Representative data from one of three mice in each group are shown. *, versus NaCl; #, versus TCL. a The experimental procedure. b Percentage of CD8+CD69+ cells. c Percentage of CD19+CD69+ cells
Discussion
In this study, we prepare the anti-lung cancer vaccine by directly mixing MHSP65 and TCL in vitro. The rationality of this method could be supported by the evidence that simultaneous immunization with the ovalbumin (OVA) antigen and MHSP65 could elicit a CTL response to an OVA-derived epitope in mice, which implied that MHSP65 engineered into a fusion protein with the antigen of interest might not be necessary [12]. Compared with the preparation of MHSP65-antigen fusion protein, the approach presented here is more simple and feasible, MHSP65, after mixing with TCL, could facilitate the multiple tumor antigens in the TCL to generate immunity to inhibit the growth of Lewis lung cancer cells in mice and prolong the survival of tumor-bearing mice. The facilitation could be partly due to the binding of MHSP65 with various tumor antigens. Our unpublished data showed that MHSP65 could bind multiple peptides in the TCL to form MHSP65-peptide complexes. The peptides in the complexes could be cross-presented to tumor specific CTLs [13–16]. As shown in the present study, MHSP65–TCL immunization generated specific CD8+ T cells that could kill Lewis lung cancer cells in vitro. The generated CD8+ T cells, as shown in this study, could be mobilized by MHSP65–TCL from the spleen to the tumor sites where tumor-infiltrating lymphocytes (TIL) were obviously increased, accompanied with the necrosis of cancer cells. Noticeably, to induce efficient immunity specific to tumor cells, MHSP65 should be used at the appropriate dosage. The MHSP65 at high dose levels, such as 10 or 100 μg, was found less active to facilitate TCL to induce anti-tumor immunity. On the other hand, MHSP65 at low dose levels, such as 0.1 or 0.01 μg, was insufficient to facilitate. Seemingly, MHSP65 at 1 μg was the most effective.
Besides the specific immunity, non-specific immunity is also responsible for part of the anti-tumor effect induced by MHSP65–TCL. In the in vitro experiment, MHSP65–TCL was shown to up-regulate CD69 expression in spleen cells from naive mice. As reported, CD69 up-regulation could indicate the activation of innate immune cells [17]. The activated naïve spleen cells could significantly kill Lewis lung cancer cells and NK sensitive cell YAC-1 cells. The nonspecific killing might be translated into in vivo benefit of facilitating the generation of tumor specific CTLs by producing tumor fragments that could be presented by APCs for activating CTL [18]. Notably, TCL and MHSP65 alone could non-specifically activate NK cells in vitro assay (Fig. 5b) but could not up-regulate CD69 expression on NK cells in ex vivo assay (Fig. 6). The discrepancy of the non-specific effect might be attributed to the assays under varied experimental conditions. In the in vitro assay, the spleen cells were isolated from naïve mice and further cultured with IL-2 for 48 h before testing their NK activities and CD69 up-regulation. In the ex vivo assay, the spleen cells were isolated from the mice received TCL or MHSP65 injection, and the NK cells in them were directly detected for their CD69 up-regulation, without further culturing with IL-2.
Collectively, MHSP65, could facilitate TCL to induce specific immune responses to lung cancer cells and to activate nonspecific anti-tumor immune response when directly mixed with TCL. The approach could be applied to generate heat shock protein based tumor vaccines of human use.
Acknowledgments
We would like to thank Guang Yang, Ran Sun, He Li for technical support.
Conflict of interest statement
The authors report no conflicts of interest.
Footnotes
B. Dong and L. Sun contributed equally to this work.
Contributor Information
Shucheng Hua, Phone: 86-431-88782258, Email: shuchenghua@eyou.com.
Liying Wang, Phone: +86-431-85619369, FAX: +86-431-85647872, Email: wlying@mail.jlu.edu.cn.
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