Tumor Cell Lysate Plus Mycobacterium Tuberculosis Heat Shock Protein 70 Induced Anti-tumor Immunity

Tumor cell lysate (TCL) is prepared by lysing tumor cells with physical and chemical methods. Theoretically, TCL could be developed into tumor vaccines that are capable of activating anti-tumor immune response targeting multiple tumor antigens. However, when used alone, TCL failed to induce an efficient immune response to eradicate tumors, showing poor immunogenicity. Mycobacterium tuberculosis derived heat shock protein 70 (TBHsp70) has been shown to assist cross-presentation of exogenously applied tumor antigens and induce the generation of antigen-specific cytotoxic T lymphocyte (CTL), indicating it might be used as an adjuvant for TCL to induce effective anti-tumor immunity. In this study, TBHsp70-B16TCL, a preparation generated by directly mixing recombinant TBHsp70 and B16 TCL (prepared from B16 melanoma cells by freeze-thaw), was tested for its anti-tumor effect in abdominal and subcutaneous B16 melanoma-bearing mouse models. In each mouse model, the TBHsp70-B16TCL was administrated in three immunization strategies, which were the prophylactic, therapeutic and prophylactic plus therapeutic (prophylactic/therapeutic) immunizations. Thereafter, the anti-tumor mechanism was investigated.1 Construction, expression and purification of recombinant TBHsp70To get recombinant TBHsp70 protein (rTBHsp70), TBHsp70 gene was amplified by PCR and inserted into vector pET28a to construct the recombinant pET28a/TBHsp70. And then the pET28a/TBHsp70 was transformed into E. coli. BL21(DE3). The rTBHsp70 was expressed in fermentor with IPTG induction. SDS-PAGE analysis showed that the rTBHsp70 was soluble and approximately accounted for 29.6% of the total bacterial proteins. Then the bacteria were disrupted by sonication. The rTBHsp70 was purified by successive applications of Nikel-affinity chromatography on Sepharose 4B, Triton-X114 washing, gel filtration on Sephadex G-25 and ion-exchange chromatography on Q-Sepharose FF. The rTBHsp70 was purified to 96.5% purity, and the concentration of it was 1.2 g/L. Endotoxin in it was less than 0.01 EU/μg. It was stable at 37℃and the natural bioactivity of TBHsp70 was observed in it.2 Preparation of TBHsp70-B16TCLB16 melanoma cells were harvested and then resuspended in PBS (2×107 cells/mL). The cell suspensions in the medium were disrupted by five cycles of freeze-thaw using -70℃and 37℃water bath. The cell lysates were verified by trypan blue dye exclusion staining and stored at -70℃for later use. 100μg of TBHsp70 was incubated with the TCL from 1×106 B16 melanoma cells in the binding buffer (PBS supplemented with 1 mmol/L ADP and 1 mmol/L MgCl2) at 37℃for 1 h. After the incubation, the mixture was immediately used in subsequent studies in mice.3 TBHsp70-B16TCL in tumor-suppressive experiments in miceThe prophylactic, therapeutic and prophylactic/therapeutic immunization strategies were used to observe the anti-tumor effect of TBHsp70-B16TCL on the abdominal and subcutaneous B16 melanoma-bearing mouse models. In each immunization strategy, mice were injected subcutaneously (s.c.) with TBHsp70-B16TCL, TBHsp70, B16 TCL or PBS. In the prophylactic protocol, mice were immunized on days -17 and -3, and challenged with B16 melanoma cells on day 0. In the therapeutic protocol, mice were inoculated with B16 melanoma cells on day 0 and then immunized on days 1, 4 and 8. In the prophylactic/therapeutic protocol, mice were immunized on days -17, -3, 4 and 11, and injected with B16 melanoma cells on day 0.The results from abdominal B16 melanoma-bearing mouse models are as follows: In the prophylactic protocol, immunization with TBHsp70-B16TCL obviously prolonged the survival of B16 melanoma bearing-mice (p=0.002 vs PBS; p=0.024 vs B16 TCL; p=0.000 vs TBHsp70). Notably,on day 29 after tumor inoculation, 37.5% of TBHsp70-B16TCL treated mice were still alive, whereas on the day all mice from other groups succumbed. Moreover, up to 60 days, 25% of TBHsp70-B16TCL immunized mice still survived; In the therapeutic protocol, administration with TBHsp70-B16TCL significantly prolonged the survival of B16 melanoma bearing-mice (p=0.038 vs PBS), whereas TBHsp70 or B16 TCL immunization couldn't exhibit such anti-tumor effect (p>0.05 vs PBS), indicating the therapeutic application of TBHsp70-B16TCL could obviously prolong the survival of abdominal B16 melanoma bearing-mice; In the prophylactic/therapeutic protocol, when all mice succumbed in PBS, TBHsp70 and TCL treated group, 42.9% of the mice in the TBHsp70-B16TCL treated group still survived by day 37, and there were 28.6% of them survived by day 60. These results showed that prophylactic/therapeutic immunization with TBHsp70-B16TCL could significantly prolong the survival of mice inoculated i.p. with B16 melanoma cells (p=0.031 vs PBS; p=0.042 vs B16 TCL).The results from subcutaneous B16 melanoma-bearing mouse models are as follows: In the prophylactic protocol, TBHsp70-B16TCL immunization obviously prolonged the tumor latency of B16 melanoma bearing-mice(p=0.020 vs PBS), whereas TBHsp70 or B16 TCL immunization didn't exhibit such inhibition (p>0.05). Additionally, TBHsp70-B16TCL significantly inhibited the melanoma growth within 15 days after tumor inoculation (p=0.046 vs PBS, p=0.032 vs B16 TCL), while B16 TCL or TBHsp70 failed to induce tumor growth inhibition (p >0.05). However, after day 15 post-tumor inoculation, no statistical differences were observed on the tumor volume in the four groups (p>0.05). Regarding the survival, by day 39 after tumor implantation, all mice immunized with PBS, TBHsp70 or TCL were succumbed. Meanwhile, 25% of mice vaccinated with TBHsp70-B16TCL were still alive, indicating a significantly prolonged survival (p=0.029 vs PBS; p=0.007 vs TCL). These data showed that prophylactic application of TBHsp70-B16TCL could significantly prolong the tumor latency, inhibit the the melanoma growth in the early stage and prolong the lifespan of the mice; In the therapeutic protocol, vaccination with TBHsp70-B16TCL remarkably prolonged the tumor latency of B16 melanoma bearing-mice (p<0.05), while no difference was shown among the TBHsp70, B16 TCL and PBS treated mice ((p>0.05). The result of tumor volume indicated that TBHsp70-B16TCL failed to induce significant tumor growth, compared with PBS, TBHsp70 or B16 TCL (p>0.05). Notably, TBHsp70-B16TCL significantly prolonged survival of the mice comparatively (p=0.020 vs PBS; p=0.044 vs B16 TCL). These results demonstrated that therapeutic vaccination with TBHsp70-B16TCL could also obviously prolong the tumor latency and the survival of B16 melanoma bearing mice. TBHsp70-B16TCL should be applied earlier to induce more effective anti-melanoma immunity; In the prophylactic/therapeutic protocol, administration with TBHsp70-B16TCL obviously prolonged the tumor latency of B16 melanoma bearing-mice (p=0.001 vs PBS; p=0.022 vs B16 TCL) whereas TBHsp70 and B16 TCL didn't show this prolangation (p>0.05). TBHsp70-B16TCL immunization profoundly inhibited the melanoma growth compared with PBS immunization (p=0.003), whereas there were no statistic differences on the tumor volume in B16 TCL, TBHsp70 and PBS treated mice (p>0.05). At the same time, a significantly prolonged survival was found in the TBHsp70-B16TCL immunized mice (p=0.028 vs PBS; p=0.038 vs TBHsp70), otherwise no significantly diffences in survival were observed in the PBS, TBHsp70 and B16 TCL immunized mice (p>0.05). These data indicated that prophylactic/therapeutic immunization with TBHsp70-B16TCL could remarkably prolong the melanoma latency, inhibit the tumor growth and prolong the survival of subcutaneous B16 melanoma-bearing mice.Taken together, immunization with TBHsp70-B16TCL could obviously inhibit the melanoma growth and metastasis in mice and more efficient anti-tumor effects were induced in the abdominal B16 melanoma-bearing mice immunized with TBHsp70-B16TCL by utilizing the prophylactic and prophylactic/therapeutic immunization protocols.4 B16 melanoma specific immunologic memory induced by TBHsp70-B16TCL in miceTo investigate whether the TBHsp70-B16TCL could induce the development of B16 melanoma specific immunologic memory, the TBHsp70-B16TCL administrated mice survived from the B16 melanoma intraperitoneal inoculation in the prophylactic and prophylactic/therapeutic immunization strategy were re-challenged intraperitoneally (i.p.) with B16 melanoma cells on day 60 after the first challenge. The result showed that all mice survived from the first tumor challenge were still alive on day 100 after the second tumor challenge, without any sign of tumor development(p=0.00), whereas all mice(challenged i.p. with B16 melanoma cells for the first time) in the control group succumbed by day 20 after the tumor inoculation. The data implied that TBHsp70-B16TCL could generate tumor specific immunologic memories. To further confirm the inference, C57BL/6 mice were prophylactically immunized subcutaneously (s.c.) with PBS, TBHsp70, B16 TCL or TBHsp70-B16TCL on day 1 and 14, and the spleens were taken out on day 17. Then, the splenocytes from each group were tested for their proliferative response induced by B16 TCL in vitro. Data revealed that when recalled by B16 TCL, immunization with TBHsp70-B16TCL significantly enhanced the splenocytes proliferation compared with those in the control groups (p<0.05), showing TBHsp70-B16TCL did generate tumor specific immunologic memories. The specificity of the anti-tumor immunity was further verified by comparing the efficacies of TBHsp70-B16TCL and TBHsp70-EL4TCL. TBHsp70-EL4TCL was prepared by mixing TBHsp70 with TCL of EL4 lymphoma cells of C57BL/6 mouse origin. Mice were injected s.c. with TBHsp70-B16TCL, TBHsp70-EL4TCL or PBS according to the prophylactic/therapeutic protocol in the abdominal and subcutaneous B16 melanoma-bearing mouse models. In the subcutaneous B16 melanoma-bearing model, immunization with TBHsp70-B16TCL markedly prolonged the tumor latency (p=0.001) and inhibited the melanoma growth compared with PBS (p=0.003), but the TBHsp70-EL4TCL showed no anti-tumor effect (p>0.05). The survival of TBHsp70-B16TCL immunized mice was also obviously prolonged compared with TBHsp70-EL4TCL (p=0.034) and PBS (p=0.028) treated mice. Analogously, in the abdominal B16 melanoma-bearing model, the survival of TBHsp70-B16TCL immunized mice was significantly prolonged (p=0.031 vs PBS), but TBHsp70-EL4TCL immunized mice showed no statistically difference in the survival (p>0.05 vs PBS). These results indicated that TBHsp70-B16TCL did induced specific anti-melanoma immunity.In order to investigate the mechanism of the specific anti-tumor immunity induced by TBHsp70-B16TCL, mice were prophylactically immunized s.c. at the right inguinal lymph node area with PBS, B16 TCL, TBHsp70, and TBHsp70-B16TCL for twice in a 14-day interval and then inoculated i.p. with B16 melanoma cells on day 3 after the second immunization. On 3 days after the tumor inoculation, the spleens, mesentery lymph nodes and greater omentums were taken out, the CD8+ and CD4+ lymphocyte subpopulations from spleen and mesentery lymph nodes of each group were analyzed, and the CD4+ cells/CD8+ cells ratio were calculated. The results showed that the CD4+ cells/CD8+ cells ratio of spleen lymphocytes from TBHsp70-B16TCL treated mice significantly decreased, suggesting the specific CD8+ T cells might be expanded to participate in the anti-tumor immunity. Interestingly, in mesentery lymph nodes from the same TBHsp70-B16TCL immunized mice, the CD4+ cells/CD8+ cells ratio significantly increased. The pathological analysis revealed marked infiltrations of lymphocytes in the omentums where inoculated B16 cells were found mainly implanted on, indicating that TBHsp70-B16TCL could induce lymphocytes migrated to the tumor site to kill the tumor cells. To further confirm our inference, B16TCL-specific CTL was analyzed. The result showed that the splenocytes from the mice immunized with TBHsp70-B16TCL could significantly kill B16 melanoma cells, compared with those from all control groups at an E:T ratio of 100 and with those from PBS group at an E:T ratio of 50 (p<0.05). No significant differences were observed in those from the mice in TBHsp70, B16 TCL and PBS groups at all three E:T ratios. The result demonstrated that TBHsp70-B16TCL induced B16 melanoma specific CTLs.In this study, the rTBHsp70 with natural activity was constructed, expressed and purified successfully. B16 melanoma cells were disrupted by using 5 cycles of freeze-thaw. Then the TBHsp70-B16TCL was prepared by directly mixing B16 TCL with rTBHsp70 in vitro. Our results showed the TBHsp70-B16TCL induced a significant inhibition on the growth and metastasis of B16 melanoma in mice and prolonged the survival of B16 melanoma-bearing mice. TBHsp70-B16TCL could induce B16 melanoma-specific CTL responses and immunologic memory. The data provides a new way for the preparation of TCL-based tumor vaccine.