Antitumor Activity Of MHSP65-TTL Combined With Low Dose Cyclophosphamide In Pancreatic Cancer-bearing Mice

Pancreatic cancer remains a lethal malignancy with less than 5% of patients alive at 5 years[Lutz E R, 2014]. Surgical resection is the only known curative treatment for pancreatic cancer, and the cancer usually recurs between 9 and 12 months after resection[Liang H, 2004]. Over the last 30 years, despite the application of chemotherapy or/and radiotherapy after the surgery, the improvement on the overall survival of the patients has still been minimal[Soares K C, 2012]. Hence, it is required to develop novel approaches for the treatment of pancreatic cancer. Immunotherapy for pancreatic cancer has been the new choice.Tumor immunotherapy is depended on activation of the anti-tumor immune system via immunization and immune regulation. Effective vaccine has two elements: effective adjuvant and tumor antigens. Mycobacterium tuberculosis heat shock protein 65(m HSP65) has widely been used as a tumor vaccine adjuvant as it is capable of facilitating cross-presentation of antigen to MHC class I molecules to induce specific CTL, activating innate immunity, promoting DC maturation and Th1 type immune response. Tumor lysate contained almost all tumor antigens, which is superior to single tumor antigens. m HSP65 plus tumor lysate has been studied in lung cancer investigation and showed potent anti-tumor activity[Dong B, 2010]. In this study, we used the m HSP65-TTL, m HSP65-TCL as candidate vaccines prepared by mixing the recombinant m HSP65 with the lysate of Panc02 pancreatic cancer tissue or cells.Recent work has highlighted that classical chemotherapeutics could stimulate tumor-specific immune responses either by inducing the immunogenic death of tumor cells or by engaging immune effector mechanisms. Among them, cyclophosphamide(CY) was shown to induce beneficial immunomodulatory effects in the context of active or adoptive immunotherapy. Administration of CY at low dose selectively induces cell death of regulatory T(Treg) cells and promotes the transformation of CD4+ T cells into Th17 cells.In this study, we used the m HSP65-TTL, m HSP65-TCL as candidate vaccine prepared by mixing the recombinant m HSP65 with the lysate of Panc02 pancreatic cancer tissue or cell to immune the C57BL/6 mice implanted with Panc02 cancer cells. To improve the efficacy of the vaccine, we select the suitable low dose chemotherapeutic drugs to combinate with the m HSP65-TTL in the mice and study the underlying mechanism.1.Preparation of m HSP65-TCL and m HSP65-TTLTo prepare pancreatic tissue lysates(TTL), mice were inoculated with Panc02 cells intraperitoneally, when the tumor growed to the appropriate size, solid tumors were isolated for preparing the tumor lyastes by repeated freezing and thawing for 5 times. To prepare pancreatic cell lysates(TCL), Panc02 cells at good condition were digested with trypsin and washed twice with saline and then prepared the tumor lyastes by repeated freezing and thawing for 5 times. The concentration of proteins in TTL was 2.24mg/ml and the concentration of proteins in TCL was 1.23mg/ml using Lowry method. m HSP65-TTL or m HSP65-TCL was prepared by mixing TTL or TCL with HSP65 and antified by SDS-PAGE for subsequent experiments in vivo or in vitro.2.Anti-tumor effect of HSP65-TTL combined with different chemotherapeutic drugsRecent studies showed that chemotherapeutic drugs at low dose could enhance the anti-tumor effect of tumor vaccines, CY, GEM, 5-Fu have been largely investigated in clinical studies. To search a suitable chemotherapeutic drug, we observed that the anti-tumor effects of m HSP65-TTL, when combined with different chemotherapy drugs. 48 healthy female C57BL/6 mice were inoculated with the tumor cell suspension made from cut debris of Panc02 cancer tissue(100mg/150μl) into their back near hind legs. All the mice survived for three days after surgery. All mice were divided into 6 groups and injected intraperitoneally with PBS, CY, 5-Fu, GEM, CY+5-Fu, CY+GEM, and than immunized with m HSP65-TTL subcutaneously at days 4,12,20,28. The results showed that CY, CY+5-Fu, CY+GEM could significantly enhance the anti-tumor effect of m HSP65-TTL, compared with m HSP65-TTL group(P=0.001, P=0.004, P=0.017). While, CY combined with 5-Fu and GEM had been no significant difference in enhancing anti-tumor effect of m HSP65-TTL, compared with CY group. The 5-Fu and GEM combined with m HSP65-TTL did not induce effective anti-tumor immunity. Therefore, CY might be an effective chemotherapeutic agent to enhance the antitumor effect of cancer vaccines.3.Antitumor Effect of m HSP65-TCL or m HSP65-TTL plus low dose CYTo explore a new immunotherapy approach against pancreatic cancer, we inoculated 1×103 Panc02 cancer cells into pancreas of C57BL/6 mice on day 0 and then immunized the mice with m HSP65-TTL or m HSP65-TCL on day 5, 13, 21, 29, with or without CY treatment(50mg/kg) on day 4, 12, 20, 28. The m HSP65-TTL(2μg m HSP65+100μg TTL/mouse) was prepared by mixing the m HSP65 and the lysate of Panc02 pancreatic cancer tissue. The m HSP65-TCL(2μg m HSP65+100μg TCL/mouse) was prepared by using the lysate of cultured Panc02 cells. The mice were monitored for their general performance, body weight, tumor incidence and survival for 140 days after the tumor inoculation, and all of the mice survived with no invisible tumors.To observe whether the mice immunized with m HSP65-TTL or m HSP65-TCL, in combination with or without CY treatment had acquired immunity against the Panc02 cancer, the mice, on day 140, were subcutaneously inoculated with the tumor cell suspension made from cut debris of Panc02 cancer tissue(100mg/mouse) into their back near hind legs, and then monitored as above mentioned. 14 days later, the inoculated tumor cell suspension began to develop into observable tumors which were then measured every 2 days. On day 31 after the tumor cell suspension inoculation, two mice injected with PBS(n=7) died and within the ongoing 10 days all of the rest 5 mice in the group were dead. Comparatively, only one mouse immunized with m HSP65-TTL(n=8) was found dead on day 53 after the tumor inoculation, and the rest 7 mice in the group survived. Noticeably, all of the mice treated with m HSP65-TTL plus CY(n=7) survived, without observable tumors. Statistic analysis showed that m HSP65-TTL alone or m HSP65-TTL plus CY significantly prolonged the survival of mice inoculated with Panc02 cancer cells suspension, compared with PBS(P<0.01, respectively). In addition, m HSP65-TTL plus CY could inhibit the incidence and growth of tumor. Taken together, these results revealed that the lysate prepared from the cultured Panc02 cancer cells was poorly immunogenic in inducing anti-tumor immunity in comparison with the lysate from Panc02 cancer tissue.4.Anti-tumor effect of m HSP65-TTL plus CY in prophylactic settingIn the above-mentioned experiments, the antitumor immunity was induced by m HSP65-TTL or m HSP65-TTL plus CY in the mice received inoculation of a small amount of cultured Panc02 cells, implying that the inoculation may be a requisite element for m HSP65-TTL plus CY to induce the antitumor immunity. To verify this, mice were directly immunized with m HSP65-TTL on day 5, 13, 21, 29, with or without CY treatment on day 4, 12, 20, 28. On day 140, the mice were inoculated with the tumor cell suspension as described above and monitored. 17 days later, tumors began to be observable in mice treated with PBS or CY. On day 33 after the inoculation, one mouse was found died in PBS group(n=8). Within the ongoing 20 days the rest 7 mice were dead. Within day 34 to 43, five mice died in m HSP65-TCL group(n=7) and the rest two survived. Only mouse was survived in CY group(n=8). Half of the mice in m HSP65-TCL plus CY group(n=8) were dead within day 33 to day 47 and the rest survived. m HSP65-TTL plus CY significantly prolonged the survival of the mice inoculated with the cell suspension of Panc02 cancer tissue(versus PBS or CY, p=0.039 or p=0.028, respectively), and that m HSP65-TTL or CY failed to induce or enhance significant antitumor immunity in the mice without previous exposure to small amount of Panc02 cancer cells. When tumors were observable, the tumor incidence of the mice was monitored and recorded. The results showed that on day 40 after the inoculation, the tumor incidence in m HSP65-TTL group or m HSP65-TTL plus CY group was only 63% or 50%, whereas the tumor incidence in the mice injected with PBS or CY reached 100% or 83.33%, respectively. When comparing the tumor volumes, it was showed that that 8, 5, 7, 4 mice in PBS, m HSP65-TTL, CY, m HSP65-TTL plus CY groups developed measurable tumors and the tumor sizes in the mice treated with m HSP65-TTL or m HSP65-TTL plus CY, tended to be smaller than those in the mice treated with PBS. Overall, m HSP65-TTL plus CY induced effective anti-tumor immunity, they prolonged the survival of mice with tumor, and tended to reduce the tumor incidence and to inhibit the growth of tumor in the mice inoculated with the tumor cell suspension.5.Effects of m HSP65-TTL plus CY on Panc02-specific CTLTo investigate the mechanism how CY enhanced the anti-tumor effect of m HSP65-TTL, we tested the effect of CY on CTL induced by m HSP65-TTL. 12 health female C57BL/6 mice were randomly divided into four groups, PBS group, m HSP65-TTL group, CY group, m HSP65-TTL plus CY group, respectively. The mice were injected intraperitoneally with PBS, PBS, CY, CY, at days 4, 12, 20, 28 and immunized PBS, m HSP65-TTL, PBS, m HSP65-TTL at days 5, 12, 21 and 29. The mice were killed on day 36 for isolating the spleen cells. The spleen cells were cultured with m HSP65-TTL(2μg/ml) at 37℃, 5% CO2 were incubated for 4 days. During the time, the cells were added with 100U/ml of rh IL-2 at 48 h, the activated spleen cells were collected and counted on days 4. The Panc02 cells were cultured in 96-well round bottom at 1×104/well and then co-cultured with activated spleen cells at effector/target ratio of 80:1, 40:1 and 20:1, for another 6h. The results showed that CY+m HSP65-TTL or m HSP65-TTL could significantly induce specific CTL at effector/target ratio of 80:1, 40:1 and 20:1 in comparison to PBS, but there were no statistical significance between the two groups. In addition, CY alone failed to induce specific CTL, but the trend to induce non-specific killing.6.Activation of m HSP65-TTL plus CY on spleen cellsTo find how the m HSP65 TTL or m HSP65 TTL plus CY could induce effective anti-tumor immunity, mice naived to the Panc02 cancer cells, were intraperitoneally administered with CY(50mg/kg) or PBS on day 0, and then immunized with(2μg m HSP65+100μg TTL/mouse) or PBS at inguinal lymph node area on the both sides on day 1. On day 4, the mice were sacrificed for isolating their spleen cells. The cells were used for detecting the expression of CD69, an activation marker of lymphocytes.5×105 spleen cells were directly stained with PE-labeled anti-CD69 m Ab, followed by flow cytometry analysis. As shown in Fig3.15, 9.51% or 10.22% of the spleen cells expressed CD69 in PBS group or CY group, respectively. In comparison, in m HSP65 TTL group or m HSP65 TTL plus CY group, 14.13% or 18.68% of the spleen cells expressed CD69, respectively. Statistic analysis showed that m HSP65-TTL or m HSP65-TTL plus CY significantly enhanced the expression of CD69, compared with PBS(P=0.003, P=0.000, respectively), indicating that m HSP65-TTL or m HSP65-TTL plus CY could activate lymphocytes in mice. Comparatively, the anti-tumor activity of m HSP65 TTL plus CY was more potent than that of m HSP65-TTL.7.Effect of m HSP65-TTL plus CY on IL-17 A and RORγt m RNA expression of spleen cellsTo test whether the anti-tumor immune response induced by m HSP65-TTL or m HSP65-TTL plus CY though induce Th17 immune response, mice were intraperitoneally administered with CY(50mg/kg) or PBS on day 0, and then immunized with(2μg m HSP65+100μg TTL/mouse) or PBS at inguinal lymph node area on the both sides on day 1. On day 4, the mice were sacrificed for isolating their spleen cells to assay for the m RNA expressions of IL-17 A and RORγt in real-time PCR. The results showed that m HSP65-TTL significantly up-regulated IL-17 A in spleen cells, compared with PBS(P=0.019, P=0.018, respectively). m HSP65-TTL plus CY could also significantly up-regulate the expression of RORγt(P=0.036), and the level was obviously higher than that up-regulated by m HSP65-TTL. Noticeably, CY alone induced much higher m RNA expressions of RORγt, compared with PBS, m HSP65-TTL, and m HSP65-TTL plus CY(P=0.007 P=0.023, P>0.05 respectively). Interestingly, CY alone or m HSP65-TTL plus CY tended to enhance the m RNA expression of IL-17 A in comparison with PBS, but there was no statistical significance in comparison with PBS.The conclusion:1.Low dose CY enhanced the efficacy of the m HSP65-TTL.2.m HSP65-TTL and CY significantly prolonged the survival of the pancreatic cancer bearing mice3.m HSP65-TTL plus CY up-regulated m RNA expressions of RORγt and IL-17 A.The innovative points:1. Low dose CY enhanced anti-pancreatic tumor activity that m HSP65-TTL plus CY immuned.2. m HSP65-TTL plus CY induced anti-pancreatic tumor immunity activity with relation of Th17.