Antitumor Immunity Of Gastric Carcinoma Total RNA Transfected Monocyte-derived Dendritic Cells Without Or With Thymosin Alpha 1 In Vivo

Malignancy has become one of the common diseases which affects human health seriously, is the major cause of death. In the worldwide, incidence of gastric carcinoma was stand in second and fourth place in male and female malignant disease, respectively. Gastric carcinoma is one of the most common maligancies in China with 400000 new cases diagnosed annually. The yearly incidence is 37.1/100000 in male and 17.4/100000 in female in China. Its high incidence keeps on rising. Gastric carcinoma is the first leading cause of various cancer death. At the time of diagnosis, most of the patients with gastric carcinoma having metastasis of the disease in local lymph nodes and adjacent organs. At present there is no therapeutic regime capable of curing unresectable metastatic disease. In spite of modern surgery, new developments in radiotherapy, and the availability of combinations of chemotherapeutic agents, some of them being newly developed, the prognosis of advanced gastric carcinoma remains substantially poor. Current treatment options for advanced gastric carcinoma are limited. In addition, many of the successful cancer therapies are associated with significant toxicity leading to long-term morbidity and an increasing second malignancy rate. It is obvious that we need to find out a better approach for more effective treatments. New therapeutic options for patients with gastric carcinoma that offer less toxicity and greater potential for cure are clearly needed.Dendritic cells (DCs) are the most professional antigen presenting cells (APCs) that take up, process and present MHC-restricted antigens to na?ve T cell. Because of unique ability of DCs to present antigen to na?ve T cells, they play a key role in responsible for antitumor immunity. DCs are attractive vectors for vaccine therapies and are being investigated for immunotherapy against various cancers. The technology of in vitro DCs differentiation out of peripheral blood monocytes or CD34+ stem cells made DCs available for use as therapeutic vaccines in clinical practice. One approach to developing immunotherapy strategies is the use of DCs loaded with various forms of antigen including tumor lysate, peptides, tumor total RNA and tumor mRNA and as a vaccine to stimulate an antitumor immune response.Several clinical trials of DCs-based vaccines were recently set up to treat patients with malignant melanoma, prostate cancer, renal cell carcinoma, breast cancer, liver cancer, ovarian cancer and colorectal cancer. There are an increasing number of reports demonstrating that DCs-based antitumor vaccine has emerged as a promising cancer immunotherapy with proven clinical efficacy. For example, Bleaumer I and colleagues (Bleaumer I, et al, 2007) did perform a phase I clinical trial in patients with progressive, cytokine-refractory renal cell carcinoma vaccinated with CA9-peptide-pulsed mature dendritic cells. The vaccination of DCs pulsed with KLH and CA9-derived peptides was well tolerated. Kyte JA and colleagues (Kyte JA, et al, 2006) developed phase I/II of malignant melanoma therapy with monocyte-derived dendritic cells transfected with autologous tumor-mRNA. Yamanaka R and co-workers (Yamanaka R, et al, 2005) have conduced a clinical phase I/II trial of autologous tumor lysate pulsed monocyte-derived DCs therapy patients with with recurrent malignant glioma. Burgdorf SK and colleagues (Burgdorf SK, et al, 2006) performed a phase I study of vaccination of patients with advanced colorectal carcinoma with autologous dendritic cells pulsed with melanoma lysate. Nakai N and colleagues (Nakai N, et al, 2006) performed a clinical trial of vaccination with peptides, tumor lysate or both peptides and tumor lysate-pulsed mature, monocyte-derived dendritic cells for advanced malignant melanoma patients that are resistant to conventional therapies. Lee WC and colleagues (Lee WC, et al, 2005) performed a clinical trial of vaccination of advanced hepatocellular carcinoma patients with autologous tumor lysates-pulsed peripheral blood monocytes-derived dendritic cells. Hus I and colleagues (Hus I, et al, 2005) perform clinical study of allogeneic monocyte-derived DCs pulsed with tumor cell lysates or apoptotic bodies as immunotherapy for patients with early-stage B-cell chronic lymphocytic leukemia (B-CLL). Su Z and colleagues (Su Z, et al, 2003) conducted a phase I trial of metastatic renal cancer patients vaccinated with renal tumor RNA-transfected dendritic cells. Caruso DA and colleagues (Caruso DA, et al, 2004) conducted a phase I study of 9 pediatric patients with recurrent brain tumors utilizing monocyte-derived dendritic cells pulsed with tumor RNA. Taken together, these clinical phase I/II trials results demonstrated that DCs vaccine are feasible and safe in cancer patients. Several other clinical trials using DCs-based vaccine are still ongoing, and will hopefully pave the way for improved DCs therapy of cancer in the future. However, DCs-based immunotherapy for gastric carcinoma is being basic research. Hopefully, our recent understanding of tumor immunology and current clinical approaches of DCs vaccine will provide us help to develop more effective vaccines against cancer including gastric carcinoma in near future.Thymonsin alpha 1(Tα1) has shown efficacy in the treatment of chronic hepatitis B, chronic hepatitis C and acquired immuno-deficiency syndrome (AIDS). Tα1 is also being develop for the treatment of non-small cell lung cancer (NSCLC), hepatocellular carcinoma and malignant lymphoma. It is evident from previous studies that Tα1 has abilities to affect the maturation , differentiation and function of T cells, promote a recovery of NK cell activity and reduce the hematological toxicity of chemotherapy, and strengthen the effects of immunomodulators in neoplastic malignancies. In contrast to the ample evidence of effects Tα1 on T cells, the effect of Tα1 on DCs is relatively unknown. The influence of Tα1 on the differentiation, maturation and function of dendritic cells differentiated from murine bone marrow in vitro in the presence of GM-CSF and IL-4. However, the effect of Tα1 on DCs from patients with maligance has not been investigated. Moreover, there is no report about the use of Tα1 in immunotherapy of cancer.This study is consisted of four chapters as follows:PartⅠTα1 affects the differentiation, maturation and function of peripheral blood monocyte-derived dendritic cells from patients with gastric carcinoma in vitroObjective: To investigate the affects of Tα1 on the differentiation, maturation and function of dendritic cells differentiated from monocytes of patients with gastric cancinoma in vitro.Methods: There were six patients enrolled in this study: two female and four male. Patients had histologically proven gastric adenocarcinoma. PBMCs were isolated by Ficoll-Paque density gradient centrifugation followed by plastic adherence for 2 hours. Human monocyte-derived DCs were generated from an adherent fraction of PBMCs and cultured in RPMI-1640 complete medium in the presence of 100ng/ml rhGM-CSF and 50ng/ml rhIL-4 for 5 days to generate immature moDCs. New medium supplemented with rhGM-CSF and rhIL-4 at the concentration previously described was added to the wells and half of the medium was changed every 2 days. On day 6 of culture, the panel were divided into five groups. Groups with the treatment of Tα1 at 0.5ng/ml, 5ng/ml and 50ng/ml final concentrations in the presence of rhGM-CSF and rhIL-4 were marked as group I, group II and group III, respectively, and cultured for a further 2 days. The group without treatment of Tα1 was as control group (group A) and the group with the treatment of 20ng/ml rhTNF-αwas as group B, cultured for a further 2 days. On day 8 of culture, the non-adherent and loosely adherent cells were harvested as DCs used for in vitro analysis. Morphology of moDCs was observed daily by an phase contrast inverted microscope during the culture period. The viability of moDCs was judged by trypan blue exclusion. The phenotype CD1a, CD80, CD86, CD83 and HLA-DR expression on moDCs was determined by flow cytometry. The capacity of moDCs in stimulating proliferation of autologous T lymphocytes in MLR was determined by MTT assay.Results: (1) Phase contrast morphology of moDCs are mostly detached from the plastic surface and exhibited an irregular shape with extensive cellular protrusions and veils extending from all aspects of the cellular body. In group I, group II and group III, the morphology of moDCs was not altered compared with to control group A. moDCs in the presence or absence of Tα1 did not alter viability (P>0.05). (2) Tα1 significantly up-regulated the expression of CD80, CD86, CD83, CD1a and HLA-DR on the surface of moDCs, in comparison with to control group A (P<0.01). This difference wasn't statistically significant among the group I, group II, group III and control group B (P>0.05). (3) In the group I, group II and group III, the capacity of moDCs in stimulating proliferation of autologous T lymphocytes was significantly increased in a Tα1 concentration-dependent manner (P<0.05, P<0.01, P<0.01), and the difference was statistically significant when compared with that in control group A (P<0.01), but no difference was found in comparison with control group B (P>0.05).Conclusion: Tα1 can effectively promote the differentiation, maturation and function of DCs derived from PBMCs of patients with gastric carcinoma. It is suggest that Tα1 has a potential as a maturation agent used in DCs-based vaccine tirals, but not as a single reagent. MoDCs is a primary target cell of the modulation of Tα1.PartⅡEstablishment of athymic nude mice model xenografts human gastric carcinoma cell line BGC-823 and reconstitution of human cellular immunity functionObjective: To investigate characteristics and establishment of athymic nude mice model xenografts human gastric carcinoma cell BGC-823 and reconstitution of human cellular immunity function.Methods: Ten 4-week-old female athymic nude mice (BALB/c nu/nu) were bred under specific pathogen free conditions. Mice were allowed to acclimatize for 1 week prior to treatment. Cultured human gastric carcinoma cells line BGC-823 were used to establish mouse model. Purified human T lymphocytes were obtained by nylon fiber column methods and were analysed by flow cytometry.Ten mice were randomly divided into experiment group and control group (5 per group). In experiment group, mice were by subcutaneous (s.c.) inoculated in their right axillary fossa with 4×106 viable gastric carcinoma cell BGC-823 and intraperitoneal (i.p.) injection of human peripheral blood T-lymphocytes (HuPBTL) (3×107). In control group, mice were s.c. inoculated in their right axillary fossa with 4×106 viable gastric carcinoma cell BGC-823 and i.p. injection of phosphate buffered solution (PBS). The tumor incidence, latency period, tumor growth and tumor volume were then monitored daily during the protocol period. Tumor size was measured once every 2 days using vernier caliper. The ratio of human CD3+ T cell, CD4+ and CD8+ T lymphocyte subset from peripheral blood of humanised athymic nude mice were detected by flow cytometry. Graft versus host disease (GVHD) after xenografts HuPBTL were observed. Cytotoxic T-lymphocyte activity against autologous tumor cells in vitro was measured by MTT method. The pathohistological examination of tumor tissue from each mouse was performed.Results:⑴Latency period were (11.2±1.9) days and (9.4±1.5) days for two groups, respectively. There was no significant difference between two groups (P﹥0.05).Tumor incidence was 100% (10/10) for two groups. At the fourth weeks after inoculation, final tumor volume for experiment group and control group were (1495.6±111.6) mm3 and (1649.6±128.0) mm3, respectively, there was no significant difference between two groups (P﹥0.05).⑵The ratio of human CD3+, CD4+ and CD8+ T cells were (46.86±9.14) %, (31.68±10.17) % and (17.91±1.38) % in experiment group, respectively, whereas they could't be found in control group. In experiment group, specific cytotoxicity were (15.37±1.58) % and (19.86±2.86) %, E/T ratio was 20:1 and 40:1, respectively, this difference was statistically significant (P﹤0.05). GVHD wasn't occurrence in each mouse during the protocol period.⑶Both experiment group and control group mice showed no liver and pulmonary metastases. The pathohistological examination showed that gastric carcinoma cell kept morphological characteristics of parental tumor cells.Conclusion: A novel athymic nude mouse model xenografts human gastric carcinoma-HuPBTL was successfully established by s.c. inoculation of human gastric carcinoma cell BGC-823 and i.p. injection of HuPBTL. The results of this study suggest that well-established human gastric carcinoma in animal models that mimic clinical patients with existing tumor burdens, may be useful for studying human gastric carcinoma immunotherapy in vivo . PartⅢAntitumor immunity in humanised athymic nude mice model by human monocyte-derived dendritic cells pulsed with gastric carcinoma total RNAObjective: To analyse antitumor immunity induced by monocyte-derived dendritic cells pulsed with human gastric carcinoma total RNA in humanised athymic nude mice model and evaluate the efficacy of DCs vaccine.Methods: PBMCs were isolated by Ficoll-Paque (density 1.077g/ml) density gradient centrifugation of heparinized peripheral blood obtained from buffy coat preparation of healthy volunteer. Tumor total RNA was extracted and isolated from gastric carcinoma cell BGC-823 using the TRIzol kit according to the protocol provided by the facturer. Quantity and purity of tumor total RNA was determined by UV spectrophotometry. All total RNA samples were routinely checked by 1.2% agarose gel electrophoresis for size distribution and integrity. Immature DCs generated from PBMCs were transfected with gastric carcinoma total RNA by passive pulsing in vitro to produce antitumor vaccine (DCRNA). A humanized athymic nude mice model were established by i.p. injection of HuPBTL. Thirty humanized nude mice were randomly divided into prophylactic panel and therapeutic panel (15 per panel). In the prophylactic panel, 15 mice were randomly divided into DCRNA group, DCs group and PBS group of 5 mice each. The mice were vaccinated (s.c.) with DCRNA, DCs and PBS on day 0 and 7, respectively. Three days after the last immunization, mice were challenged s.c. with tumor cell BGC-823. In the therapeutic panel, as nude mice tumor are just palpable, 15 mice were randomly divided into DCRNA group, DCs group and PBS group of 5 mice each. The mice were s.c. with DCRNA, DCs and PBS, respectively, twice, at weekly interval. Tumor growth, tumor volume, tumor weight were observed. IL-12 and IFN-γsecretion were detected by ELISA kit.Results:⑴The amount of tumor total RNA was about 600μg. The ratio of 28S/18S was over 1.8 in all cases. Tumor total RNA wasn't degradated.⑵In the prophylactic panel, final tumor volume in DCRNA group (574.20±95.73) mm3 was significantly less than that in DCs group (847.76±113.91) mm3 and PBS (969.25±131.8) mm3 (P﹤0.05). Tumor dry weight in DCRNA group (0.464±0.156)g was significant less than that in DCs group (0.750±0.220)g and PBS group (1.002±0.138)g (P<0.05, P<0.01), but there was no significant difference between DCs group and PBS group (P﹥0.05). Inhibition tumor rate for DCRNA group (53.7%) was significant higher than that for DCs group (25.1%) (P﹤0.05). Production of IL-12 in DCRNA group (387.0±53.6) pg/ml were significantly higher than that in DCs group (279.2±42.7) pg/ml and PBS group (24.7±5.1) pg/ml (P﹤0.01), there was significant difference between DCs group and PBS group (P﹤0.01). Production of IFN-γin DCRNA group (986.4±163.7) pg/ml were significantly higher than that in DCs group (713.2±121.8) pg/ml and PBS group (57.9±9.3) pg/ml (P﹤0.05, P﹤0.01), there was significant difference between DCs group and PBS group (P﹤0.05).⑶In the therapeutic panel, final tumor volume in DCRNA group (814.7±173.6) mm3 was significantly less than that in DCs group (1089.3±196.8) mm3 and PBS (1258.6±257.3) mm3 (P﹤0.05), but there was no significant difference between DCs group and PBS group (P﹥0.05). Tumor weight in DCRNA group (1.027±0.236)g was significant less than that in DCs group (1.458±0.390)g and PBS group (1.679±0.358)g (P﹤0.05, P﹤0.01), but there was no significant difference between DCs group and PBS group (P﹥0.05). Inhibition tumor rate for DCRNA group (38.8%) was significant higher than that for DCs group (13.2%) (P﹤0.05). Conclusion: Gastric carcinoma total RNA pulsed dendritic cells vaccine pulsed inducing enhanced antitumor immunity in humanised athymic nude mice model.PartⅣCombined thymosin alpha 1 and dendritic cells based vaccine antitumor immunity against gastric carcinoma in athymic nude mice modelObjective: Thymosin alpha 1 is a immunoregulatory function that induces antitumor activity and activates immune cells such as DCs and T cells. Combination of thymosin alpha 1 with chemotherapy for patients of cancer is currently the most promising option. We hypothesized that a combined Tα1 and DCs vaccine would have some advantage in vivo, whether thymosin alpha 1 show enhanced antitumor activity.Methods: PBMCs were isolated by Ficoll-Paque density gradient centrifugation of heparinized peripheral blood obtained from healthy volunteer. Tumor total RNA was isolated from gastric carcinoma cell BGC-823 using the TRIzol kit. Quantity and purity of tumor total RNA was determined by UV spectrophotometry. All total RNA samples were checked by 1.2% agarose gel electrophoresis for size distribution and integrity .Immature DCs generated from PBMCs were transfected with tumor total RNA by passive pulsing in vitro to produce DCRNA. Humanized athymic nude mice xenografts gastric carcinoma was successfully established by s.c. inoculation of human gastric carcinoma cell and i.p. injection of HuPBTL. As nude mice tumor are just palpable, twenty mice were randomly divided into PBS group, DCRNA group, Tα1 group and DCRNA combined with Tα1 group (5 per group). The mice were immunized twice at weekly intervals s.c. with PBS, DCRNA , Tα1 and DCRNA combined with Tα1, respectively. Tumor volume, tumor weight were recorded. IL-12 secretion was detected by ELISA kit.Results:⑴Tumor volume inⅡgroup (814.7±173.6) mm3 andⅣgroup (672.1±126.8) mm3 were significantly less than that inⅠgroup (1258.6±257.3) mm3 (P﹤0.05, P﹤0.01).⑵Tumor weight inⅡgroup (1.027±0.236) g andⅣgroup (0.758±0.223) g were significantly less than that inⅠgroup (1.679±0.358) g (P﹤0.01). Inhibition tumor rate forⅣgroup (54.85%) was higher than theory inhibition tumor rate (53.99%).⑶IL-12 inⅡgroup(387.0±53.6) pg/ml,Ⅲgroup (182.3±36.2) pg/ml, andⅣgroup (543.9±91.8) pg/ml, were significantly higher than that inⅠgroup (24.7±5.1) pg/ml (P﹤0.01). This difference was statistically significant betweenⅡgroup andⅣgroup (P﹤0.05).Conclusion: It was showed that DCs vaccine could be induced antitumor immunity against gastric carcinoma and Tα1 augmented antitumor activity in vivo. In addition, Tα1 in combination with DCs vaccine have synergism for antitumor. These results will help in the development of immunotherapy against tumor based on DCs vaccine with Tα1.