Tracking Mouse Dendritic Cells Migration In Vivo And The Antitumor Efficacy Of Tumor-derived Dribble-pulsed Dendritic Cells Vaccine On Oral Squamous Cell Carcinoma-bearing Mice

Background:Currently, It’s hard to further improve 5-year survival rate in patients with oral squamous cell carcinoma by carrying out traditional therapy, such as surgery, radiotherapy and chemotherapy, etc. As a result, clinicians and researchers are paying more and more attention to biotherapy, such as immunotherapy, gene therapy and targeted therapy, etc. Immunotherapy is one of the major means of cancer biotherapy, which attempts to induce and enhance the antitumor immunity of cancer patients. Dendritic cells are considered the most powerful professional antigen presenting cells and at the centre of the immune system. However, impaired function, phenotype changes and decrease of dendritic cells in cancer patients are demonstrated in many studies. Therefore it has become a research hotspot that ex vivo-generated dendritic cells vaccines are injected back into cancer patients. The efficiency of dendritic cells vaccines against tumor pivotally relies on dendritic cells migrate to the draining lymphoid organs and present tumor antigens to naive T cells to induce anti-tumor immune responses.After dendritic cells vaccines are injected back into cancer patients, the efficiency of their migration to lymphoid organs plays a key role in their induction of anti-tumor immune responses. Synthetic superparamagnetic iron oxide (SPIO) nanoparticles were applied to label and track dendritic cells in our previous studies. It was demonstrated that SPIO were highly biocompatible for labeling dendritic cells, but only a few labeled dendritic cells migrated to the draining lymph nodes after injected back into mice. Consequently, the influence of SPIO on dendritic cells’ migratory capability in vivo should be further investigated, and it should be defined whether SPIO could be used to track dendritic cells for clinical application.The methods of tumor antigen preparation are extremely crucial for dendritic cells vaccines’ induction of anti-tumor immune responses. Currently, it is the most traditional and common priming strategy that dendritic cells are primed with tumor lysates. However, its efficacy is unsatisfactory. Autophagy is a general and conservative physiological process of cells, which involves degradation of unnecessary or dysfunctional cellular components to maintain homeostasis of cells. Tumor antigens are degraded either by the ubiquitin-proteasome system or the autophagy-lysosome pathway in tumor cells. Most peptide antigens presented by MHC class Ⅰ molecules on tumor cells mainly derive from the tumor antigens degraded by the ubiquitin-proteasome system (particularly including defective ribosomal products, DRiPs). Tumor cells were treated with autophagy inducer, proteasome inhibitor and lysosomal inhibitor to package undegraded tumor antigens in autophagosomes and those autophagosomes were isolated from both tumor cells and culture medium. The DRiPs-containing autophagosome-rich blebs were named DRibbles and regarded as a novel tumor antigens source. Even though DRibbles were defined effective carriers of tumor antigens, there were controversies about the nature of their tumor antigens which could be cross-presented by antigen-presenting cells. It still needs further investigation whether DRibbles derived from oral-squamous-cancer cell lines could improve the efficiency of dendritic cells vaccines against tumor as a novel tumor antigens source. Furthermore, whether DRibbles need dendritic cells as carrier to generate anti-tumor immunity and the mechanisms of DRibbles-pulsed dendritic cells vaccines inducing anti-tumor immunity remains to be defined.Objective:1. To observe the migration of SPIO labeled dendritic cells in vivo.2. To establish the protocol of preparating DRibbles from SCC7 and observe the ability of DRibbles to induce the proliferation and activation of T cells and B cells.3. To evaluate the influence of DRibbles on the antigen presentation of DCs and observe the efficacy of DRibbles-pulsed DCs on tumor-bearing C3H/HeJ mice.4. To evaluate the protective efficacy of DRibbles-pulsed DCs on C3H/HeJ mice challenged with SCC7.Methods:1. Mice bone marrow-derived DCs were induced and their purity was detected. After EGFP-DCs were labeled with different concentrations of SPIO, the uptake SPIO of DCs was detected by Prussian blue staining and quantification of internal iron. Surface molecules, cell apoptosis and fluorescence intensity of EGFP-DCs were displayed by flow cytometry. After EGFP-DCs, labeled with SPIO, were injected into footpads for 24 hours, the mice were examined in vivo by optical imaging (OPI). Meanwhile, confocal imaging and flow cytometry were applied, respectively, to detect the migration of labeled DCs into draining lymph nodes. After SPIO-EGFP-DCs and EGFP-DCs were injected into footpads respectively for 0,2,4, 7,14 days, the mice were examined in vivo by optical imaging (OPI). Meanwhile, confocal imaging and flow cytometry were applied, respectively, to detect the migration of SPIO-EGFP-DCs and EGFP-DCs into draining lymph nodes.2. DRibbles were prepared and their morphological and autophagic properties were characterized. The influence of DRibbles on proliferation and activation of T cells and B cells was detected by Cell Counting Kit-8 and flow cytometry. After mice was vaccinated with DRibbles in different dose, CD4+IFN-y+T cells and CD8+IFN-7+T cells of inguinal lymph nodes were detected by flow cytometry. IFN-y in supernatant was detected by ELISA.3. Dendritic cells (DCs), generated from the bone marrow monocytes of mice, were cocultured with DRibbles, then surface molecules of DCs as well as apoptosis of DCs were determined by flow cytometry. Meanwhile, functional properties of the DRibble-DCs were examined by mixed lymphocyte reaction and animal experiments.4. DCs pulsed with different dose of DRibbles were injected to mice. Immune response were detected by cytotoxicity test, flow cytometry and ELISA.7 days after vaccinated with DRibbles, DRibble-DCs, Lysate-DCs or DCs, mice were challenged with SCC7. The survival and tumor volume were measured. Treg and IFN-y were detected by flow cytometry and ELISA.Results:1. When the DCs were induced for 7 days, CD11c+ cells was more than 80%. Nearly 100% of cells were labeled by the SPIO, in which the intracellular blue color gradually deepened and the iron contents rose with the increase of labeling iron concentrations. In addition, cell apoptosis and the expressions of surface molecules, including CD40, CD80, CD86, MHC class Ⅱ molecules and MHC class Ⅰ molecules on DCs were at similar levels after SPIO labeling (P>0.05). Moreover, the expressions of CCR7 on DCs were gradually decreased with the increase of labeling iron concentrations. After confirming that the fluorescence intensity of EGFP on DCs was not influenced by SPIO, the homing ability of EGFP-DCs labeled with SPIO displayed that the fluorescence intensity and the ratios of EGFP-DCs in draining lymph nodes were gradually decreased with the increase of labeling iron concentrations.7 days after injection, the fluorescence intensity and the ratios of EGFP+ cells in popliteal lymph node from both groups reached peak, while 14 days after injection, the fluorescence intensity and the ratios of EGFP+ cells in popliteal lymph node from both groups decreased notably. But there were no significant differences between the two groups.4 and 7 days after injection with SPIO labeled EGFP-DCs, the fluorescence intensity and the ratios of EGFP+ cells in inguinal lymph nodes was obvious. On the contrary, SPIO unlabeled EGFP-DCs weren’t observed in inguinal lymph nodes at all time points.2. Treated with autophagy inducer, proteasome inhibitor and lysosomal inhibitor for 24 hours, SCC7 expressed LC3-Ⅱ 2.82 times that of control group and higher than 12 hours group and notably higher than 36 hours group (P<0.01). The diameter of autophagic nanoparticles with a spherical and double-membrane structure was between 200nm and 900nm.10μg/ml DRibbles were more capable of stimulating the proliferation of T cells than other concentrations (P< 0.001). Spleen cells were stimulated with 10μg/ml DRibbles, and the ratios of T cells increased to 68.12%, which was notably higher than other concentration groups (P<0.05); the ratios of CD4+ cells were about 41.26% and that of CD8+ cells were about 28.66%, which were both higher than other concentration groups. Compared with tumor lysates and control group, DRibbles significantly decreased the ratios of Treg cells of spleen cells to 1.08%(P<0.01). Compared to tumor lysates, DRibbles significantly upregulated the expressions of CD40, CD80, CD86 and MHC classⅡ molecules on B cells (P< 0.05). Mice were given 2.5μg DRibbles via intranodal injection, and the ratios of CD4+ IFN-γ+ T cells of lymph node cells increased to 1.16% and that of CD8+ IFN-γ+ T cells increased to 0.53%, which were both higher than other concentration groups; IFN-γ secreted by the restimulated lymph node cells and spleen cells reached to 1086.45 pg/ml and 142.04 pg/ml respectively, which were both higher than other concentration groups.3. Incubated for 6 hours, most of DCs had taken CFSE labeled DRibbles. Cell apoptosis of DCs were at similar levels after different concentrations of DRibbles or different time of pulsing (P>0.05). There were no significant differences between apoptosis of DRibbles-pulsed DCs and that of tumor lysates-pulsed DCs (P>0.05). DRibbles significantly upregulated the expressions of CD40, CD86, MHC class Ⅰ molecules but not MHC class Ⅱ molecules (P>0.05) on DCs; the ratios of DCs with positive expression of costimulatory molecules increased in a dose-dependent manner. Compared with tumor lysates and control group, DRibbles significantly upregulated the expressions of CD40, CD86, MHC class Ⅰ molecules (P<0.05) but not MHC class II molecules (P>0.05) on DCs. DRibbles-pulsed DCs were more capable of stimulating the proliferation of T cells in vitro than lysates-pulsed DCs (P <0.05); the differences were more significant after added with pro-mature cytokine cocktails (P<0.01). Compared with tumor lysates-pulsed DCs and control group, DRibbles-pulsed DCs significantly improved survival rate of tumor-bearing mice to 87.5% and inhibited tumor growth.4. Mice were inoculated with 2.5μg/ml DRibbles-pulsed DCs, and cytotoxicity of spleen or lymph node-derived T cells reached 52.85% and 66.14% respectively, which were higher than other concentrations of DRibbles-pulsed DCs groups; IFN-y in plasma was 7.04 pg/ml and higher than other concentrations of DRibbles-pulsed DCs groups; IFN-y secreted by the restimulated spleen cells and lymph node cells were 7.59 pg/ml and 13.89 pg/ml respectively, which were higher than other concentrations of DRibbles-pulsed DCs groups; the ratios of CD8+IFN-γ+T cells of peripheral blood mononuclear cells were 3.68%, and the ratios of CD4+IFN-γ+cells of T cells were 7.35%, which were both higher than other concentrations of DRibbles-pulsed DCs groups; the ratios of CD4+IFN-yγ+cells of spleen-derived T cells were 2.19% and that of lymph node-derived T cells were 5.44%, and the ratios of CD8+IFN-y+ cells of lymph node-derived T cells were 2.38%, which were both higher than other concentrations of DRibbles-pulsed DCs groups. DRibbles-pulsed DCs significantly improved survival rate of mice challenged with malignant tumor cells to 90%(P< 0.05) and inhibited tumor growth (P<0.05); DRibbles-pulsed DCs significantly decreased the ratios of Treg cells of peripheral blood mononuclear cells and that of lymph node cells to 7.28% and 7.77% respectively. Mice inoculated with DRibbles-pulsed DCs were challenged with malignant tumor cells, and IFN-y secreted by the restimulated lymph node cells and spleen cells were 862.9 pg/ml and 309.5 pg/ml respectively, which were both significantly higher than other vaccine groups (P<0.05).Conclusions:1. DCs labeled with different dose of SPIO had different iron contents, however their apoptosis and the surface molecules, including CD40, CD80, CD86, MHC class II molecules and MHC class I molecules, were at similar levels. From the short-term (24 hours) observation, the expressions of CCR7 on DCs and the ratios of DCs migrating to the primary draining lymph nodes were gradually decreased with the increase of labeling iron concentrations. However, from the long-term (14 days) observation, there was no significant difference between the ratios of SPIO labeled DCs and SPIO unlabeled DCs migrating to the primary draining lymph nodes. The ratios of SPIO labeled DCs migrating to the secondary draining lymph nodes were significantly higher than that of SPIO unlabeled DCs. The ratios of SPIO labeled DCs and SPIO unlabeled DCs migrating to the primary draining lymph nodes were both at peak 7 days after inoculation and reduced significantly 14 days after inoculation.2. The protocol of preparating DRibbles from SCC7 was established. The prepared DRibbles were autophagosome-rich blebs. As a novel tumor antigens source, DRibbles was able to induce the proliferation and activation of T cells and B cells as well as reduce the ratios of Treg cells in vitro. Mice were given low dose of DRibbles via intranodal injection and cellular immune responses were induced effectively. On the contrary, cellular immune responses induced by high dose of DRibbles were weaker than that induced by low dose of DRibbles in vivo.3. Stimulating DCs with different concentrations of DRibbles or for different time have no significant effect on their apoptosis. The expressions of CD40, CD86 and MHC class I molecules, but not MHC class II molecules, on DCs stimulated with DRibbles had a significantly dose-dependent increase. Compared with tumor lysates, DRibbles were more capable of facilitating antigen presentation and stimulation proliferation of T cells of DCs. Compared to tumor lysates-pulsed DCs, DRibbles-pulsed DCs vaccines significantly improved survival rate of tumor-bearing mice and inhibited tumor growth.4. Inoculated with DCs vaccines pulsed with low dose of DRibbles effectively induced antitumor immune responses. However, DCs vaccines pulsed with high dose of DRibbles demonstrated less effective. Boosting with DRibbles-pulsed DCs vaccines effectively induced antitumor immune responses and had a distinguished efficacy on protecting mice against malignant tumor cell challenge.