| Objective:DC-based tumor vaccines have shown to be one of the promising methods for inducing therapeutic antitumor response. However, DCs alone rarely cause curative antitumor effects and the immunosuppressive microenvironment may be a cause that compromise DC vaccine functions. Xirradiation could enhance the immunogenicity by inducing tumor cells producing several dangerous singals including HMGB-1and HSP70. Consequently, irradiation in combination with DCs has been shown to promote immune-mediated tumor destruction in preclinical studies. However, little is known about how irradiation alters the tumor microenvironment and what host pathways modulate the activity of administrated DCs. In this study, we want to know if X-irradiation could modulate an inflammatory microenvironment within irradiated tumors that is adaptive to DCs injection. Additionally, we want to test whether treatment with local tumor irradiation plus i.t. immature DCs administration could provide better outcomes than other single treatment with a tumor-bearing tumor model.Methods: 1 DC generation, the activity and the ability of antigen presentation detecting Bone marrow cells were collected from femoral bones of BALB/c mice. The differentiation of DCs was induced by IL-4(10ng/ml) and GM-CSF(10ng/ml) for 5 days. DCs cultured at 5 days were gathered again and the number of cells was adjusted at 1×106/ml in 24-well plates. A final concentration of 50 μg/ml of the lysate of 4T1 tumor cells treated with X-ray(30Gy) was added to 24-well plates, which were placed in 5% CO2 incubator with 37℃ water-saturated air for 16 hours. DCs treated with LPS(100ng/ml) and IFN-γ for maturation. DCs treated with mitomycin C at final concentration of 25μg/ml were used as stimulating cells,while response cells were spleen cells obtained from female C57BL/6 mice. DCs were serially diluted in a round-bottom 96-well plate, then one hundred microliters naive splenic T cells(0.1 million) were added the plate according to the ratio of 200:1, 100:1, 50:1(T:DC) for 68 hours in 5% CO2 incubator with 37℃ water-saturated air. Then 20 μl Cell Titer 96® AQueous One Solution Reagent(Cell Proliferation Assay, promega) was added to every well prior to 4 hours of terminating the incubation. The proliferation of response cells was analyzed by MTS colorimetry. The culture supernatant of antigen loaded and LPS plus IFN-γ maturated DCs was collected to detect IL-12p70 level. For surface marker analysis, DCs were incubated with FITC-conjugated monoclonal antibodies(m Abs) against CD11c(BD Pharmingen,clone:G235-2356), PE-conjugated anti-CD86(BD Pharmingen,clone:R35-95), ICAM-1(BD Pharmingen,clone:3E2) and MHC II(e Bioscience) for 30 minutes at 4°C. Analyses of fluorescence intensity were performed using a Becton Dickinson FACScan(San Jose, CA). 2 Dynamic analysis of cytokines from tumor tissues and tumor infiltrating immune cells after Xi-irradiaton 2.1 X-irradiation treated tumor-bearing mice Mouse mammary carcinoma 4T1 cells were injected into the right side of the fifth mammary fat pads in order to establish the tumor-bearing mice model. At the 7 days of tumor injection, the mice were divided into 3 groups(8 mouse/group), including untreated group, 10Gy×3 group and 30 Gy group, respectively. 2.2 Dynamic analysis of IL-6 IL-10 from tumor tissues and tumor infiltrating immune cells after X-irradiation by Real-time PCR 2.2.1 Isolation of tumor-infiltrating leukocytes At the 2,11 and 21 days postirradiation, the tumor-bearing mice were killed, tumor tissues were isolated. Tumors were weighted and dissected carefully by removing surrounding normal tissue, minced into ~ 1-mm pieces, and digested with an enzymatic solution containing 0.2 mg/ml DNase, 850 U/ml collagenase type II in unsupplemented RPMI 1640 at 37°C for 45 min, then passed through a 40-μm filter, and washed several times. Then, the homogenate was resuspended in serum-free RPMI 1640, the suspension was underlaid with 75%-100% Ficoll and centrifuged for 20 min at 2,000 g. Leukocytes were collected from the interphase, washed, and counted. 2.2.2 Quantitative real time RT-PCR Total RNA was extracted from 100 mg s.c. 4T1 tumors, harvested from treated and untreated after 2,11,or 21 days after the last dose of radiation, or from aliquots of 106 tumor-derived white cells using TRIzol(Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Primers for β-actin, IL-6,IL-10 were in the following table 1 and were synthesized by Invitrogen. Data were analyzed using the 2-ΔΔCT method. 2.3 Detecting TGF-β and TNF-α levels by ELISA To quantify levels of extra-cellular TGF-β and TNF-α, tumors(500 milligrams/tumor) were homogenized in 0.5 ml PBS containing protease inhibitors(Sigma). Tumors from 4 mice were analyzed individually. The amount of cytokines in the collected supernatants was determined using ELISA. 3 Tumor challenge and treatment with X- radiation in combination with DC vaccine 4T1 cells were inoculated into the right side of the fifth mammary fat pads of BALB/c mice with 1×105 in 0.1 ml of serum-free RPMI 1640, 7 days later, the mice were randomly divided into 6 groups. Untreated group, 10Gy*3 group( X-ray was adopted to treat the 4T1 tumor-bearing mice at a dose of 10 Gy for sequential 3 times interval of 24 hours), 30 Gy group( at a dose of 30 Gy only one time), 10Gy*3+DC group( after 48 h of receiving 10Gy*3 irradiation, DC vaccines were injected intratumorally at 1×106/mouse/100μl), 30Gy+DC group(after 48 h of receiving 30 Gy irradiation, DC vaccines were injected intratumorally at 1×106/mouse/100μl,). One week later, the micereceived another DCs injection. Tumor volumes were measured 2 or 3 times a week with a caliper. Tumor volume was calculated using the formula(A×B2)/2, where A is the largest of the two measurements and B the shortest and the survival curve was drawn. 4 In vivo neutralization experiments 4T1 tumor-bearing mice were treated with indicated irradiation doses(as described in 3). Two days after irradiation animals were treated i.v. with antiTNF-α antibody(20 μg/mouse, 500-P64, peprotech, USA) or PBS and then DCs were injected intratumorally as mentioned in 3. 5 Observation of long-lasting and systemic immunity in mice treated with Xirradiation plus DCs 5.1 Rechallenge 4T1 cells for “cured” tumor-bearing mice Some tumor-bearing mice treated with X-irradiation plus DCs could survive and the survival period exceeded 15 weeks with no or small measureable tumors were rechallenged with 4T tumor cells injected at sites contralateral mammary fat pads distant from the original tumors. 5.2 Analyzing the number of CD8+ T expressing IFN-γ, CD127 and CD44 by FCM At the 28 days of tumor cells rechallenged, the mice were killed and the splenic lymphocytes were restimulated with tumor antigen for 24 h. For the analysis of IFN-γ, CD127 and CD44 expression in CD8+T, activated Agspecific T cells were labeled with anti-IFN-γ-FITC, anti-CD127-FITC, antiCD44-FITC, anti-CD8-PE. 5.3 Analyzing the number of CD8+ T and T-Reg in tumor tissues by FCM At the 28 days of tumor cells rechallenged, the mice were killed and the tumor infiltrated immune cells were isolated. The anti-CD4-FITC and antiCD25-PE antibodies were used for T-Reg cells detection; anti-CD8-PE was used for CD8+T detection. 5.4 CTL activity of tumor infiltrating lymphocytes measuring At the 28 days of tumor cells rechallenged, the mice were killed and the tumor infiltrating lymphocytes were restimulated with tumor antigen and IL-2 for 7d and used as effector cells. 4T1 and irradiated 4T were used as the target cells. Then CTL responses were assessed with a standard colorimetric assay. Percentage of lysis was calculated as(experimental release-spontaneous release)/(maximum release- spontaneous release) ×100.Results: 1 Higher antigen presentation to T cells by DCs was observed when DCs pulsed with X-irradiated 4T1 lysate In order to obtain the X-irradiated 4T1 lysate, 4T1 cell line in logarithmic growth phase was irradiated with 10 Gy X-ray for sequential 3 times interval of 24 hours. The X-irradiated 4T1 cells were collected 24 h after the final irradiation and total protein was extracted by cell freeze-thaw. To determine whether X-irradiated 4T1 lysate had some advantage on DCs activity over non-irradiated 4T1 lysate, day 5 DCs from mouse bone morrow induced with GM-CSF/IL-4 were loaded with X-irradiated 4T1 lysate(100μg/ml), nonirradiated 4T1 lysate(100μg/ml) or PBS for 8h, respectively. Then the DCs were analyzed by FACS for surface expression of MHC II, CD86 and ICAM-1. While culturing DCs in the presence of LPS up-regulated MHC class II, CD86, and ICAM-1, exposure of DCs to 4T1 lysate had no significantly detectable effect on the expression of these markers. Furthermore, ELISA assays performed on supernatants of the above mentioned DC cultures detected IL-12p70 only in the presence of LPS(Fig.1). To determine whether 4T1-lysate loading could enhance the Ag presentation function for DCs, we evaluated the capacities of DCs to stimulate allogenic T cells. The results showed irradiated 4T1-lysate pulsed DCs induced stronger allogenic T cell proliferation than that of non-irradiated 4T1 lysate and PBS(Fig.1). 2 TNF-α level increased in irradiated tumor microenvironment In order to know whether local X-irradiation could form a proinflammatory microenvironment in the irradiated tumor beds and to seek exactly right timing window for DCs injection, we assessed the dynamic m RNAs expression of IL-10 and IL-6 from tumors or white cells withintumors of treated versus untreated mice after 2,11 and 21 days of local irradiation. Additionally, the levels of TGF-β and TNF-α in irradiated tumor tissues also were analyzed. As shown in Figure 3(A-E), IL-10 and IL-6 m RNAs expression, as well as TGF-β level have not shown significantly change in tumors or tumor infiltrating white cells compared with that of untreated mice. TNF-α level enhanced markedly after 2 and 11 days of irradiation compared with untreated mice. However, on day 21 after irradiation, TNF-α production decreased and there is no difference to the control(Fig.2F). 3 Antitumor immunity of 4T1-lysate pulsed DCs was enhanced by Xirradiation We next examined whether X-irradiation potentiates the anti-tumor ability of 4T1-lysate loaded DC vaccine with the therapeutic tumor-bearing models. 7 days after tumor inoculation, X-ray was adopted to treat the 4T1 tumor-bearing mice at a dose of 10 Gy for sequential 3 times interval of 24 hours or at a dose of 30 Gy only one time. Then, DC vaccine was injected intratumorally at 1×106/mouse/100μl at day 11 and day 18 after tumor inoculation, respectively. The results showed that irradiation only or combined with DCs significantly temporarily inhibited tumor growth compared with PBS or DCs. All of the mice untreated and treated with DCs only died at week 7 after tumor establishment and 20%-50% of mice treated with local tumor irradiation plus i.t. DC administration survived with tumors that size was less than 300 mm3 for more than 15 weeks. The tumors in mice treated with irradiation only were inhibited obviously compared with untreated mice and tumor volume kept unvaried until it showed obvious augmented at the ninth week and died out at the twelfth week after tumor inoculation(Fig.3C). 4 Inhibiting TNF-α with neutralization antibody significantly relieving the inhibitory effect on tumors of the combined treatment As shown in figure 4A, neutralization of TNF-α abolished the inhibition tumor growth tumor-bearing mice that were treated with irradiation combined DCs injection. Moreover, TNF-α decreased following TNF-α neutralization intumors from the irradiated beds(Fig.4B). 5 Combination of DC with X-irradiation induce long-lasting and systemic immunity in mice After a long period with no signs of progression, rapid tumor out-growth occurred in some of the mice that were given irradiation plus DCs combination therapy(Fig.2C). However, the other 40-60% of mice in this treatment group had no or small measureable tumors even at week 10. Since local irradiation enhanced i.t. DC therapy with triggering a derepressed proinflammatory microenvironment, we next assessed whether the microenvironment could be good for establishing a more long-term, systemic anti-tumor immunity. To this end, the survived mice in which the survival period exceeded 10 weeks with no or small measureable tumors were rechallenged with 4T tumor cells injected at sites posterior to the flank distant from the original tumors. Sixty percent of these mice were resistant to rechallenge for 10 weeks in X-ray plus i.t. DCs injection mice; whereas 100% naive mice inoculated with the same tumor cells appeared recurrence after one week of tumor inoculation and all died of tumors within 6 weeks(Fig.5A). 5.2 Immunologic memory generated in “cured”tumor-bearing mice after treatment with X-irradiation combining with DCs In order to analyze the immunological function status, the mice were killed at the fourth week of tumor rechallenge. The data indicated the CD8+T cells from rechallenged mice showed high levels of CD127, and IFN-γ expression, whereas the CD8+T cells from naive mice expressed low levels of CD127 and IFN-γ, CD44 expression had no change in naive and the treated mice(Fig.5C). 5.3 DC vaccination promotes accumulation of effector T cells and increases the ratio of CD8+T/T-reg and tumor specific CTL activity within tumors after irradiation Finally, the long-lasting and systemic immunity induced by treating with local tumor irradiation plus i.t. DC administration urged us to observe if the derepressed pro-inflammatory microenvironment might affect trafficking ofeffector T cells into solid tumors. So, using flow cytometry, we characterized the immune infiltrate within treated versus untreated s.c.4T1 at day 19 after final irradiation(i.e. day 10 after final DCs injection). A dramatic statistically significant increase occurred after irradiation with or without combining DCs injection in the mean number of CD8+T lymphocytes(P<0.05 or P<0.01), whereas no substantial change was observed in the mean number of T-reg lymphocytes((Fig.6E, 6F). The mean value of the CD8+T/CD4+CD25+ T ratio significantly increased after irradiation with combining DCs injection(P<0.01, Fig.6G). Furthermore, tumor-infiltrating cytotoxic T showed a higher cell lysis on 4T1 and irradiated 4T1(Fig.6H).Conclusions: 1 Within 2 weeks of irradiation, TNF-α level increased in tumor tissues and this is favor of DCs injection. 2 A long-lasting antitumor effect could be induced by combination X-ray with DCs i.t. injection. 3 Inhibiting TNF-α with neutralization antibody significantly relieving the inhibitory effect on tumors of the combined treatment. |
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