Effects Of Tumor Microenvironment On Dendritic Cells Migration As Well As Concerned Singnaling Pathway

BackgroundMalignant glioma represent about one-third of all adult primary brain tumors. The prognosis of human malignant glioma remains poor with an overall 5-year survival rate of less than 3% and a median survival time of about 1 year for higher grade tumors such as glioblastoma. Alternative therapeutic approaches to surgery, radiotherapy and chemotherapy are required to improve patient survival. Recent insights into neuroimmunology provide evidence of the immunocompetence of the brain even in the case of tumor development, thus opening new hopes for immunotherapy.Dendritic cells (DCs) play a crucial role during the priming of antitumor-specific immune response. The DCs immunotherapy for inducing specific immunity against tumor has shown promising activity in both preclinical models and clinical observations. Two main DC-based immunotherapies are intensively used for tumor bearing patients. One is injecting DCs directly into tumor or surgical resection region of tumor; the other is Loading DCs with tumor antigens ex vivo and transfusing back to body. Intratumorally injected DCs can acquire and process tumor antigens in situ, migrate via lymphoid vessels to regional lymphoid organs, and initiate significant tumor-specific immune responses, even in the CNS. A cumulating data indicate that intratumoral injection of DCs may be a more promising strategy for the therapy of tumor patients. A major advantage of stimulating DCs with tumor antigen in vivo instead of ex vivo is that DCs encounter the tumor antigen in their natural environment. Other co-factors involved in antigen processing and presentation are already available, simplifying the manipulation required to maximize anti-tumor immune responses. In addition, this therapy can be administered to patients at the same time as tumor resection by surgery, while ex vivo impulse antigen of DCs require a week or more before the patient can receive the immunotherapy. This means that the tumor is rapidly hit with the anti-tumor immune response, is not given any chance to recover from surgical resection and the therapy can take advantage of any inflammation naturally associated with the tumor resection.However, clinical trials have produced contrasted results and no definitive conclusion can be drawn about the real efficacy of this approach. Basic questions, concerning functional deficiency of DCs in tumor microenvironment, are currently unresolved. A recent study asserted that the migration of in vitro generated DCs to the regional lymph nodes was very few observed when injected into tumor tissues. This suggested that DCs lose their migrating activity in tumor tissues. Migration of DCs from the site of antigen recognition to regional lymphoid tissues is crucial for DCs to function as potent antigen presenting cells (APCs). DCs migration is known to be highly sensitive to microenvironmental changes. A number of immunosuppressive factors in tumor supernatant (TSN) and regional low oxygen (Hypoxia) are particular phenotypes in tumor microenvironment. But to date, it remained unclear which factor in tumor microenvironment play a crucial role in hindering the migration of DCs. So it is very interesting to find which factor in tumor microenvironment can impair the migration of DCs and how to effect on it.In our study, we compared the migrating activities of human monocyte-derived DCs under hypoxic in the present or absent of TSN to their normoxia counterparts in vitro and assessed the effect of these two factors on the migration of human monocyte-derived DCs. Whereafter, we further investigate the mechanisms leading to the suppression of DCs migration under tumor microenvironment, and find a signaling pathway to inhibit DCs migration. So as to provide a new insight into the understanding of molecular mechanisms on how tumor cells escape host immune surveillance in tumor local-tissue microenvironments.Objective1. In vitro generation of monocyte-derived DCs from peripheral blood monocytes (PBMCs) under normoxic and hypoxic conditions and identification of their morphology and phenotype.2. To study the effect of TSN and hypoxia in tumor microenvironment on the migration of DCs.3. To study how to affect the migration of DCs by TSN and/or hypoxia.Methods1. In vitro generation and culture of monocyte-derived DCsPBMCs were isolated by density gradient centrifugation with Ficoll-Paque Plus of buffy coats. PBMC were resuspended in 2% FCS-RPMI-1640 medium at 10⁶/mL and distributed in 6-well plates (2 mL/well). The plates were incubated in a 37℃incubator for 2h before nonadherent cells were removed. The remaining adherent cells were cultured in the presence of GM-CSF (1000 U/mL) and IL-4 (500 U/mL) in 10% FCS-RPMI-1640 medium. Fresh medium containing GM-CSF and IL-4 was added on Day 3 (2 mL/well). To induce maturation, LPS (1μg/mL) was added to cells on day 5 of culture for 48h.2. Preparation of TSN and TSN-exposed DCsTSN were prepared by seeding 25cm² flasks with 1×10⁶ U251 glioma cells in 10 ml of complete medium. The culture supernatants were collected after 24 h under hypoxia, centrifuged to remove cells, and stored at -80℃. The effects of TSN on DCs were examined by replacing 50% (vol/vol) of the culture medium with TSN.3. Normoxic and hypoxic culture conditionsFor normoxic conditions, cells were incubated in a regular incubator (21% O₂, 5% CO₂, and 74% N2). Hypoxic incubation was performed in a sealed, anaerobic work station, where the hypoxic environment (1% O₂, 94% N₂ and 5% CO₂), the temperature (37℃), and humidity (90%) were kept constant. 4. To investigate the effect of hypoxia and/or TSN on the DCs migration.â‘ DCs cultured under normoxic or hypoxic conditions were treated with or without50% TSN in medium for 24h and were evaluated for their migratory activity througha Matrigel-coated filter in a Transwell system.â‘¡In gene level, using Real-time quantitative RT-PCR to detect the changes of CCR7gene expression in every one of experimental groups, In protein level, using Flowcytometric analysis to detect CCR7 on membrane of DCs.â‘¢Using Real-time quantitative RT-PCR and Western blot to detect the MMP-9 ofDCs separate from gene and protein level.5. To investigate the mechanisms of hypoxia suppressing the produce of MMP-9 by human DCs.â‘ To profile the relative expression of hypoxia signal pathway proteins related genes in normoxic and hypoxic DCs by gene microarray analysis.â‘¡To verify the expression pattern of adenosine receptor subtypes on DCs in response to hypoxia by real-time PCR.â‘¢To evaluate the function of adenosine receptors by pharmacological approach using adenosine receptors agonists and antagonists.â‘£To study the cAMP/PKA signaling pathway by forskolin (a direct activator ofadenylate cyclase), SQ22536 (a specific adenylate cyclase inhibitor) and the specificPKA inhibitor H89.Result1. Effect of hypoxia and/or TSN on the DCs migration.(1) After 8 h of incubation in the presence of MIP-3β, it was evident that DC-H(94±16.1 cells/HPF) displayed a lower migratory activity by twofold than that ofDC-N (185±23.6 cells/HPF, P < 0.05). The migratory activity of DC-T (163±18.1cells/HPF) had no significant change when compared with DC-N. DC-H-T (92±15cells/HPF) was the similar with DC-H on migratory activity. Interestingly, themigratory capacity of DC-H was partially recovered when cultured with thesupernatant from DC-N. â‘¡Stimulation with hypoxia induced a twofold increase in the steady-state levels of MMP-9 mRNA (P < 0.01), but no significant differences were found in the induced levels stimulated with TSN. Both DC-N and DC-T exhibited a distinct band corresponding to MMP-9 in Western blot. In contrast, DC-H and DC-H-T presented a faint band corresponding to MMP-9.â‘¢Stimulation both with hypoxia and TSN had no significant differences in the levels of CCR7 mRNA were found compared with DC-N. CCR7 protein was slightly increased by hypoxia.2. Hypoxia induces adenosine receptor A_2b in expression pattern of adenosine receptor subtypes in DCsThe experiment data demonstrated that adenosine receptor A_2b was selectively induced by hypoxia (3-fold in imDCs and 21-fold in mDCs) and other subtypes were not changed. According to this clue, we verified the expression pattern of adenosine receptor subtypes on DCs in response to hypoxia by real-time PCR. The results consistently revealed that the A₁, A_2a and A₃ receptors expression levels in hypoxia remained unchanged compared with them in normoxia. In contrast, A_2b receptor on mDCs was increased by as much as 6±0.11-fold compared with it in normoxia (P <.01), whereas on imDCs it was not changed. What's more, the ratio of expression of A_2b receptor to others changed to predominance under hypoxic condition. Taken together, our results suggest that adenosine receptor A_2b on mDCs can be induced by hypoxia, which consistent with previous studies that hypoxia prominently induces A_2b receptor.3. Inhibition of MMP-9 produce by DCs in hypoxia is mediated by A_2b receptormDC-N was treated with or without adenosine receptor agonist (NECA, 1μM) under normoxia for 8 hours. mDC-H was treated with or without A_2b receptor specific antagonist (MRS 1754, 1μM) under hypoxia for 8 hours. Cultured supernatant Gelatin zymography showed that mDC-N exhibited a distinct band corresponding to MMP-9. In contrast, mDC-H presented a faint band corresponding to MMP-9, in agreement with our previous publication. MRS 1754 (1μM) was able to counteract the inhibition of hypoxia on MMP-9 in mDC-H. Under normoxic conditions, however, NECA had no effect on the secretion of MMP-9 in mDC-N incubated for 8 hours. The results obtained by zymography were confirmed by ELISA. To detect whether inhibition of MMP-9 is mediated by other receptors subtypes of DCs, we use each of specific receptor antagonists in different concentration (0, 10, 100, 1000μM) to incubate with mDC-H in hypoxia. Cultured supernatant ELISA showed that the A_2b specific antagonist MRS 1754 at the concentration of 100 nM was able to abrogate the inhibition of hypoxia on MMP-9. The A₁ specific antagonist DPCPX, A_2a specific antagonist SCH58261 and A₃ specific antagonist MRS 1220 were essentially without effect. To rule out the possibility that this suppression on hypoxia could be a result of an increase in MMP-9 secretion by MRS 1754 itself, The A_2b antagonist MRS 1754 (1000 nM) used in DCs under normoxia did not modify the MMP-9 secretion. Among the four adenosine receptors agonists tested (The A₁ specific agonist CPA, A_2a specific agonist CGS21680, A_2b agonist NECA and A₃ specific antagonist IB-MECA); none of them was able to mimic the inhibitory effect of hypoxia on MMP-9. Taken together, these results suggest the suppression of MMP-9 secreted by DCs in hypoxia is involved in activation of adenosine receptor A_2b.4. Inhibition of MMP-9 produce by DCs in hypoxia is cAMP/PKA relatedFirstly, the effect of forskolin, a direct activator of adenylate cyclase, was determined. Forskolin (10μM) significantly decreased MMP-9 secreted by DC-N. Secondly, direct measurements of cAMP levels in DCs showed that hypoxia can increase cAMP production. These results suggested that forskolin can mimicked the action of hypoxia on MMP-9. Finally, the effect of SQ22536, a specific adenylate cyclase inhibitor, on the action of hypoxia was studied. In control cultures, hypoxia decreased MMP-9 production (as measured by ELISA in 8-hour supernatants) by 51±2% (mean±SD). In the presence of SQ22536 (1 mM), hypoxia decreased MMP-9 production by only 20±3% (P<.01 compared with hypoxia alone, n=4;). These findings suggest that the effects of hypoxia on MMP-9 are mediated through increasing of cAMP levels. When added to DCs under hypoxia, H89 abrogated the inhibition of MMP-9. Taken together, the cAMP/PKA pathway could be responsible for the observed effects of hypoxia on MMP-9 through binding to the A_2b receptor. 5. The inhibition of MMP-9 by hypoxia due to the elevation of A_2b receptor activity not adenylate cyclaseWe have showed that stimulation of A_2b receptor was not able to replicate the inhibitory effect of hypoxia on MMP-9 under normoxia, whereas the inhibition of A_2b receptor can counteract the MMP-9 under hypoxia. To explain the phenomena, our studies focused on subsequent signal transduction molecular cAMP. To rule out the effect of adenosine produce by DCs under hypoxia on cAMP, DC-N and DC-H were cultured with interventional agents under normoxia. Our results showed that NECA only mildly increased cAMP levels in DC-N compared with the significant increase in cAMP production in DC-H. The A_2b selective antagonist MRS 1754 eliminated the stimulatory effect of NECA only in DC-H, indicating that A_2b activity is high in DC-H and low in DC-N. Administration of forskolin demonstrated that adenylate cyclase activation augmented cAMP levels in both DC-N and DC-H, suggesting that adenylate cyclase activity was not affected by hypoxia. We noted that A_2b receptor antagonist MRS 1754 can not inhibit the stimulatory effect of forskolin, whereas specific adenylate cyclase inhibitor SQ22536 can inhibit the stimulatory effect of NECA. The observation demonstrated that the inhibition of MMP-9 mediated by A_2b receptor in hypoxia is via cAMP.Conclusion1. We can successfully generate DCs under hypoxic and normoxic conditions in vitro.2. Hypoxia can hinder the migratory of DCs through suppression of DCs-induced MMP-9, whereas TSN has no effect on their migration activity.3. The inhibitory effect of hypoxia on MMP-9 by DCs requires the activation of A2b in a cAMP/PKA-dependent signaling pathway.