Activated T Cell Exosomes Promoted Tumor Invasion Via Fas Signaling Pathway

To this day, the clinical effect of cancer immunotherapy is far from as we expected. The poor therapeutic effect may mainly result from complexity of tumor immunity and immune evasion. Tumor escape and recurrence set obstacle to tumor immunotherapy. The most important reason of tumor recurrence and difficult to cure is tumor distance metastasis. Therefore, it makes scientific sense to intensive study the related mechanism to explore an effective protocol to cure tumor.Tumor invasion indicates tumor cells can break basement membrane and transfer to connective tissue and the level of metastasis ultimately depend on the tumor invasive ability. MMP9, a member of the matrix metalloproteinases (MMPs), plays a critical role in breakdown of extracellular matrix and facilitating tumor invasion. It has been reported that Fas on peripheral blood myeloid cells or tumor cells stimulated by Fas ligand (FasL) can induce the expression of MMP9on these cells during inflammatory response and tumor development, subsequently lead to cell infiltration surrounding tissue and invasion increase. These results suggest there is a close relationship between Fas signal and the expression of MMP9.Fas is a classic death receptor, which can induce apoptosis triggered by FasL to maintain systematic homeostasis. However, under certain condition, Fas signal can exert non-apoptotic function, including inflammatory response, liver regeneration, increase branching of developing neurons, migration of cells, angiogenesis, fibrosis, proliferation and differentiation of cells and advancing the cell cycle. Therefore, although almost tumor cells express Fas receptor, Fas pathway may sever as a beneficial function for tumor cells survival rather than apoptosis.Activation of T cells is a pivotal step in the progress of host anti-tumor immunity. During this course, T cells increase the express of FasL. The tumor environment is infiltrated by tumor antigen-specific cytotoxic T lymphocytes (CTLs) that are, however, in an unresponsive status. It is not clear that FasL on CTL plays inhibitory or promoting role in tumor development via Fas signal. The exosomes secreted by activated T cells express bioactive FasL and can induce apoptosis of T cell and dendritic cells (DCs), suppressing immune response.Exosomes are nanoscale membrane vesicles (50-100nm) released by various live cells, which have a structure of lipid bi-layer. They are formed by membrane budding into the lumen of an endocytic compartment, leading to the formation of multivesicular bodies (MVB). Fusion of MVB to the plasma membrane leads to the extracellular release of exosomes. Exosomes, derived from various kinds of cells, have different bioactive function. FasL positive exosomes have reported to be an inhibitory effector in immune system, and these researches mostly focus on the roles in regulating T cells and DC function via apoptosis. However, the potential effects of expressed FasL in activated T cell exosomes on Fas resistant tumor cells in tumor metastasis are still less understood. To illustration this issue, we isolated activated CD8+T cell exosomes (termed as EXO). The effect and related mechanism of promoting tumor invasion were investigated and the counterpart of EXO in vivo was identified, which may provide a novel view to understand the tumor escape in immune system.Firstly, we purified FasL positive exosmoes (EXO) derived from OVA-specific active CD8+T cells of OT-I mice. Then we detected whether EXO could induce the apoptosis of Fas expressing B16or3LL tumor cells. The results showed that FasL positive EXO has little effects on B16or3LL tumor cells apoptosis. In addition, prolonged treatment time or increased concentration, EXO did not affect the proliferation of B16or3LL compared with control. We further investigated whether EXO can cause other functional changes in tumor cells, and we performed the invasiveness assays in vitro. These results suggest that EXO may increase invasive ability of tumor cells via Fas signaling in vitro. It has been reported that CD95L mediates migration of myeloid cells via MMP9activation. We assume that the effect of EXO on B16tumor cells invasion is also related to MMP9activation. The results of real time PCR and western blot provide solid evidence that up-regulation of MMP9induced by EXO is Fas/FasL dependent. To further confirm the relation between EXO mediated promotion of tumor cell invasion and MMP9up-regulation, MMP9inhibitor was added to B16tumor cell invasiveness assays in vitro. These results indicate that the tumor cell invasion induced by FasL positive EXO was associated with MMP9expression via Fas signaling.The stimulation of CD95ligand (CD95L) on tumor cells can activate NF-κB and ERK pathways and the activation of NF-κB and ERK increases MMP9expression. Similarly, EXO stimulation activated the NF-κB and ERK pathways in B16cancer cells. To further reveal the roles of activated NF-κB and ERK in MMP9expression and tumor cell invasion, specific inhibitors for NF-κB and ERK signaling pathways were used to pre-treat B16cancer cells before EXO stimulation and then MMP9expression was examined. We found both NF-κB and ERK inhibitors (PDTC and U0126) markedly suppressed the EXO-induced MMP9expression and tumor cell invasion. Collectively, these results showed that FasL positive EXO activated NF-κB and ERK signaling pathway was responsible for the elevated MMP9expression and tumor cell invasion. Cellular FLICE inhibitory proteins (c-FLIP) is an endogenous inhibitor of death receptor-induced apoptosis and Fas signal can up-regulate the lever of c-FLIP in apoptosis-resistant tumor cells. In B16tumor cells, we found that Fas engagement with EXO led to the increased accumulation of c-FLIPL-The increase of c-FLIPL could be detected at5minutes after EXO stimulation and the high level of c-FLIPL could maintain until to24h. It has been reported that c-FLIP can promote activation of ERK and NF-κB signaling pathways and NF-κB signals can induce the expression of c-FLIP, which is related to the expression of MMP9. We therefore investigated the relation of c-FLIP and ERK or NF-κB signaling and the effect of c-FLIP on MMP9in B16tumor cells upon EXO stimulation. Results showed that the phosphorylation of ERK and NF-κB was significantly decreased when the expression of c-FLIPL was inhibited by siRNA, in turn, the inhibition of NF-κB and ERK signaling pathways decreased the expression of c-FLIPL on B16cells induced by EXO. Meanwhile, the expression of MMP9and the ability of cell invasion were also inhibited when c-FLIPL expression was knocked down. We found that the apoptosis of c-FLIPL knock down B16cells showed no difference to control cells. These results suggest that c-FLIPL play an important role in the increase of MMP9and tumor invasion induced by EXO. In B16cells stimulated by EXO, we found that the expression of Bcl-2obviously increased and specific inhibitor of ERK and NF-κB could inhibit the up-regulation of Bcl-2.After inhibited by Bcl-2inhibitor, we found that EXO could induce apoptosis of B16cells by FasL. These results suggest that Bcl-2is closely related to the Fas resistance of B16tumor cells induced by FasL expressed EXO.These results in vitro indicated that activated CTLs could release FasL positive exosomes (EXO). Furthermore, the high level of c-FLIPL could maintain until24h, the NF-κB and ERK signaling pathways were actived, the expression of MMP9was up-regulated and the ability of invasion was improved on B16tumor cells induced by EXO via FasL/Fas system, facilitating tumor cells invasion. In the non-apoptotic Fas signaling pathway, c-FLIPL actived NF-κB and ERK signaling, in turn, NF-κB and ERK signaling was involved in up-regulated the expression of c-FLIPL and the expression of Bcl-2was increased leading to tumor cells resistant apoptosis and improving cells invasion.To further clarify the effect of EXO on tumor invasion in vivo, we performed tumor invasion analysis with the B16tumor cell and EXO inducing murine lung cancer model through tail vein injection. We found that EXO greatly increased the tumor cell lung metastasis after14days. After pre-incubated with FasL mAbs, the ability of EXO to promote tumor lung metastasis was markedly decreased. These results strongly support that EXO increase the tumor cell lung invasion via Fas signaling in vivo.To further confirm the existence of CD8and FasL positive exosomes and their effects on tumor invasion in tumor mice, we established B16tumor model. Firstly, the result of immunofluorescence showed that there were CD8and FasL positive cells in spleens, draining lymph nodes and tumor tissue of tumor mice, indicating it exists the such cells which releasing CD8+and FasL+exosomes in vivo. Then we isolated CD8positive exosomes from spleens and draining lymph nodes in control and tumor mice by CD8positive magnetic beads. The CD8positive exosomes from tumor tissues were also isolated. After stimulated with exosomes isolated from tumor but not control mice, B16tumor cells showed increased expression level of MMP9and enhanced ability of invasion. These results indicate that CD8and FasL positive exosomes are naturally generated with tumor progression and exerted an exacerbated effect in tumor invasion. Meanwhile, we established3LL tumor model to test such the phenomenon. And the results suggested CD8and FasL positive exosomes may not be limited in particular tumor.Our results reveal a novel mechanism of tumor immune escape and suggest that strategies to inhibit rather than to promote Fas activity should be considered during cancer therapy.