Effect Of Tumor Cell Microparticles On Tumor Growth And Metastasis And Its Mechanism

Objective:In response to stress, tumor cells change their cytoskeleton, leading to encapsulation of cytosolic contents by cellular membrane to form vesicles that are subsequently released into the extracellular space. These specialized subcellular vesicles with a diameter of 100 to 1,000 nm are called microparticles, which are essential for cell-cell interaction by their packages such as protein, lipid, cytokine and nuclear molecules (DNA, microRNAs, mRNAs). Whether tumor cell-derived microparticles (T-MPs), carrying tumor self-antigens, can be used as cancer vaccines remains unknown. Success of a tumor vaccine lies in its ability to provide both tumor antigens and immune-stimulatory signals. In the present study, we provide evidence that T-MPs may represent a novel tumor vaccine due to their possession of both tumor antigens and innate DNA signals.Methods:(1) To determine the immunogenicity of T-MPs, we immunized BALB/c mice with H22 hepatocarcinoma cell-derived microparticles (H22-MPs) followed by a challenge with 3×105 H22 cells. As a comparison, different groups of mice received tumor-cell lysates or tumor cell-released exosomes. Splenocytes harvested from mice pre-immunized with H22-MPs, H22 exosomes, or H22 lysates 20 days after tumor implantation were used to perform an invitro tumor-specific CTL killing assay. For the immune-deficient model, nude mice were immunized with H22-MPs or PBS and then challenged with H22 tumor cells. In lymphocyte-depletion experiments, mice were injected i.p.with 0.1-mg anti-CD4, anti-CD8, or anti-asialo GMl antibody. To determine that immunogenicity of microparticles is not limited to one tumor type or a specific mouse strain, we immunized C57BL/6 or Balb/c mice with B16 melanoma cell-derived microparticles, Hepal-6 hepatocarcinoma cell-derived microparticles and CT26 colon carcinoma cell-derived microparticles. To test the specific of T-MPs, we immunized mice with H22-MPs and the subsequent challenge with CT-26 or Hepal-6. (2) To investigate the therapeutic potency of T-MPs, BALB/c mice were inoculated s.c. with 3 ×105 H22 tumor cells 5 days before vaccination with H22-MPs and then analyze the number and IFN-γ expression of intratumoral CD8+T cells. (3) We analyze the uptake efficiency of T-MPs by DCs using FACS and fluorescence microscopy. DCs maturation and T cell proliferation assay were performed after T-MPs treatment in vitro. DCs maturation and immune cell type were assayed in draining lymph node after T-MPs treatment in vivo. (4) We detect DCs maturation related signaling pathways such as Erk, P38, IKK and IRF3 using western blot. Analyze the mRNA and protein expression of IFN-α/β in DCs treated with T-MPs us RT-PCR and Elisa. Furthermore, we detected DCs maturation and T cell proliferation after block the IFN-α/β signaling with IFNAR blocking antibody. (5) HSP70, HSP90 and HMGB1 in T-MPs were detected using western blot. Semi-quantitative PCR were performed to detected the expression of DNA in T-MPs. Myd88-/-DCs maturation were analyzed after treatment with T-MPs. DCs were silenced for RIG-I, cGAS or STING expression using siRNA, respectively. After siRNA transfection, expression of IFN-a/(3 was measured in DCs after incubated with H22-MPs.Results:(1) 100% tumor formation was seen in mice immunized with PBS or tumor-cell lysates, whereas 50% of microparticle-immunized mice and 12.5% of exosome-immunized mice remained tumor free, suggesting that T-MPs are more immunogenic than the other tumor cell-derived materials. Consistent with the observed antitumor activity in mice, in vitro cytolytic analysis indicated that splenic T cells from mice immunized with H22-MPs were more potent in killing H22 targets than those from exosome-or cell lysate-immunized mice. In T-cell-deficient nude mice, H22-MPs failed to induce the antitumor effect. Depletion of CD4+or CD8+T cells, but not natural killer cells in wild-type mice, also diminished tumor protection of T-MPs. Immunized animals were protected against subsequent challenge with B16 tumor cells via s.c. or i.v. delivery. Similarly, BALB/c mice immunized with CT26 colon carcinoma cell-derived microparticles prevented CT26 tumor growth. Interestingly, mice immunized with H22-MPs did not affect CT26 colon carcinoma growth, but resulted in protection against Hepal-6, another murine hepatocarcinoma tumor cell line. (2) T-MP vaccine alone failed to affect the growth and vaccination with H22-MP-loaded DCs significantly inhibited H22 tumor growth. Intratumoral cellular analysis revealed that the number of infiltrating CD8+T cells was three times higher in the H22-MP-loaded DC group than in the other groups, and they were more capable of producing IFN-y. (3) T-MPs were more efficiently taken up by DCs than apoptotic tumor cells. An in vitro T-cell proliferation assay indicated that the presence of DCs was required because T-MPs alone were not sufficient to stimulate T-cell proliferation. Upregulation of CD80, CD86, MHC class II, and CCR7 demonstrates that the uptake of T-MPs triggered DC maturation. Expression of IL-12 and IFN-y were induced by T-MPs. (4) No further activation of Erk, P38 or IKK was seen in DCs treated with T-MPs. However, phosphorylation of IRF-3 was evident 1 hour after exposure to T-MPs. Consistent with this finding, the increase of IFNa and IFNb expression by DCs was detected in mRNA and protein levels after incubation with T-MPs. Upregulation of CD80, CD86, and MHCII during incubation with T-MPs was attenuated by IFNAR-1 blockade, leading to reduced capacity of microparticle-loaded DCs to stimulate T-cell proliferation. (5) HSP70, HSP90 and HMGB1 were not detectable by Western blot in T-MPs. Abundant nuclear gene GAPDH fragments and mitochondrial DNA fragments were included in T-MPs. Furthermore, DCs deficient for MyD88 underwent maturation during incubation with T-MPs. Our results show that knockdown of RIG-I did not influence DC production of type I IFN by T-MPs stimulation. Knockdown of cGAS/STING strongly inhibited type I IFN production.Conclusion:T-MPs are more immunogenic than tumor-cell lysates and tumor cell-derived exosomes in eliciting T-cell-dependent antitumor immunity. T-MP vaccines can achieve therapeutic effects by using DCs as a carrier. Uptake of T-MPs is a necessary step for DC maturation and antigen presentation. T-MPs activate DCs via cGAS-STING pathway, leading to IFN-α/β expression. IFN-α/β induced DCs maturation through autocrine and paracrine pathway.Objective:The tumor microenvironment is often hypoxic, leading to tumor growth and tumor metastasis. Pre-metastatic niche formation is a key step in the development tumor metastasis. Hypoxia has been reported to induce the release of extracellular vesicles by tumor cells, including microparticles. However, it is still unknown whether microparticles released from tumor tissue could reach the lung tissue and initiate lung pre-metastatic niche. This study focused on the impact of hypoxia-induced tumor microparticles on the lung pre-metastatic niche formation and lung metastasis. The potential mechanism was explored by analyzing the change of cells, inflammatory cytokines and extracellular matrix in lung treated with microparticles.Methods:(1) Microparticles isolated from tumor cells cultured in hypoxic conditions (1% 02) for 24h. The quantity, size and protein content were analyzed; (2) To analyze the tissue distribution of the microparticles, we injected PKH26-labeled B16F10-MPs intravenously into C57/B6L mice.2h after injection, liver, spleen and lung were removed and performed the frozen sections and fluorescence microscope analysis; (3) To further examine the role of MPs in primary tumor growth, we intravenously injected mice with MPs three times a week, starting 7 d after orthotopic injection of tumor cells(B16F10, 4T1 and Lewis), and tumor growth were calculated. To test whether MPs were involved in promoting tumor cell escape from the primary tumor, we measured the number of circulating tumor cells; (4) Mice bearing orthotopic tumor (B16F10,4T1 and Lewis) were treated with MPs three times a week intravenously, and lung metastatic burden were measured; (5) Mice bearing subcutaneous B16F10 tumor were treated with hypoxia-induced MPs, and AMs were selectively depleted by intranasal administration of clodronate liposomes; (6) To analyze vascular leakiness properties in the lung, we injected hypoxia B16F10-MPs into mice, followed by injection of fluorescently labeled dextran 24 h later. FITC-dextran infiltration were analyzed using fluorescence microscope; (7) 24 h after B16F10-MPs injection, S100A8, S100A9 and serum amyloid A3 (SAA3) expressions were examined by RT-PCR and inflammatory monocyte recruitment were analyzed by flow cytometry; (8) To study whether MPs initiate lung pre-metastatic niche formation, we injected B16F10-derived MPs intravenously into mice every other day over 3 weeks, and then i.v. implanted B16F10 cells, lung metastatic burden were analyzed 21 days later; (9) CCL2 expression in lung tissue, lung macrophage, bone marrow macrophages were examined after B16F10-MPs treatment; To study the effect of MPs-induced CCL2 increase on inflammatory monocyte recruitment and lung tumor metastasis, CCL2 was neutralized using a CCL2-specific antibody; (10) fibrin expression in lung were analyzed by fluorescence microscope after B16F10-MPs treatment.Results:(1) Hypoxia increased the release of microparticles with no change in their mean particle diameter and size distribution. Greater number and mass of MPs were obtained in conditioned media from hypoxic tumor cells; (2) MPs distributed in the interstitium of the lungs, livers and spleens of the mice. In addition, we found that over 60% of lung cells that take up MPs were F4/80+CDllc+by flow cytometry or F4/80+ by immunofluorescence, a phenotype consistent with alveolar macrophages (AMs); (3) MPs did not affect tumor migration and intravasation in the primary tumor; (4) Hypoxia-induced MPs increase spontaneous tumor metastasis; (5) Macrophage depletion significantly blocked the effect of B16F10-MPs on lung metastasis; (6) The B16F10-MPs enhanced the lung endothelial permeability in the mice, as judged by the presence of extravasated FITC-dextran; (7) S100A8, S100A9 and serum amyloid A3 (SAA3) were upregulated in lung tissue 24 h after B16F10-MPs injection; CD11b+Ly6C++ inflammatory monocytes were preferential recruited to the lung 48h after B16F10-MPs injection; (8) Mice injected with B16F10 MPs had a greater metastatic burden in the lungs; (9) CCL2 neutralization markedly inhibited the recruitment of inflammatory monocytes to lungs and attenuated the lung metastasis burden induced by B16F10-MPs; (10) A marked increase of fibrin expression was observed in lung tissue induced by B16F10-MPs.Conclusion:(1) Hypoxia increased the release of microparticles, which significantly increase spontaneous tumor metastasis; (2) Hypoxia-induced tumor microparticles enhance pulmonary vascular permeability and increased S100A8, S100A9 and SAA3 expression in lung tissue. After taking up microparticles, alveolar macrophages secreted CCL2, which then recruited inflammatory monocytes. Hypoxia-induced tumor microparticles increased the fibrin expression in lung. All above finding suggested that Hypoxia-induced tumor microparticles initiate pre-metastatic niche formation and promote tumor lung metastasis.