Innate Immune Cell-generated Microparticles Facilitate Liver Cancer Cell Metastasis

【Objective】Tumor cell metastasis is a complex succession of cell-biological events. To date, this metastatic process and mechanism still remains one of the most enigmatic aspects of the malignant disease. The old cell-cell fusion theory, which proposed that metastatic cells are generated by fusion of tumor cells with tumor-associated leukocytes, is conferring a malignant cell with the ability of immune cells to move through the bloodstream. An alternative explanation is stemmed from immune cell-released microparticles. Besides, there are many similarities in extravasations between immune cells and metastatic tumor cells. Adhesion molecules such as integrins are known to be the major contributor for immune cell extravasation through mediating immune-endothelial cell interactions. However, tumor cells usually do not express immune-related integrins, such as LFA-1 and Mac-1, raising a possibility that tumor cells usurp immune elements to cross blood vessels. In the present study, we explored innate immune cell-generated microparticles facilitate liver cancer cell metastasis and then defined underlying molecular mechanisms by cell line and animal models.【Methods】1. Generation and isolation of MPsBriefly, mouse splenic cells were cultured in the presence or absence of 50 ng/ml PMA for 12 hours at 37℃in 6-well plates. The supernatants from activated or resting cells were harvested and centrifuged to pellet MPs.2. Two-photon confocal microscope Isolated MPs were labeled with a red-fluorescent cell linker and then co-incubated with CFSE-stained H22 cells at 37℃for 20 hours. At last H22 cells taking up MPs were washed three times and observed by two-photon confocal microscope.3. Flow cytometryMPs were suspended in 250μl PBS with 1μ13μm non-fluorescent beads. After mixing, beads were run, to control for MPs size and count the number of MPs by a flow cytometer. The forward and side scatters were set at logarithmic gain. Fluorescent dye-conjugated antibodies were added at the appropriate dilution to prewashed MPs or H22 cells respectively. MPs or H22 cells were kept on ice for 30 min, resuspended in 0.3 ml of the same buffer, and then MPs suspensions were analyzed by flow cytometry, using Cell Quest software.4. In vitro migration AssayMigratory activity was quantified using 24-well Trans-well inserts. The Matrigel was added to the filter to form a thin gel layer and dried in a hood overnight. Tumor cells were suspended in 200μ1 of RPMI 1640 medium and then added to the upper chamber. The lower chamber was filled with 600μl RPMI 1640 medium containing 20%FBS. After 20 h of incubation at 37℃, B16 cells on the upper surface of the filter were removed by using a cotton swab. B16 cells that penetrated to the lower surface of the filter were fixed in methanol, and then sections were stained with hematoxylin and observed under a microscope. CFSE-H22 cells that transferred to the lower chamber were directly counted using a fluorescence microscope in three random×100 fields.5. Animal experimentsH22 tumor cells were injected to BALB/c mice via tail vein. After tumor formation, take photos for the mice and the left mice were fed for the long-term survival study. To study the blockade effects of CD 18 and CD11b molecular, part mice were i.v. injected with 30μg of anti-CD18 or CD11b antibody after the tail vein injection of H22 cells.【esults】1. Generation of MPs by activated immune cellsCFSE-labeled splenic cells were stimulated with 50 ng/ml PMA for 12 hours. The released MPs were showed□6-fold increases in the production of MPs after PMA stimulation. Then we analyzed the expression of immune phenotype, and found that MPs from the normally cultured splenic cells showed a low proportion of CD3, CD 19, Gr-1, F4/80 and MHC-II expressions. Whilst, PMA stimulation significantly increased, the proportion of Gr-1+or F4/80+MPs but not CD3+or CD19+MPs, suggesting that relative to T or B cells, myeloid cells probably are the major donors of MPs under PMA stimulation.2. Immune phenotypes can be transferred to H22 cells through MP pathwaysAfter 20 h co-incubation of the CFSE-labeled MPs with H22 cells, it was found that 37% H22 cells were CFSE positive in PMA-MP group and implicated the transfer of immune features to tumor cells. The confocal yellow dots were observed in cells, suggesting that MPs are taken up by H22 tumor cells but not adhered to them. The above H22 cells were stained with fluorophore-conjugated antibody and found that MPs indeed conferred H22 tumor cells to express surface immune markers, which was enhanced by PMA stimulation-induced MPs, suggesting that immune cell-derived MPs may transfer immune phenotypes to tumor cells.3. Activated immune cell-derived MPs favor H22 cell metastasis MP-treated H22 tumor cells were injected to mice via tail vein. Surprisingly, PMA-MP-treated H22 cells formed metastatic tumors in various forms in all the mice. The tumor could grow in thorax, abdomen, four limbs and even genitalia. In contrast, the injection of untreated H22 cells did not cause the tumor formation. Mice in both control MP and untreated groups showed a very well long-term survival, but most mice in PMA-MP group died in a short time. PMA-MPs were also found to effectively promote tumor cell migration and invasion in vitro. These data suggest that immune cell-originated MPs confer certain immune traits to H22 tumor cells, leading to tumor metastasis.4. CD11b/CD18 act as a key player in mediating H22 metastasis via MP pathwaysTo test immune MPs mediating tumor metastasis is through transferring immune integrins CD11b/CD18 to H22 cells, a blocking strategy was applied to the previous trans-well assay. It was found that the addition of either anti-CD 18 or CD1 1b blocking antibody to the upper chamber caused the inhibition of H22 tumor cell migrating to the low chamber. Thus, an in vivo study was followed. As a result, although the injection of MP-treated H22 cells resulted in tumor formation and mice death, the administration of each antibody reduced the tumor formation in most mice and resulted in the long-term survival observation, suggesting that CD11b/CD18 is essential for the above MP mediated H22 cell metastasis.5. Innate immune cells are the source for MPs promoting H22 cell migrationTo investigate which immune cell subset-generated MPs mediated H22 tumor cell metastasis, MPs were generated from CD3+T cells, CD19+B cells and CD3-CD19- innate immune cells with the stimulation of PMA, respectively. Such MPs were used for H22 cell trans-well assay above. Compared to the bulk MPs, both T and B cell-generated MPs only had an minimal effect on H22 cell migration, in contrast, the non-T, non-B innate MPs still maintained the ability to promote H22 cell migration. Although the expression of CD 18 was upregulated in both innate MPs and T or B cell-MPs, the expression of CD1 1b was only upregulated in innate MPs but null in T or B cell-MPs. Consistently, H22 cells, only when incubated with innate MPs rather than T or B cell-MPs, could express CD11b and CD 18 on their surfaces.6. TILs generating MPs promotes H22 cell metastasisTILs, isolated from H22 liver tumors, were cultured in the presence or absence of frozen-thaw H22 cell supernatants to generate MPs, which also expressed and resulted in CD11b and CD 18 transfer to H22 cells after incubation. In Matrigel assay, the rest TIL-derived MPs had a minimal influence on H22 cell migration, but the stimulated TIL-generated MPs significantly increased H22 cell migration, which could be impaired by the addition of CD11b or CD 18 blocking antibody. The pro-metastasis effect of TIL-derived MPs was verified in vivo and found that the rest TIL-derived MPs did not cause H22 cells to form metastatic tumors, but the stimulated TIL-derived MPs promoted H22 cells to grow tumors at indefinite sites. The long term survival experiment showed that mice in the rest TIL-MP group survived very well, but those in the stimulated TIL-MP group all died within 50 days. Nevertheless, either anti-CD 18 or CD11b antibody could rescue the survival of 50% those mice.【Conclusions】Our study explored liver cancer cells usurp immune cell migratory components or signal molecules for their metastasis in vitro and vivo. Then we demonstrated immune cells generated MPs could carry adhesion molecules CD11b and CD 18 to H22 cells. When mAb directed against such molecules could reduce cell migratory in vitro and block tumor formation in mice resulted in the long-term survival observation, suggesting that CD11b/CD 18 is essential for the above MPs mediated H22 cell metastasis. Third, we found that myeloid innate immune cells subset that express CD11b and CD 18 molecules involving cells migration. Last, we suggested that activated TILs also release MPs that could be taken up by H22 cells and act on tumor migration and metastasis. In summary, this study possibly opens a new aspect of MPs biology and may lead to discovery of a new mechanism in tumor migration and metastasis.