| [Objective] Other than killing tumor cells, chemotherapy drugs can also interfere with the immune system. The reconstruction of immune situation in tumor-bearing host after chemotherapy is quite important for preventing tumor recurrence and progression. Myeloid derived-suppressor cells (MDSCs) are a most important group of cells which has become one of the research hotspot of tumor-induced immune suppression for its immunosuppressive effects to some of the immune cells. It has been found that MDSCs could accumulate in most tumors including lung cancer. Moreover, many factors can affect this process. When damaged, the live cells could release Damage Associated Molecular patterns (DAMPs), which have important immune regulation effects. As one important member of the lectin family, Galectin-3 also belongs to DAMPs. Although numerous chemotherapy drugs can directly affect the accumulation of MDSC, no one has focused on the influence of cisplatin on MDSCs:The objection of this research is to observe the changes of MDSCs and Galectin-3 after cisplatin treatment in the host using a Lewis lung cancer model. Then we aimed to explore the influence of galectin-3 to the differentiation, activity and migration of MDSCs in vitro.[Methods] (1) The Lewis lung cancer(LCC) cells were incubated to the C57BL/6 mice subcutaneously. After 5 days treatment of different concentration of cisplatin (0.4mg/kg,4mg/kg) intraperitoneally, the tumor tissues, spleens and peripheral blood of the mice was obtained. The number of MDSC in the tumor sites and the spleens was tested by using immunofluorescence (IF) and Flow cytometry (FCM) respectively. Immunohistochemistry was conducted to test the Galectin-3 expression in the tumor sites and Enzyme linked immunosorbent assay (ELISA) to check the Galectin-3 concentration in the plasma. (2)During the culture of Lewis lung cancer cells, immunofluorescence, Western Blot and FCM were used to detect the Galectin-3 expression on LLC cells. After the administration of 2 uM cisplatin for 24 hours, the concentration of Galectin-3 in the cell culture supertantanants was tested by ELISA.(3) The MDSC was separated from the spleens of the tumor-bearing mice by CDllb magnetic bead and was put on the upper class of the Transwell cabinet, and the lower class was the Lewis lung cancer cells culture medium which had been certified to have Galectin-3 protein. In some cases, anti-mouse Galectin-3 monoantibodies were added. Galectin-3 was also tested in MDSC by Western Blot. (4) The MDSC was separated from the spleens of tumor-bearing mice by CDllb magnetic bead. Galectin-3(1uM、50uM) was added to the culture medium, the apoptosis of MDSC was calculated after 12 hours or 24 hours by AnnexinV/PI. (5) GM-CSF (20ng/ml) was added to the monocytes from the bone marrow of the C57BL/6 mice, some species were added Galectin-3(100ng/ml), non-adhesive cells in the supernatants was collected and checked for the expression of Gr-1 and CD11b and CD11c on day 2 and day 4 to measure the mature of the progenitor cells and indicate the effect of Galectin-3 on the differentiation of MDSC.[Results] (1) MDSC infiltrated in tumor sites in the entire Lewis lung cancer tumor bearing mice and the cisplatin dramatically increased the number of MDSC. There was significantly statistical significance of numbers of MDSC in different concentration groups of tumor bearing mice (P<0.05). After being treated with 0.4mg/kg、4mg/kg cisplatin for 5 days, the percentage of MDSC in the tumor bearing mice spleens was increased with the increasing concentration of cisplatin, and the MDSC percentage was 4.1%、13.6% and 26.7%(P<0.05). (2) In the control group, the staining of Galectin-3 was positive in the tumor tissues and the buffy grains could be seen in endochylema, and the buffy grains was very thin; cisplatin strengthened the stain degree of Galectin-3 in the tumor tissues, the stain and the buffy grains become thicker and masculine cell population was more than control group, and there was statistically significant difference between control group and every treatment group(P<0.05). The concentration of Galectin-3 in the plasma of tumor bearing mice treated with 0.4mg/kg、4mg/kg cisplatin for 5 days was decreasing respectively. The concentration was 1.58±0.87ng/ml、3.6±1.1ng/ml、4.7±0.56ng/ml and the difference was significant between 4mg/kg cisplatin treatment groups and the control groups although no significant between the 0.4mg/kg groups and the control groups or the two tested groups. (3) Galectin-3 was expressed both in the cell membranes and cytoplasm of the Lewis lung cancer cells. The expression percentage was 78.3% and 95.8% respectively. Galectin-3 could be released to the cell culture supernatants, and the concentration of Galectin-3 in the supernatants significantly increased after the cisplatin treatment with 14.26±2.16ng/ml compared to 9.23±2.54ng/ml. (4) The Lewis lung cancer cells culture medium was chemotatic to MDSC and when the Galectin-3 was blocked by the monoantibodies, the chemotactic impact was significantly decreased. Western Blot result showed that Galectin-3 exited in the MDSC. (5) Cisplatin neither inhibited MDSC apoptosis nor induced them apoptosis. (6)When the bone marrow progenitor cells was cultured with 20ng/ml Galectin-3 for 4 days, the percentage of MDSC and DC in the culture cells was 46.7% and 14.7% respectively, compared to the control group of 22.8% and 21.3%.[Conclusions]1. Cisplatin increaced the number of MDSCs in both the spleen and tumor tissue and increased the expression of Galectin-3 in the tumor tissue as well as the peripheral blood in the tumor-bearing mice.2. Lewis lung cancer cells could express Galectin-3 and the expression level increased in response to cisplatin.3. Tumor-derived Galectin-3 could induce the accumulation of MDSC in vitro.4. It seems that there was no effect of galectin-3 on MDSCs apoptosis.6. Galectin-3 could inhibit the immature bone marrow cells differentiate to mature cells, thereby promoting the inducement of MDSC level.7. Galectin-3 may act as one of the members to influence the accumulation of MDSCs after cisplatin treatment in Lewis lung cancer bearing mice. This would provide new target for reversing the immunosuppression of tumoral hosts after chemotherapy. |
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