Cellular immunity is the most important and universal immune system against cancer cells
Cellular immunity is the most important and universal immune system against cancer cells. inflammation in mice injected with cells that were silenced for STAT3 and injected with PD-L1 antibody. TUNEL assay showed low level of apoptosis in mice injected with cells silenced for STAT3 or injected with PD-L1 antibody, and higher level of apoptosis following combined treatment of STAT3 silencing Anlotinib and PD-L1 antibody injection. Immunohistochemistry, PCR, and Western blot analyses revealed that the expression of C-met, PD-L1, and STAT3 was significantly reduced in tumors following the combined treatment. Compared with treatment of STAT3 silencing or PD-L1 antibody injection, the combined treatment enhanced apoptosis. Conclusions Silencing STAT3 and PD-L1 antibody injection in combination increased apoptosis in tumor cells and thus offers better anti-cancer activity. (Hs03929097_g1) was used as an internal reference. The PCR analysis was carried out in a total volume of 10 l reaction containing 1.5 l diluted and pre-amplified cDNA and 10 l UltraSYBR mixture. The cycling conditions were 50oC for 2 min, 95oC for 10 min, followed by 40 cycles, each consisting of 15 s at 95oC and 1 min at 60oC. Primer sequences are listed in Table 1. Table 1 Primers for PCR. test. control HE staining Compared with mice in the control group, tumor cells in the NC group were arranged more tightly, while they were relatively loosely arranged in the mice treated with anti-PD-L1 antibody and sh-STAT3 (Figure 3), where small vacuoles were visible and evident and cells were arranged irregularly, particularly in the mice that received combined treatments, where cell morphology was extremely irregular and Anlotinib inflammation was strong. Open in a separate window Figure 3 HE staining of tumor tissues following sh-STAT3 and anti-PD-L1 antibody treatment alone or in combination. Arrows indicate inflamed and vacuolated tissues. Apoptosis As shown in Figure 4, normal cells were regularly-shaped and only the nuclei were stained blue by DAPI. In contrast, apoptotic cells were chaotically arranged and were green-colored. Compared with the control group, few apoptotic cells were observed in the NC group. Green fluorescence was enhanced in the sh-STAT3 and PD-L1 groups, and was the strongest in the sh-STAT3+PD-L1 group, suggesting that apoptosis was significantly increased in that group. Open in a separate window Figure 4 TUNEL assay results of apoptosis in cancer cells following sh-STAT3 and anti-PD-L1 antibody treatment alone or in combination. (A) Control; (B) NC; (C) sh-STAT3; (D) anti-PD-L1 antibody; (E) NC+PD-L1; (F) sh-STAT3+PD-L1. Expression of C-met, PD-L1, and STAT3 Immunohistochemistry showed that cells in the NC and control groups had similar and more brown-yellow labeling for the 3 proteins, while there were obviously fewer brown-yellow colored granules in the other 3 groups, indicating that the expression of C-met, PD-L1, and STAT3 decreased following sh-STAT3 and anti-PD-L1 treatments (Figure 5). The reduction was more remarkable following combined sh-STAT3 and anti-PD-L1 treatment. qPCR data and Western blot analysis showed similar results for mRNA and protein levels of these genes (Figure 6). Open in a separate window Figure 5 Immunohistochemistry assays of Anlotinib C-met, PD-L1, and STAT3 following sh-STAT3 and anti-PD-L1 antibody treatment alone or in combination. (A) C-met; (B) PD-L1; (C) STAT3. Open in a separate window Figure 6 Relative mRNA and protein levels of C-met, PD-L1, and STAT3 following sh-STAT3 and anti-PD-L1 antibody treatment alone or in combination. (A) Relative mRNA level; (B) Left panel: representative Western blot, right panel: relative protein level. * Denotes control. Discussion For tumors, particularly malignancy, treatment is still challenging. Radiotherapy or drug therapy often cause a number of adverse effects and complications, such as nausea, hair loss, and reduced health. Anti-PD-LI antibody has been demonstrated to be safe, with few adverse effects . We therefore investigated its effect on immune response in the tumor microenvironment in combination with sh-STAT3 and anti-tumor activity. The results of the present study showed that treatment with PD-L1 antibody or silencing STAT3 expression inhibited the growth of tumors, and IL5RA it was found that the Anlotinib combined use of PD-L1 antibody and sh-STAT3 had better anti-tumor activity; specifically, the weight and volume of the tumors decreased significantly after the combined treatment. We also found that treatment with PD-L1 antibody or sh-STAT3 promoted apoptosis of tumor cells, and the effect of combined treatment was more obvious. Studies have shown that.