Sorafenib could inhibit the upregulation of PD-L1 induced by doxorubicin in Osteosarcoma cells in vitro.
For the first time, we found that sorafenib could inhibit the doxorubicin-induced upregulation of PD-L1 in Osteosarcoma cells. We used flow cytometry, PCR, and Western blotting analysis to analyze the total and surface PD-L1 expression in the human Osteosarcoma cell line MG63 and the mouse Osteosarcoma cell line K7 in vitro.
As shown in Fig. 1A-E, compared with the control group, doxorubicin significantly induced PD-L1 expression in Osteosarcoma cells in vitro. PD-L1 expression was decreased in the sorafenib plus doxorubicin group compared with the doxorubicin group. In addition, sorafenib therapy alone did not alter PD-L1 expression compared with the control group.
The effect of combined therapy on Osteosarcoma is better than therapy with sorafenib or doxorubicin alone in vivo.
Previous studies revealed that sorafenib can enhance the efficiency of other therapeutic agents (Jayson et al. 2016, Coventon 2017). Our previous study has shown that doxorubicin-induced PD-L1 upregulation on Osteosarcoma cells could suppress the proliferation of CD8 + T Lymphocytes and induce apoptosis in CD8 + T Lymphocytes, thus causing immunosuppressive status in Osteosarcoma (Wang et al. 2019). On the basis of these findings, the combination of sorafenib and doxorubicin may theoretically enhance the antitumor response of the immune system by inhibiting the doxorubicin induced overexpression of PD-L1.
Therefore, we studied whether the combination of sorafenib and doxorubicin is better than treatment with each agent alone. As shown in Fig. 2A, doxorubicin alone and sorafenib alone inhibited tumor progression compared with the control group; however, the tumor volume increased during the two-week treatment period. In contrast, sorafenib plus doxorubicin dramatically inhibited tumor progression and reduced the tumor volume during the two-week treatment period. As shown in Fig. 2B and Fig. 2C, after two weeks of treatment, the tumor volume and tumor weight in the doxorubicin plus sorafenib group were significantly smaller than those in the sorafenib, doxorubicin, and control groups.
Besides this, isobologram analysis was used to evaluate the possible synergistic interaction between sorafenib and doxorubicin. As shown in Fig. 2D, the combination of doxorubicin and sorafenib exhibited tiny synergistic effects in the Osteosarcoma cells, which is not consistent with the in vivo effectiveness.
Sorafenib could inhibit the upregulation of PD-L1 induced by doxorubicin in Osteosarcoma cells in vivo.
To confirm whether sorafenib could inhibit the doxorubicin-induced upregulation of PD-L1 on Osteosarcoma cells in vivo, we used flow cytometry to analyze PD-L1 expression on the surfaces of tumor cells from the tumor tissues. Meanwhile, IHC analysis was performed using subcutaneous tumor tissue to analyze PD-L1 levels in the tumor bed.
As shown in Fig. 3A-C, compared with the control group, doxorubicin significantly increased PD-L1 expression on Osteosarcoma cells in vivo. PD-L1 expression was decreased in the sorafenib plus doxorubicin group compared with the doxorubicin group. In addition, sorafenib therapy alone did not alter PD-L1 expression compared with the control group.
Proportion of CTLs among CD8 + T cells was significantly elevated in the combined group.
PD-L1 has been shown to play critical roles in immune escape by interacting with PD-1, which is expressed on lymphocytes or other host immune cells (Francisco LM 2009). The interaction between PD-L1 and PD-1 can lead to the apoptosis of lymphocytes including CTLs (Zhang et al. 2008). Our previous study has shown that doxorubicin-induced PD-L1 upregulation on Osteosarcoma cells could decrease the proportion of CTLs in Osteosarcoma tissue (Wang et al. 2019).
To analyze the T lymphocytes in the tumor tissue, cells from tumor tissues of the abovementioned four different groups were analyzed for the proportion of T lymphocytes, Tregs and CTLs.
There was no significant difference among the four groups in the proportion of CD4+, CD8 + T cells, and Tregs (Fig. 4A-B). Doxorubicin and sorafenib therapy alone did not significantly alter the proportion of CTLs among CD8 + T lymphocytes, while the proportion significantly increased in the sorafenib plus doxorubicin group (Fig. 4C).
Sorafenib and doxorubicin regulate PD-L1 expression via the ERK pathway in Osteosarcoma cells.
To investigate the mechanism by which sorafenib and doxorubicin regulated PD-L1 expression, we analyzed different pathways. As shown in Fig. 5A-D, compared with the control group, doxorubicin activated phospho-ERK (p-ERK) in Osteosarcoma cell lines, while sorafenib decreased p-ERK levels. The change in ERK phosphorylation level was consistent with the regulation of PD-L1 expression. In addition, no significant association was observed between either the mTOR or Stat3 pathway and PD-L1 expression in Osteosarcoma cells.
To confirm the relationship between the ERK pathway and PD-L1 expression, we used the ERK1/2-specific inhibitor PD98059. As shown in Fig. 6A-B, PD98059 significantly decreased the ERK phosphorylation. Western blot analysis indicated that basic PD-L1 expression in Osteosarcoma cells could not be decreased by PD98059, while doxorubicin-induced PD-L1 upregulation was inhibited by PD98059, which was consistent with the effect of sorafenib. Moreover, flow cytometry analysis of cell surface PD-L1 expression showed similar results (Fig. 6C-D). These data demonstrated that the ERK pathway participates in doxorubicin-induced PD-L1 upregulation on Osteosarcoma cells.