肺癌是全世界造成最多死亡的癌症，T細胞的PD-1及其位於腫瘤細胞的配體PD-L1，在免疫檢查哨治療中扮演重要角色，並已應用於肺癌治療。細胞遷移及侵襲是構成癌細胞轉移的要件；之前實驗結果已知在肺腺癌細胞株, CL1-0和CL1-5, PD-L1過度表現和癌細胞遷移有關連性，同時也有較多的上皮間質轉化(epithelial mesenchymal transition, EMT)調節因子及間質標記蛋白表現。根據文獻，PD-1 可促進自身腫瘤細胞的生長。因此，我們將探討肺腺癌細胞中，PD-L1和PD-1的交互作用及細胞生長率、化療敏感性的影響以及臨床預後。
我們實驗使用肺腺癌細胞株CL1-0; CL1-5為較具侵犯性的細胞株，源自CL1-0細胞。我們並從CL1-0細胞中找出PD-L1過度表現之細胞。細胞遷移及侵犯能力使用transwell assay評估；EMT標記蛋白及調節因子使用西方墨點法評估，並且觀察細胞型態。我們並使用MTT assay和 colony formation assay來測試化療敏感性及細胞生長率。另外，藉由在手術檢體上，以IHC染色，了解肺癌細胞PD-L1表現量和臨床預後的關係。最後，為了解PD-1和PD-L1是否有交互作用，將抗PD-1抗體加入CL1-0, CL1-5, CL1-0-PDL1細胞，比較其遷移及侵襲性及細胞型態；並用siRNA抑制PD-1觀察其影響。
我們實驗結果確認PD-L1促使人類肺腺癌細胞遷移及侵犯性，並且和EMT有相關。在PD-L1表現較多之細胞，有較強的遷移能力、較多的間質標記蛋白及EMT標記蛋白表現。我們同時觀察到，CL1-5和CL1-0-PDL1細胞, 為較多PD-L1表現之細胞株，其細胞型態類似紡錘型；而CL1-0細胞則接近圓形。於收集肺腺癌的手術檢體，發現淋巴轉移之PD-L1表現量明顯多於沒有淋巴轉移的患者，此一臨床結果配合前述實驗結果，認為PD-L1表現量較多的細胞是較具有侵犯性的。在細胞生長率方面，PD-L1表現減少細胞生長率，但對化療的敏感度，沒有很大差異。
在加入抗PD-1抗體至CL1-5, CL1-0, and CL1-0-PDL1細胞後，其遷移及侵襲能力都減弱了，這代表抗PD-1抗體阻斷了癌細胞本身的PD-1和PD-L1連結。這現象，我們也以PD-1 siRNA再次證實。因此我們認為肺腺癌細胞內生的PD-1/PD-L1互相作用，促使細胞遷移及侵襲性。
此一研究證實肺腺癌細胞合併較高PD-L1表現，會促使細胞遷移、侵襲、EMT及少量化療抗性。PD-L1表現會減少細胞生長率。而肺癌細胞本身PD-1和PD-L1交互作用亦造就細胞遷移及侵襲性。

Lung cancer is the most frequent cause of cancer death. Programmed death 1 (PD-1) in T cells and its ligand PD-L1 in tumor cells play a key role in immune checkpoint therapy and had applied to advanced stage lung cancer. Migration and invasion of tumor cells is a prerequisite for tumor cell metastasis. Our previous study found that in CL1-5 cells, derived from CL1-0 cells, with high PD-L1 expression possessed higher cellular migration ability than the parental CL1-0 cells with less PD-L1 expression. CL1-0 cells with PD-L1 overexpression had more expression of EMT (epithelial mesenchymal transition) regulator and mesenchymal marker. Since intrinsic PD-1 receptor functions promote tumor growth was reported, we will investigate the interaction between PD-1 and PD-L1 in lung adenocarcinoma cell lines, the impact on chemosensitivity, and clinical outcome.
In vitro experiments, lung adenocarcinoma CL1-5 cells, derived from CL1-0 cells. We prepared PD-L1-overexpression human lung adenocarcinoma cell line, derived from CL1-0 cells (CL1-0-PD1). Migration and invasion ability were assessed by transwell assay; EMT marker and regulator were evaluated by Western blotting. We also observed the morphology of cells. Further, we evaluated cellular proliferation and chemosensitivity by MTT assay and colony formation assay. We correlate PD-L1 expression in lung cancer cells with clinical outcome by IHC stain clinically.To explore interaction between PD1 and PD-L1, we added anti-PD-1 antibody into CL1-0, CL1-5, and CL1-0-PDL1 cells, and then test migration, invasion and cellular morphology. We also suppressed PD-1 by siRNA to test whether PD-1/PDL-1 interaction contributed to the EMT change.
Our study confirmed that PD-L1 up-regulated cell migration and invasiveness in human lung adenocarcinoma cells and promotes EMT. We observed that CL1-5 and CL1-0-PDL1, which had more PD-L1 expression, are shaped like spindles; while CL1-0 cells are more rounded. PD-L1 expression also decreased cellular proliferation and had little influence on chemsensitivity. Finally, we found that higher PD-L1 expression was correlated with lymph node metastasis in clinical specimen.
After adding anti-PD1 antibody in CL1-5, CL1-0, and CL1-0-PDL1 cells, migration and invasion ability decreased. These result indicated anti-PD-1 antibody block the link between PD-1 and PD-L1 in cancer cells. The phenomenon was confirmed by PD-1 siRNA. Therefore, PD-1/PD-L1 axis regulated cancer cells migration and invasiveness.
Taken together, our study showed lung adenocarcinoma cells with higher PD-L1 expression promote cell migration, invasiveness, EMT, and little chemoresistance. PD-L1 expression lowers proliferation rate. PD-1 and PD-L1 interaction on lung adenocarcinoma cells contribute cellular migration and invasiveness.

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