Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 May 23;24(1):633.
doi: 10.1186/s12885-024-12390-8.

PD-L1 knockdown suppresses vasculogenic mimicry of non-small cell lung cancer by modulating ZEB1-triggered EMT

Wenjuan Li #  1   2 , Jiatao Wu #  1   2 , Qianhao Jia #  2 , Yuqi Shi  2 , Fan Li  1   2 , Linxiang Zhang  1   2 , Fan Shi  2 , Xiaojing Wang  3 , Shiwu Wu  4   5   6
Affiliations

PD-L1 knockdown suppresses vasculogenic mimicry of non-small cell lung cancer by modulating ZEB1-triggered EMT

Wenjuan Li et al. BMC Cancer. .

Abstract

Background: PD-L1 overexpression is commonly observed in various malignancies and is strongly correlated with poor prognoses for cancer patients. Moreover, PD-L1 has been shown to play a significant role in promoting angiogenesis and epithelial-mesenchymal transition (EMT) processes across different cancer types.

Methods: The relationship between PD-L1 and vasculogenic mimicry as well as epithelial-mesenchymal transition (EMT) was explored by bioinformatics approach and immunohistochemistry. The functions of PD-L1 in regulating the expression of ZEB1 and the EMT process were assessed by Western blotting and q-PCR assays. The impact of PD-L1 on the migratory and proliferative capabilities of A549 and H1299 cells was evaluated through wound healing, cell invasion, and CCK8 assays following siRNA-mediated PD-L1 knockdown. Tube formation assay was utilized to evaluate the presence of VM structures.

Results: In this study, increased PD-L1 expression was observed in A549 and H1299 cells compared to normal lung epithelial cells. Immunohistochemical analysis revealed a higher prevalence of VM structures in the PD-L1-positive group compared to the PD-L1-negative group. Additionally, high PD-L1 expression was also found to be significantly associated with advanced TNM stage and increased metastasis. Following PD-L1 knockdown, NSCLC cells exhibited a notable reduction in their ability to form tube-like structures. Moreover, the levels of key EMT and VM-related markers, including N-cadherin, MMP9, VE-cadherin, and VEGFA, were significantly decreased, while E-cadherin expression was upregulated. In addition, the migration and proliferation capacities of both cell lines were significantly inhibited after PD-L1 or ZEB1 knockdown.

Conclusions: Knockdown PD-L1 can inhibit ZEB1-mediated EMT, thereby hindering the formation of VM in NSCLC.

Keywords: Epithelial-to-mesenchymal transition; Non-small cell lung cancer; PD-L1; Vasculogenic mimicry; ZEB1.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
PD-L1 was upregulated in NSCLC cells and related to unfavorable prognosis in NSCLC patients. (A and B) Western blotting analysis compared PD-L1 levels between NSCLC cells and normal lung epithelial cells. (C and D) IHC analysis of PD-L1, PAS-CD31, used to detect the presence of VM in NSCLC tissue (black arrows: endothelium-dependent vessels, PAS-CD31+; red arrows: vasculogenic mimicry, PAS + CD31-; magnification, ×400). (E and F) Patient survival analysis demonstrated varying OS and DFS rates depended on PD-L1 expression levels
Fig. 2
Fig. 2
PD-L1 knockdown attenuated VM formation in vitro. (A) VM formation ability was assessed in all of three types cells. (B and C ) The efficacy of PD-L1 knockdown using siRNA was evaluated through Western blotting and q-PCR. (D) The correlation between angiogenesis and PD-L1 levels was analyzed using the TCGA-Lung Adenocarcinoma (TCGA-LUAD) dataset, which comprised 465 samples. (E and F) VM-related genes’ expression in mRNA and protein levels was assessed through Western blotting and q-PCR analyses. (G) The number of VM structures decreased considerably following PD-L1 siRNA transfection
Fig. 3
Fig. 3
Proliferation, invasion and metastasis of A549 and H1299 were inhibited with PD-L1 knockdown. (A) CCK-8 assays assessed the proliferation capacity of A549 and H1299 following PD-L1 knockdown. (B) Flow cytometry was conducted to explore the rate of apoptosis in A549 and H1299 after transfection of siPD-L1. (C, D and E) Down-regulation of PD-L1 drastically inhibited migration and invasion ability in A549 and H1299 cells, as observed in all three assays (migration, invasion, and scratch healing)
Fig. 4
Fig. 4
PD-L1 exhibited a relationship with ZEB1 and the EMT process. (A and B) Bioinformatic analysis revealed a correlation between PD-L1 and ZEB1 expression, as well as other EMT-related genes. (C and D) The expression of ZEB1 and EMT-related markers was assessed using Western blotting and q-PCR analysis in NSCLC cells transfected with PD-L1 siRNA
Fig. 5
Fig. 5
Cell lines construction and ZEB1 knockdown decreased EMT markers expression and VM structures formation in vitro. (A, B and C) Western blotting and q-PCR analysis of PD-L1 knockdown efficiency of shRNA. (D and E) Immunohistochemistry was employed to detect the presence of VM in both the ZEB1-positive and ZEB1-negative groups (magnification, ×400). (F) ZEB1 knockdown reduced the VM structure formation in vitro. (G and H) EMT-associated markers in NSCLC cells transfected with shRNA targeting ZEB1 were assayed at the protein and mRNA levels using Western blotting and q-PCR. (I) CCK8 assay demonstrated that the proliferation of NSCLCL cells was inhibited following ZEB1 knockdown. (J, K and L) Results from scratch healing assay, migration, and invasion experiments consistently demonstrated significant suppression of migratory and invasive capacities in A549 and H1299 cells upon ZEB1 knockdown
Fig. 6
Fig. 6
The distribution of PD-L1 exhibits concordance with the ZEB1 distribution. (A) Exploration of ZEB1 expression in distinct PD-L1 groups through immunohistochemistry. (B) Immunohistochemistry exhibited a remarkable concordance in the distribution patterns of PD-L1 and ZEB1 (magnification, ×200)
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Chen P, Liu Y, Wen Y, Zhou C. Non-small cell lung cancer in China. Cancer Commun (Lond) 2022;42:937–70. doi: 10.1002/cac2.12359. - DOI - PMC - PubMed
    1. Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66:115–32. doi: 10.3322/caac.21338. - DOI - PubMed
    1. Luo YH, Chiu CH, Scott Kuo CH, et al. Lung Cancer in Republic of China. J Thorac Oncol. 2021;16:519–27. doi: 10.1016/j.jtho.2020.10.155. - DOI - PubMed
    1. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023;73:17–48. doi: 10.3322/caac.21763. - DOI - PubMed
    1. Sung H, Ferlay J, Siegel RL et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021; 71: 209–249. - PubMed

MeSH terms