Shandong Cancer Hospital Affiliated to Shandong First Medical University Jinan, Shandong
J. Wang, J. Liu, W. Wen, T. Zhong, X. Zhang, F. Sun, J. Yu, and D. Chen; Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shangdong, China
Purpose/Objective(s): Immune checkpoint blockade (ICB) therapies targeting programmed cell death-ligand 1 (PDL1) have been approved for the treatment of non-small cell lung cancer (NSCLC). However, the mechanisms of resistance causing these problems remain unclear. To gain a better understanding of the molecular biological mechanisms, we constructed a cellular model of PDL1 immunotherapy resistance. Materials/
Methods: This study involved the construction of anti-PDL1 immunotherapy-resistant lung cancer cell lines and a mouse subcutaneous tumor animal model. The percentage of immune cells in NSCLC tumor tissues and immunotherapy-resistant tumor tissues was compared using flow cytometry. The levels of various cytokines in mouse serum were measured using ELISA. Additionally, we screened for key molecules and signaling pathway changes in the immunotherapy resistance process using transcriptome sequencing (RNAseq), identifying CC motif chemokine ligand 20 (CCL20) as one of the key molecules. Additionally, we performed functional validation in in vivo and in vitro models through gene overexpression technique and intratumoral backfill of CCL20 recombinant protein. Results: Our study found that PDL1 immunotherapy resistance induces a suppressive tumor immune microenvironment in mice. There was a significant decrease in the proportions of CD3+, CD8+, Granzyme B+, IFN-, and CD8+CD44+CD62L- effector T cells in the lymphoid lineage. Furthermore, our findings indicate that PDL1 expression was downregulated in drug-resistant lung cancer cell lines. The results of transcriptome sequencing indicated that the expression of CCL20 was down-regulated in drug-resistant lung cancer cells. Further in vivo experiments showed that overexpression of CCL20 and intratumoral infusion of CCL20 recombinant protein effectively controlled the growth of PDL1-immunotherapy-resistant tumors in NSCLC. The analysis of single-cell sequencing data revealed the effects of CCL20 on the tumor microenvironment. Through protein interaction network analysis, a group of macrophages that scored the highest for interactions with CCL20 was identified. These macrophages highly expressed interferon-stimulated genes and tended to be M1-polarized. Additionally, CCL20 backfilling induced the expression of the macrophage surface molecule CCR5. Cellular communication has been shown to reveal enhanced Alcam-CD6 signaling. This is mainly due to elevated Alcam expression on macrophages. These results suggest that CCL20 may enhance anti-tumor immune responses and overcome PDL1 immunotherapeutic resistance. This is achieved by promoting macrophage M1 polarization as well as activation of CD8+ T cells through the Alcam-CD6 axis. Conclusion: Our study has revealed that the chemokine CCL20 plays a critical role in regulating the tumor microenvironment. CCL20 may activate CD8+ T cells through the Alcam-CD6 axis, enhancing anti-tumor immune responses and overcoming PDL1 immunotherapy resistance.
