Shinjiro Sakamoto

Supplementary Figure S11 shows schematic illustration of our study.

Immune checkpoint inhibitors (ICI) have improved the prognosis of patients with non–small cell lung cancer (NSCLC), but the cure rate remains low because tolerant persister cancer cells can survive within the tumor during ICI treatment. We have previously reported that plasminogen activator inhibitor-1 (PAI-1) is involved in tolerance acquisition to osimertinib in EGFR-mutated NSCLC. This study aimed to examine the role of PAI-1 in ICI tolerance and whether PAI-1 may be a therapeutic target to overcome this tolerance. In a mouse subcutaneous tumor model using Lewis lung carcinoma or KLN205 cells, cancer cells surviving within the tumor 7 days after anti–PD-1 (aPD-1) antibody treatment were defined as aPD-1 antibody–tolerant persister cells (aPD-1-TP). PAI-1 and mesenchymal gene expression levels were higher in aPD-1-TPs than in control cells. IHC analyses showed higher numbers of tumor-associated macrophages, expression of PD-L1 in cancer cells, and degree of angiogenesis. In contrast, the number of tumor-infiltrating lymphocytes was lower in aPD-1 antibody–tolerant tumors than in control tumors. Combination treatment with an aPD-1 antibody and the PAI-1 inhibitor, TM5614 decreased mesenchymal gene expression, PD-L1 expression, tumor-associated macrophage numbers, and angiogenesis and increased tumor-infiltrating lymphocyte counts in tolerant tumors. Furthermore, it resulted in prolonged inhibition of tumor growth. In conclusion, this study underscores the involvement of PAI-1 in the survival of aPD-1-TPs via epithelial–mesenchymal transition and alteration of the tumor microenvironment. Combination treatment with an aPD-1 antibody and TM5614 can be a new therapeutic strategy for NSCLC.

Supplementary Figure S9 shows tumor microenvironment of tumors composed of LLC and KLN205 cells.

Supplementary Figure S8 shows relationship of TGF-β secretion with TAMs and CAFs.

Supplementary Figure S4 shows that SK-216 significantly increases the antitumor efficacy of aPD-1.

Supplementary Figure S10 shows degree of angiogenesis in the tumors treated with aPD-1 antibody or aPD-1 antibody with TM5614.

Supplementary Figure S1 shows the experimental scheme for aPD-1 antibody-tolerant cells and the induction of PAI-1 expression during tumor regrowth.

Supplementary Figure S2 shows tumor expression level of PAI-1 in surgically resected NSCLC samples.

Supplementary Figure S5 shows tumor expression of mesenchymal and epithelial genes.

Supplementary Figure S3 shows PAl-1 protein levels in the culture medium of control and TM5614 groups, and Effect of TM5614 on the lung cancer cells.