Macrophages can exhibit a spectrum of activation states ranging from a pro-inflammatory and antitumorigenic M1 (CD80+; IL-12high; iNOShigh) to a pro-tumorigenic M2 [CD163+; IL-10high; CD206+; Arg+ (mouse)] phenotype. Plasminogen activator inhibitor-1 (PAI-1) is overexpressed in many cancers and although it acts as a protease inhibitor it is paradoxically correlated with poor outcome. PAI-1 exerts its pro-tumorigenic role via pro-angiogenic and anti-apoptotic functions. Increasing evidence points towards the importance of PAI-1 in inflammation and tumorigenesis, but its influence on the immune component of the tumor microenvironment has not yet been investigated. Here we report a novel observation that PAI-1 is a regulator of macrophage polarization. We observe that in PAI-1 KO mice xenotransplanted with 3 human cancer cell lines (HT1080, A549, HCT116) in which PAI-1 was knocked down (KD), tumors were less infiltrated with macrophages with lower Arg expression compared to WT mice implanted with PAI-1 pos. tumors. We demonstrate that recombinant PAI-1 through its uPA interactive domain increased the expression of M2 polarization markers (CD163, IL-10) and decreased or did not affect the expression of M1 polarization markers (iNOS, CD80) in human peripheral blood monocytes. Further investigation demonstrated that treatment of monocytes with PAI-1 induced a rapid (2 h.) increase in IL-6 mRNA and secretion (4 h.) of the protein that was followed by phosphorylation (8-24 h.) of signal transduction and activation of transcription 3 (STAT3) in monocytes. Further linking STAT3 activation to PAI-1, we observed a decreased pSTAT3 in monocytes co-cultured with HT1080 cells upon PAI-1 downregulation. We found that STAT3 activation was downstream of IL-6 and responsible for M2 polarization, as the blockage of the IL-6 receptor with a function blocking antibody (tocilizumab) or inhibition of STAT3 activation by a JAK2/STAT3 inhibitor (ruxolitinib) prevented M2 polarization of monocytes by PAI-1. The contribution of PAI-1 to M2 polarization of macrophages was tested in PAI-1 KO mice implanted with human tumors in which PAI-1 expression was controlled by doxycycline. These experiments demonstrated that induction of PAI-1 in established tumors increased the presence of tumor associated macrophages (TAM) (F4/80+) and decreased the presence of M1 (iNOS+) TAM. Further supporting the presence of an autocrine PAI-1/IL-6 pathway in macrophage polarization in human cancers, a meta-analysis of gene expression array data indicated strong correlations between PAI-1 and IL-6 expression in breast (P=<0.05) and colon (P<0.01) cancers and between PAI-1 and CD163 in colon cancer (P<0.0001). The data thus identify a new pro-tumorigenic function for PAI-1 in the communication between tumor cells and macrophages where tumor-derived PAI-1 activates an autocrine IL-6/STAT3 pathway that promotes M2 polarization.

Citation Format: Marta H. Kubala, Veronica R. Placencio, Yves A. DeClerck. Tumor-derived PAI-1 promotes macrophage M2 polarization by stimulating an autocrine IL-6 /STAT3 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3061. doi:10.1158/1538-7445.AM2017-3061