Background: A recent study reported that tumor-infiltrating neutrophil (TIN) has potential on malignant tumor progression leading to metastasis. In our study, to control the effect of TIN, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) was used. PLAG has shown its efficacy in modulating the neutrophil counts in chemotherapy-induced neutropenia by attenuating neutrophil extravasation via down-regulation of adhesion molecules, inflammatory cytokines, and chemokines. In this study, we investigated whether PLAG has an anti-metastatic effect on A549 lung cancer orthotopic implantation model via modulating TIN. Methods: To investigate the anti-metastatic effect of PLAG on lung cancer metastasis, RFP-labeled A549 cells were implanted into the lungs of BALB/c nude mice via intratracheal injection and bred for 12 weeks. After 6 weeks from tumor implantation, PLAG with three concentrations (25/50/100 mpk) were daily administrated for another 6 weeks. Each image of the growth of the primary tumor and colonization of metastatic tumor to other sites were acquired using CT and IVIS. TIN was analyzed in the primary tumor using immunohistochemistry (IHC). To identify the mechanism of PLAG on preventing the neutrophil-associated metastasis of cancer cells, direct or non-direct co-culture methods were used. Results: In the orthotopic implantation model, metastatic tumors to GI-track and brain were detected by IVIS. It was confirmed that metastasis to GI-track was decreased by 73% in 100 mpk PLAG-treated group, while metastasis to the brain was decreased by about 92% in PLAG-diet compared with positive control. Primary lung tumor growth was retarded by PLAG in a dose-dependent manner. The size of the primary tumor was much smaller than the positive control and alveolar tissue was similar to that of normal mice in 100 mpk PLAG-diet. In contrast, massive neutrophil infiltrations in positive and delivery groups, but significantly reduced upon PLAG administration. Moreover, p-EGFR was concomitantly detected with a similar pattern of neutrophil infiltration. During the direct contact in vitro assay, the A549 lung cancer spheroid cells were scattered by neutrophils, but it was effectively hindered by PLAG treatment. Neutrophil-stimulated cancer in the non-direct contact method showed enhanced metastatic activity. However, such a phenomenon was significantly reduced with the PLAG-treated group. Moreover, neutrophil stimulated the expression of EMT markers, whereas it was effectively down-regulated by PLAG administration. Transactivation between tumor cells and neutrophil was mediated by neutrophil elastase (NE). NE binds to and activates protease-activated receptor 2 (PAR2) on cancer cells and subsequently activated EGFR for metastasis to occur by cleaving HB-EGF through the ARR2/clathrin complex. On the other hand, the suppression of metastasis by PLAG was mediated through the regulation of the NE-PAR2 pathway and acceleration of intracellular trafficking and degradation of PAR2. Conclusion: In this study, TIN was observed in the primary lung tumor and metastasis into GI-track and brain, but PLAG dramatically reduced infiltration of neutrophil. The collective results suggest that TIN educates tumor cells for enhancing metastasis by EGFR transactivation. However, PLAG effectively interfered the transactivation. Having not met the medical needs for treating metastasis, PLAG may be a promising candidate as an anti-metastatic drug by modulating the TIN.

Citation Format: GUEN TAE KIM, SUN YOUNG YOON, KI-YOUNG SOHN, MYUNG-HWAN KIM, JAE WHA KIM. Anti-metastatic effect of PLAG via interference of neutrophil elastase/PAR2/EGFR signaling on A549 lung cancer orthotopic implantation model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C037. doi:10.1158/1535-7163.TARG-19-C037