Nintedanib (BIBF 1120) blocks the tumor promoting signals of lung fibroblast soluble microenvironment

Rationale: Nintedanib is a potent, triple angiokinase inhibitor of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor, and has been recently approved for the treatment of non-small cell lung cancer (NSCLC), following first-line chemotherapy. It is well established that microenvironment plays an important role in tumor progression. Therefore, targeting tumor microenvironment-cancer cell interaction may provide a significant therapeutic target. In this study we tested the effect of Nintedanib on NSCLC cells directly and in the presence of normal and tumor soluble microenvironment. Methods: Primary fibroblast cultures derived from NSCLC tumors and normal lung tissues were established and their supernatants were collected. These supernatants were added to NSCLC cell lines (H1299, H460 and A549) cultured with/without Nintedanib (0.1-10 μM) for 24 and 48 h. Cell death (AnnexinV-PI, flow-cytometry), cell number, proliferation (PCNA), protein expression (immunoblotting) and cell migration (scratch test), were tested. Expression of 10 pro-angiogenic cytokines was measured by ELISA-based quantitative array. Results: Tumor and normal supernatants demonstrated similar pro-metastatic effects on the NSCLC phenotype: both elevated cancer cell number, PCNA levels, reduced total and apoptotic cell death and facilitated cell migration. Nintedanib had limited but significant effects on the NSCLC cell number, cell death and migration, but required high doses. However, at lower doses Nintedanib caused cell detachment and elevated integrin-alpha 5 and EGFR levels, both markers of anoikis resistance. This suggests them as possible targets in combination with Nintedanib. Moreover, Nintedanib completely blocked the supernatants ability to facilitate the aggressive cancer cell characteristics. While cytokine array analysis showed no significant changes in FGF, PDGF or VEGF, we found that both supernatants contained high HGF levels, suggesting it as the facilitator of cell migration and proliferation. Conclusion: Our results demonstrate that tumor microenvironment-cancer cell interaction is a therapeutic target and should be considered when new drugs are tested.