Abstract A49: Anti-DLL4 antibodies inhibit cancer stem cells in small cell lung cancer.

Small cell lung cancer (SCLC) is a very aggressive lung cancer with features suggesting enrichment in cancer stem cells (CSCs). Delta-like ligand 4 (DLL4) is a membrane bound ligand for Notch receptors critical for functional angiogenesis. Blocking DLL4 signaling increases the density of nonfunctional blood vessels and hypoxia of tumors, and thereby inhibits growth of tumor xenografts in mice. In addition, growing evidence implicates DLL4 Notch signaling pathway in the maintenance of CSCs. Here we investigated the anti-CSC activity of anti-DLL4 mAbs using in vitro and in vivo models of SCLC. Biochemical and flow cytometry analyses revealed that multiple SCLC cell lines express DLL4, and notably, SCLC spheres cultured under CSC-enriching conditions express higher levels of DLL4. Prior to determining the ability of anti-DLL4 mAb in inhibiting CSC in vivo, we first characterized the phenotype of CSCs in the SCLC cell line NCI-H69. NCI-H69 cells enriched for high CD133 expression were more tumorigenic and expressed higher levels of Nanog, Oct 3/4, and EZH2, which are genes crucial for maintenance of CSCs, than cells with low or negative levels of CD133. Moreover, RNA level of DLL4 was found to be three fold higher in CD133 high cells compared to CD133 low cells, suggesting that expression of DLL4 correlates with the CSC phenotype. In vivo, an anti-DLL4 antibody leads to inhibition of NCI-H69 tumor growth when the antibody is administered as a single agent, or in combination with cisplatin+etoposide or topotecan, chemotherapy agents commonly used in treatment of SCLC. To determine if blockade of DLL4 inhibits CSCs in vivo, NCI-H69 tumor xenografts treated with anti-DLL4 mAb were analyzed by flow cytometry. A subset of dissociated tumor cells expressed CD133 and DLL4, and a reduction of these populations of cells was observed in the anti-DLL4 antibody treated groups. In summary, these studies highlight that, in addition to vascular expression, (1) DLL4 is frequently expressed in SCLC cells, (2) DLL4 expression correlates with a CSC phenotype, and (3) that DLL4 blockade using an anti-DLL4 mAb results in inhibition of CSCs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A49. Citation Format: Patrick Strout, Martin Korade, Ching Ching Leow, Ivan Inigo, Suneetha Thomas, Elaine Hurt, Jon Chesebrough, Adeela Kamal, Song Cho. Anti-DLL4 antibodies inhibit cancer stem cells in small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A49.