Background: Neuroendocrine tumors (NETs) are among the most understudied types of human cancer. This is partially a result of their uncommon occurrence, but is mainly related to the lack of models that represent the human disease. Spheroid cultures effectively recapitulate the physiology of these tumors. For the current treatment of patients with NETs everolimus, an mTOR inhibitor, is commonly used, but with limited efficacy. A means by which to enhance the response of NETs to everolimus is a great clinical need. Therefore, we sought to determine the potential of the proapoptotic agent ABT263 (a BCL-2, BCL-xL, and BCL-W inhibitor) to enhance the response to everolimus in NET spheroids.
Methods: NET spheroids derived from patient biopsies or surgical samples (n=6) were digested, plated for spheroid culture in Matrigel, and covered with media supplemented with epidermal growth factor. Cultures were treated with control (normal feeding media), 200 nM everolimus, 250 nM ABT263, or the combination. Brightfield images (4x) of the spheroids were taken pre- and 48 hours post-treatment and changes in spheroid diameter measured. Additionally, optical metabolic imaging was performed, examining the inherent auto-fluorescence of NADH and FAD+, as a marker of the metabolic activity of the cancer cells and an optical redox ratio measured. Spheroid culture whole mounts were stained pre- and post-treatment using immunofluorescence.
Results: A total of 5 different patient-derived NET lines were generated from surgical specimens. In all instances the Ki67 index and synaptophysin/chromogranin A staining demonstrated that these cultures are similar to the tumors from which they are derived. Everolimus and the combination of everolimus and ABT263 resulted in a significant reduction in median spheroid diameter compared to the control and ABT263-only groups in 3 of the 5 NET spheroid lines with a change in median spheroid diameter of -30.16%, -15.94%, and -3.34% (p<0.001). Optical metabolic imaging demonstrated similar efficacy to that seen with the diameter measurements with significant responses especially in the setting of the combination treatment. Reduction of cell proliferation and induction of apoptosis in the combination treatment were both confirmed using immunofluorescence imaging for Ki-67 and cleaved caspase 3, respectively.
Conclusion: This study establishes the utility of NET spheroids as a more representative model of this otherwise difficult to study cancer. The addition of ABT263 to everolimus enhances the treatment response for a subset of NETs. Future investigations will identify the molecular subset most likely to respond and explore this combination regimen clinically.
Citation Format: Christopher P. Babiarz, Amani Gillette, Mitchell G. Depke, Cheri A. Pasch, Susan N. Payne, Carley M. Sprackling, Linda Clipson, Kristina A. Matkowskyj, Melissa C. Skala, Dustin A. Deming. Everolimus in combination with ABT263 in patient-derived organotypic neuroendocrine tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5019.