Abstract LB-218: Validation of a novel microfluidic device for screening of immune checkpoint inhibitors using 3D organotypic tumor spheroids
Authors: Ivanova Elena , Aref Amir , Jenkins Russel , Lizotte Patrick , Huang Wei , Palakurthi Sangeetha , Kirschmeier Paul , Barbie David , Paweletz Cloud , Janne Pasi , Wong Kwok-Kin 
Published: July 2017

Abstract
Immune checkpoint blockade, including anti-PD-1 and PD-L1 therapies, is revolutionizing cancer care for melanoma, lung and other malignancies. However, pre-clinical models in which to rapidly and robustly evaluate the efficacy of these treatment approaches, including the development of combination therapies and/or relevant biomarkers, are lacking. We have developed a novel approach for evaluating ex vivo response to immune checkpoint blockade using murine- and patient derived organotypic tumor spheroids (mDOTS/pDOTS) cultured in a new 3D microfluidic system. We have demonstrated that spheroids isolated from fresh mouse and human tumor samples retain autologous lymphoid and myeloid cell populations.
Methods: Using MC38 and B16F10 syngeneic mouse cancer models with reported responsiveness or resistance to PD-1 blockade respectively, we treated mDOTS in the 3D microfluidic device with either control IgG or increasing concentrations of anti-PD-1 for up to 6 days and quantified cell viability by dual labeling fluorescence microscopy. To confirm tumor cell-specific death and survival of mDOTS immune component we applied multi-color immunofluorescent staining of CD8 as well as CD11b+ and CD11c+ myeloid cells in parallel with viability dye calcein AM. Tumor cell-specific death was assessed by absence of calcein AM and pyknotic nuclear staining in CD45 negative mDOTS component.
Results: We demonstrated dose-dependent killing following treatment with anti-PD-1 in MC38 spheroids. We showed presence of viable cytotoxic lymphocytes as well as other immune cells for up to 5 days of ex vivo growth, when we observed the beginning of tumor but not immune cell killing. In contrast, mDOTS derived from B16F10 melanoma, which is reported to be minimally sensitive to single agent PD-1 blockade, exhibited little cell death despite identical treatment and comparable immune profiles (at highest anti-PD-1 dose of 10ug/mL viability was 98.2%+0.04 versus 29.7%+1.68 in MC38) . No significant cell death (viability 88.2%+2.18) was also noted in spheroids generated from MC38 cell line alone, conceivably due to the absence of autologous cytotoxic T cells. Additional analysis of immune dynamics will be discussed.
Conclusions: These data demonstrate the ability to recapitulate sensitivity and innate resistance to PD-1 blockade ex vivo. By being able to study the impact of immunotherapy and targeted therapy in a rapid ex vivo system the development of drug combinations and their associated biomarkers can be significantly enhanced. The preclinical studies can be used to develop and guide the next generation clinical trials for lung cancer patients, and hopefully improving their success rate.
Citation Format: Elena Ivanova, Amir Aref, Russel Jenkins, Patrick Lizotte, Wei Huang, Sangeetha Palakurthi, Paul Kirschmeier, David Barbie, Cloud Paweletz, Pasi Janne, Kwok-Kin Wong. Validation of a novel microfluidic device for screening of immune checkpoint inhibitors using 3D organotypic tumor spheroids [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 LB-218. doi:10.1158/1538-7445.AM2017-LB-218