Abstract 1501: Employing 3D-ACT platform to assess the ability of stromal-targeting agents to improve penetration and efficacy of cell therapy
Authors: Tong Zhisong , Iwanowycz Stephen , Ehrhart Jared , Pabón Mibel M. , Pastoor Tina , Altiok Soner 
Published: July 2021

Background: Adoptive T cell therapy (ACT) has demonstrated great clinical success for the treatment of hematological malignancies. However, the unique challenges presented by solid tumors has significantly limited their utility. The primary characteristic of an effective ACT is the ability to selective identify tumor cells. In case of solid tumors, the ACT faces the additional hurdle requiring penetration into the tumor microenvironment (TME) while retaining function. Tumors develop highly immunosuppressive stroma which serves as a mechanical hinderance for the penetration and function of activated effector cells. In this study we used our 3D-ACT platform to investigate the ability of focal adhesion kinase (FAK) inhibitors to enhance the penetration of engineered cells into the intact patient tumor stroma and their efficacy within the tumor immune microenvironment.

Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma (RCC), and colorectal carcinoma (CRC) using proprietary technology developed at Nilogen Oncosystems. Engineered T cells were labeled with different cell tracker fluorescent dyes to monitor cell movements and locations within tumoroids by confocal analysis. 3D tumoroids were treated with vehicle only or FAK inhibitors and exposed to CAR-T cells at 10:1 E:T ratio. Cell penetration within tumoroids and tumor cell killing was measured by high-content confocal imaging combined with 3D-image analysis. ACT cell activation was monitored using multiparameter flow cytometry analysis and multiplex cytokine release assays to assess changes in the TME.

Results: Our studies demonstrated that the confocal-based high-content real time imaging platform, combined with custom image analysis algorithms, allowed for monitoring of treatment-mediated tumor cell killing with structural analysis of engineered T-cells in the intact 3D tumoroids. Our data revealed that CAR-T cell penetration into tumoroids greatly varied between different tumor types and was heavily influenced by the tumor's stromal components. Treatment with FAK-inhibitors led to alterations within the TME, which subsequently impacted penetration and efficacy of CAR-T cells as assessed by flow cytometry and confocal imaging.

Conclusion: The ex vivo model described here (3D-ACT) retains the TME and replicate the heterogeneity of tumor stroma to provide provides a clinically relevant platform to evaluate the influence of the tumor stromal components on engineered T cell functions. We believe the 3D-ACT platform can effectively be used to screen for drugs that can promote ACT penetration into the tumor stroma and survival within the tumor microenvironment.

Citation Format: Zhisong Tong, Stephen Iwanowycz, Jared Ehrhart, Mibel M. Pabón, Tina Pastoor, Soner Altiok. Employing 3D-ACT platform to assess the ability of stromal-targeting agents to improve penetration and efficacy of cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1501.