Abstract 4224: The application of sophisticated image analysis to a T cell killing assay in 3D deciphers the dynamics of the different components of the tumor microenvironment
Authors: Kerwin Ryan , Rohleff Ina , Blanchard Martin , Lashuk Kanstantsin , Walker Bryan , Schueler Julia B. 
Published: March 2024

Abstract
We developed an in vitro protocol to generate tumor-specific cytotoxic lymphocytes (CTL) by priming T cells from healthy donors on HER2+ breast cancer cell line SKBR-3. Subsequently, these primed CTLs were tested in a 3D immune cell killing assays in a life cell imaging device. An image analysis workflow was applied to understand the T cell movement in spatial relation to the tumor and the kinetic of tumor cell killing. The activity of the CTLs was compared with non-activated CD8+T cells, staurosporin and CD8+ T cells, unspecifically activated by αCD2/αCD3/αCD28 beads. The tumor cells were expressing mKate whereas the T cells were label-free. By analyzing a combination of different masks, we could computationally measure tumor spheroid characteristics and dynamic attributes such as changes in shape, percent of cells expressing red fluorescentce, and relative average distance of T cells from the spheroid in each timelapse assay. This process was applied at each frame to determine the average T cell distance from its corresponding spheroid center and subsequently calculate the average T cell flux over the course of each assay by linking data between image frames. Additional measurements allowed for the quantification of tumor debris accumulation over time both relative to the primary tumor mass and in absolute terms. Our analyses revealed that bead-activated T cells approached the tumor spheroid with the highest speed and infiltrated the spheroid homogeneously. In contrast the CTLs approached the spheroid slower and started tumor cell killing from the rim of the spheroid towards the center. The reduction of spheroid area over time displayed a very similar kinetic when co-cultured with CTL or bead-activated T cells. At the end of the experiment the CTLs induced a more pronounced shrinkage of spheroid size. Interestingly the size of the spheroid and the red fluorescence intensity were not necessarily aligned. The positive control Staurosporin induced a fast reduction of the mKate signal, whereas the spheroid area did not change over time. In summary, the bead-activated T cell approached the spheroid fast, infiltrated the complete area and induced tumor cell killing homogenously across the tumor area. The CTL approached the spheroids slower but started killing earlier from the outside towards the center of the spheroid. The observed differences in infiltration and killing characteristics of the investigated T cell types lead to a better understanding of T cell biology in the context of the tumor biology, which can be translated into applications during dug development. Taken together, with the applied image analysis algorithm we were able to deconvolute the biological processes in the assay in much more detail. The additional read-outs increase the translational value of the assay tremendously in the context of drug development as well as precision medicine.
Citation Format: Ryan Kerwin, Ina Rohleff, Martin Blanchard, Kanstantsin Lashuk, Bryan Walker, Julia B. Schueler. The application of sophisticated image analysis to a T cell killing assay in 3D deciphers the dynamics of the different components of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4224.