DOI: 10.5061/dryad.3n5tb2rp6
title: BEHAV3D: A 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response

description: The use of patient-derived material and immune cell co-cultures in
 modeling immune-oncology has gained significant interest for understanding
 and manipulating immune cell tumor targeting in a patient-specific
 context. However, current protocols have limitations in visualizing and
 analyzing the dynamic cellular features of these living culture systems.
 We recently developped a workflow names BEHAV3D,  that combines
 multi-color live 3D imaging and computational tools to analyze cell death
 dynamics, classify T cell behavior, and generate data-informed 3D images
 and videos. Here we provide some example pre-processed dataset of videos
 of two co-culture set ups: breast cancer Patient Derived Organoids with αβ
 T cells engineered to express a γδ TCR (TEGs) and Acute Lymphoblastic
 Leukemia cells with CD19 CART cells.
description: Our approach involves the development of a co-culture assay that
 allows for 3D live imaging to simultaneously monitor tumor cell response
 and T cell dynamic behavior. We tested and designed different combinations
 of live cell dyes to label viable, dead cells, and up to two types of
 immune cells. This method does not require genetic manipulation or the use
 of fluorescent markers, making it fast, widely applicable, and preserving
 cellular heterogeneity. We also determined the optimal proportions of
 tumor cells to therapeutic cells for solid and liquid tumors, and
 optimized the imaging medium to ensure compatibility with live
 imaging. For image acquisition was performed on a Zeiss
 LSM880 confocal system. This allows for the acquisition of four spectrally
 resolved fluorophores in a single scan, enabling fast imaging with minimal
 photobleaching and phototoxicity. Image processing is performed with
 Imaris (Oxford Instruments) sofware and an image processing pipeline was
 developed to extract statistics on cellular and organoid tracks. Single
 cells and tumor cells are segmentated and tracked in these example
 datasets.
description: Applications used: Imaris file converter, Imaris 9.2 or higher,
 Imaris Tracking module and Imaris XT module.
description: # Title of Dataset: This dataset contains example imaging datasets
 acquired with the pipeline BEHAV3D on Zeiss LSM880, in the related
 manuscript (Alieva et al, Nat Prot, in revision, 2023). These datasets
 exemplify the image processing procedure that has to be followed to
 segment and track cells. The extracted data can be processed with
 https://github.com/AlievaRios/BEHAV3D/ ## Description of the Data and file
 structure Two datasets are provided exemplifying two different
 experimental set ups and co-culture preparation, see below.
 **LSM880\_example\_dataset\_TEG\_BC\_organoids\_2019\_06\_26 dataset**
 This dataset consists of a co-culture of engineered T cells called TEGs in
 co-culture with breast tumor derived organoids. Both cell types are human.
 Different cells are marked with different colors as follows: -Channel 1:
 efluor 450 labels CD8+ TEGs -Channel 2: NucRed/TOPRO3 marks all live cells
 -Channel 3: NucRed/TOPRO3 marks all dead cells -Channel 4: Calcein AM
 marks CD4+ TEGs. **LSM880\_example\_dataset\_CART\_ALL\_Exp28 dataset**
 This dataset consists of a co-culture of engineered T cells called CART in
 co-culture with Acute lymphoblastic Leukemia cells. Both cell types are
 human. Different cells are marked with different colors as follows:
 -Channel 1: CellTrackerGreen marks CD4+ CART -Channel 2: efluor 450 labels
 CD8+ CART -Channel 3: NucRed/TOPRO3 marks all live cells -Channel 4:
 NucRed/TOPRO3 marks all dead cells ## Sharing/access Information Links to
 other publicly accessible locations of the data: Was data derived from
 another source? If yes, list source(s): Unprocessed data can be found
 here: https://www.ebi.ac.uk/biostudies/studies/s-biad448