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Emulate recently presented a webinar discussing a novel workflow that models the unique and critical mechanisms of CAR T-cell trafficking, recruitment, migration, and killing in a unified Organ-Chip assay. The assay and workflow were designed to help researchers develop new mechanisms to interrogate CAR T cells and discover new pathways to improve the efficacy of these cells in solid tumors.
The vast majority of cancer deaths are associated with solid tumors. Yet, efforts to adapt CAR T-cell therapy to treat solid tumors have proven challenging. Solid tumors are extremely heterogeneous and express a myriad of antigens, making targeting difficult. The immune-suppressing microenvironment and CAR T-cell exhaustion also play major roles.
“But the T cells have to reach the tumor first,” said Anita Mehta, PhD, Principal Scientist and Immunology Team Lead at Emulate. CAR T cells need to migrate out of the bloodstream and make a long, demanding, well-orchestrated journey.
“To effectively model what is happening in vivo, we have to understand critical components of T-cell recruitment and migration into the tumor,” Mehta noted.
The Workflow
Emulate Organ-Chips, which were used to model this entire journey, have a top and bottom channel separated by a porous membrane. “In the top channel, we seed tumor cells, and in the bottom channel, we seed tissue-relevant endothelial cells” Mehta detailed. “To develop the workflow, Emulate used non-small cell lung cancer (NSCLC) cells, A549 cells, which express HER2 as a target antigen. Expression of the HER2 antigen was analyzed by flow cytometry, and the mean fluorescence intensity demonstrated expression of HER2 but not CD19 (a target antigen for blood cancer).
“At 24 hours prior to CAR T-cell administration, the bottom channel is primed with cytokines, and the top channel with tumor-relevant chemokines. The cytokines prime the endothelial cells to express adhesion molecules, which are critical for the CAR T cells that we flow through the Organ-Chip at a high rate to attach in an inflammation-specific manner.”
Once administered in the vascular channel, HER2+ CAR T cells attach to the adhesion molecules and travel through the membrane into the top channel, where they recognize the antigen expressed by the tumor cells and begin antigen-specific killing.
Confocal imaging was coupled with image quantification to analyze the complex datasets. “You can see the attachment of the CAR T cell, either in the endothelium, inside the membrane, or in the epithelial
channel, to quantify attachment and migration,” Mehta said.
To demonstrate the antigen-specific killing response, CD19+ CAR T cells were administered as a negative control and recruited into the top channel but did not kill the tumor cells. Meanwhile, the HER2+ CAR T-cell killing of the tumor cells, with the expressed antigen, was visually striking, facilitating quantification.
“The assay allows you to track the killing in real time or in a kinetic manner,” Mehta said. “This is a powerful tool to understand different CAR designs and provides mechanisms of action over time to enhance efficacy.”
IL-2 was used experimentally as a co-treatment. Although IL-2 did not affect recruitment significantly, the fitness of the CAR T cells was drastically improved. They were capable of killing more tumor cells compared to a monotherapy approach.
Summary
The robust, validated, and flexible CAR T-cell Organ-Chip workflow for solid tumors allows modeling of CAR T-cell vasculature attachment, recruitment, and killing in one assay. Co-therapeutics testing, CAR T-cell extraction, and immunophenotyping analysis can also be accomplished, and the complexity of the model can be expanded as experimentally desired.
The model was built using Emulate’s Basic Research Kits. The protocol provides guidance on how to adapt the workflow to specific needs.
To view the webinar, visit: emulatebio.com.
