A dual-luciferase bioluminescence system for the assessment of cellular therapies.

Bioluminescence imaging is a well-established platform for evaluating engineered cell therapies in preclinical studies. However, despite the discovery of new luciferases and substrates, optimal combinations to simultaneously monitor two cell populations remain limited. This makes the functional assessment of cellular therapies cumbersome and expensive, especially in preclinical in vivo models. In this study, we explored the potential of using a green bioluminescence-emitting click beetle luciferase, CBG99, and a red bioluminescence-emitting firefly luciferase mutant, Akaluc, together to simultaneously monitor two cell populations. Using various chimeric antigen receptor T cells and tumor pairings, we demonstrate that these luciferases are suitable for real-time tracking of two cell types using 2D and 3D cultures in vitro and experimental models in vivo. Our data show the broad compatibility of this dual-luciferase (duo-luc) system with multiple bioluminescence detection equipment ranging from benchtop spectrophotometers to live animal imaging systems. Although this study focused on investigating complex CAR T cells and tumor cell interactions, this duo-luc system has potential utility for the simultaneous monitoring of any two cellular components-for example, to unravel the impact of a specific genetic variant on clonal dominance in a mixed population of tumor cells.

Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform.

The majority of clinically approved drugs target proteins that are secreted or cell surface bound. However, further advances in this area have been hindered by the challenging nature of receptor deorphanization, as there are still many secreted and cell-bound proteins with unknown binding partners. Here, we developed an advanced screening platform that combines CRISPR-CAS9 guide-mediated gene activation (CRISPRa) and high-avidity bead-based selection. The CRISPRa platform incorporates serial enrichment and flow cytometry-based monitoring, resulting in substantially improved screening sensitivity for well-known yet weak interactions of the checkpoint inhibitor family. Our approach has successfully revealed that siglec-4 exerts regulatory control over T cell activation through a low affinity trans-interaction with the costimulatory receptor 4-1BB. Our highly efficient screening platform holds great promise for identifying extracellular interactions of uncharacterized receptor-ligand partners, which is essential to develop next-generation therapeutics, including additional immune checkpoint inhibitors.

Engineering Tumor Stroma Morphogenesis Using Dynamic Cell-Matrix Spheroid Assembly.

The tumor microenvironment consists of resident tumor cells organized within a compositionally diverse, three-dimensional (3D) extracellular matrix (ECM) network that cannot be replicated in vitro using bottom-up synthesis. We report a new self-assembly system to engineer ECM-rich 3D MatriSpheres wherein tumor cells actively organize and concentrate microgram quantities of decellularized ECM dispersions which modulate cell phenotype. 3D colorectal cancer (CRC) MatriSpheres were created using decellularized small intestine submucosa (SIS) as an orthotopic ECM source that had greater proteomic homology to CRC tumor ECM than traditional ECM formulations such as Matrigel. SIS ECM was rapidly concentrated from its environment and assembled into ECM-rich 3D stroma-like regions by mouse and human CRC cell lines within 4-5 days via a mechanism that was rheologically distinct from bulk hydrogel formation. Both ECM organization and transcriptional regulation by 3D ECM cues affected programs of malignancy, lipid metabolism, and immunoregulation that corresponded with an in vivo MC38 tumor cell subpopulation identified via single cell RNA sequencing. This 3D modeling approach stimulates tumor specific tissue morphogenesis that incorporates the complexities of both cancer cell and ECM compartments in a scalable, spontaneous assembly process that may further facilitate precision medicine.