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Self-assembly is a fundamental property of living matter that drives the three-dimensional organization of cell collectives such as tissues and organs. Here, the co-assembly of synthetic and natural cells is leveraged to create hybrid living 3D cancer cultures. We screen a range of synthetic cell models for their ability to form augmented tumoroids with artificial but controllable micro-environments, and show that the balance of inter- and extracellular adhesion and synthetic cell surface tension are key material properties driving integrated co-assembly. We demonstrate that synthetic cells based on droplet-supported lipid bilayers can establish artificial tumor immune microenvironments (ART-TIMEs), mimicking immunogenic signals within tumoroids and eliminating the need to integrate complex living immune cells. Using the ART-TIME approach, we identify a AhR-ARNT-mediated co-signaling mechanism between PD-1 and CD2 as a driver in immune evasion of pancreatic ductal adenocarcinoma. Our study advances the field of hybrid organoid engineering, offers opportunities for the construction and modelling of artificial tumour environments, and marks a step towards the design of functional living/non-living cytomimetic materials. Synthetic cells have huge potential in model systems. Here, the authors engineer synthetic–living hybrid tumoroids that replicate tumour-immune interactions in 3D, study synthetic cells integration, and demonstrate systematic studies of immune evasion and T cell engager therapies.

Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges, including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer, functional, and tunable alternative for gene silencing without the need for DNA editing. As a proof of concept for multiplex gene silencing, we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3ζ and β2-microglobulin (β2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors, enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models. Silenced (S) M5CAR T cells retained antitumor functionality comparable to, and in some cases exceeding, that of KO cells. In vivo , S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs). Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells, with potential advantages in potency, persistence, metastasis prevention, and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks.

The scaffold protein AMBRA1, which participates in the autophagy pathway, also promotes CD4+ T cell differentiation to Tregs independent of autophagy through its interactor PP2A. Here we have investigated the role of AMBRA1 in CD8+ T cell differentiation to cytotoxic T cells (CTL). AMBRA1 depletion in CD8+ T cells was associated with impaired expression of the transcription factors RUNX3 and T-BET that drive CTL differentiation and resulted in impaired acquisition of cytotoxic potential. These effects were recapitulated by pharmacological inhibition of the AMBRA1 activator ULK1 or its interactor PP2A. Based on the ability of PP2A to activate TFEB, we hypothesized a role for TFEB in the CTL differentiation program regulated by AMBRA1. We show that TFEB modulates RUNX3 and T-BET expression and the generation of killing-competent CTLs, and that AMBRA1 depletion, or ULK1 or PP2A inhibition, suppresses TFEB activity. These data highlight a role for AMBRA1, ULK1 and PP2A in CTL generation, mediated by TFEB, which we identify as a new pioneering transcription factor in the CTL differentiation program.