Bridging live-cell imaging and next-generation cancer treatment.

By providing spatial, molecular and morphological data over time, live-cell imaging can provide a deeper understanding of the cellular and signalling events that determine cancer response to treatment. Understanding this dynamic response has the potential to enhance clinical outcome by identifying biomarkers or actionable targets to improve therapeutic efficacy. Here, we review recent applications of live-cell imaging for uncovering both tumour heterogeneity in treatment response and the mode of action of cancer-targeting drugs. Given the increasing uses of T cell therapies, we discuss the unique opportunity of time-lapse imaging for capturing the interactivity and motility of immunotherapies. Although traditionally limited in the number of molecular features captured, novel developments in multidimensional imaging and multi-omics data integration offer strategies to connect single-cell dynamics to molecular phenotypes. We review the effect of these recent technological advances on our understanding of the cellular dynamics of tumour targeting and discuss their implication for next-generation precision medicine.

Single-cell transcriptomics reveals immune suppression and cell states predictive of patient outcomes in rhabdomyosarcoma.

Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin that is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we use single-cell mRNA sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche reveals evidence of an immunosuppressive microenvironment. We also identify a putative interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a potential cause of tumour-induced T-cell dysfunction. In malignant RMS cells, we define transcriptional programs reflective of normal myogenic differentiation and show that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the less aggressive fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.

Uncovering the mode of action of engineered T cells in patient cancer organoids.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.