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.

Multispectral confocal 3D imaging of intact healthy and tumor tissue using mLSR-3D.

Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements 'on-the-fly' linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.

Rapid tissue prototyping with micro-organospheres.

In vitro tissue models hold great promise for modeling diseases and drug responses. Here, we used emulsion microfluidics to form micro-organospheres (MOSs), which are droplet-encapsulated miniature three-dimensional (3D) tissue models that can be established rapidly from patient tissues or cells. MOSs retain key biological features and responses to chemo-, targeted, and radiation therapies compared with organoids. The small size and large surface-to-volume ratio of MOSs enable various applications including quantitative assessment of nutrient dependence, pathogen-host interaction for anti-viral drug screening, and a rapid potency assay for chimeric antigen receptor (CAR)-T therapy. An automated MOS imaging pipeline combined with machine learning overcomes plating variation, distinguishes tumorspheres from stroma, differentiates cytostatic versus cytotoxic drug effects, and captures resistant clones and heterogeneity in drug response. This pipeline is capable of robust assessments of drug response at individual-tumorsphere resolution and provides a rapid and high-throughput therapeutic profiling platform for precision medicine.

3D imaging for driving cancer discovery.

Our understanding of the cellular composition and architecture of cancer has primarily advanced using 2D models and thin slice samples. This has granted spatial information on fundamental cancer biology and treatment response. However, tissues contain a variety of interconnected cells with different functional states and shapes, and this complex organization is impossible to capture in a single plane. Furthermore, tumours have been shown to be highly heterogenous, requiring large-scale spatial analysis to reliably profile their cellular and structural composition. Volumetric imaging permits the visualization of intact biological samples, thereby revealing the spatio-phenotypic and dynamic traits of cancer. This review focuses on new insights into cancer biology uniquely brought to light by 3D imaging and concomitant progress in cancer modelling and quantitative analysis. 3D imaging has the potential to generate broad knowledge advance from major mechanisms of tumour progression to new strategies for cancer treatment and patient diagnosis. We discuss the expected future contributions of the newest imaging trends towards these goals and the challenges faced for reaching their full application in cancer research.

Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D.

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning-based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor's spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors.

LGR6 marks nephron progenitor cells.

BACKGROUND: Nephron progenitor cells (NPCs) undergo a stepwise process to generate all mature nephron structures. Mesenchymal to epithelial transition (MET) is considered a multistep process of NPC differentiation to ensure progressive establishment of new nephrons. However, despite this important role, to date, no marker for NPCs undergoing MET in the nephron exists. RESULTS: Here, we identify LGR6 as a NPC marker, expressed in very early cap mesenchyme, pre-tubular aggregates, renal vesicles, and in segments of S-shaped bodies, following the trajectory of MET. By using a lineage tracing approach in embryonic explants in combination with confocal imaging and single-cell RNA sequencing, we provide evidence for the multiple fates of LGR6+ cells during embryonic nephrogenesis. Moreover, by using long-term in vivo lineage tracing, we show that postnatal LGR6+ cells are capable of generating the multiple lineages of the nephrons. CONCLUSIONS: Given the profound early mesenchymal expression and MET signature of LGR6+ cells, together with the lineage tracing of mesenchymal LGR6+ cells, we conclude that LGR6+ cells contribute to all nephrogenic segments by undergoing MET. LGR6+ cells can therefore be considered an early committed NPC population during embryonic and postnatal nephrogenesis with potential regenerative capability.

Quantifying single-cell ERK dynamics in colorectal cancer organoids reveals EGFR as an amplifier of oncogenic MAPK pathway signalling.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity.

Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine.

Snake Venom Gland Organoids.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.