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Congratulations to Prof Christian Eggeling, Prof Simon Davies and Dr Jan Rehwinkel, MRC Human Immunology Unit, WIMM, who have just been awarded a Multi-user Equipment Grant from the Wellcome Trust.
Phenoscaping Reveals Multimodal γδ T-cell Cytotoxicity as a Strategy to Overcome Cancer Cell-Mediated Immunomodulation.
UNLABELLED: γδ T cells can kill cancer cells via antibody-independent cytotoxicity (AIC) and antibody-dependent cellular cytotoxicity (ADCC). A better understanding of how these cytotoxic mechanisms are affected by different cancer cells and different T-cell donors could help identify improved immunotherapeutic strategies. To test the combinatorial interactions among T cell interdonor heterogeneity, cancer cell intertumor heterogeneity (ITH), and multimodal γδ T-cell killing, we performed a systematic single-cell phenoscaping analysis of more than 1,000 γδ T-cell and colorectal cancer patient-derived organoid cultures. Single-cell analysis of posttranslational modification (PTM) signaling, cell cycle, apoptosis, and T-cell immunophenotypes revealed that whereas unmodified γδ T cells have limited antitumor activity, IL15Rα-IL15 fusion protein [stabilized IL15 (stIL15)]-engineered γδ T cells can kill patient-derived organoids via AIC without exogenous cytokine support. However, when stIL15 γδ T cells only killed via AIC, cancer cells reciprocally rewired γδ T-cell PTM signal networks in an ITH-specific manner to suppress anticancer cytotoxicity. stIL15 γδ T cells could overcome this cancer cell immunomodulation by also engaging B7-H3-targeted ADCC independent of B7-H3 checkpoint activity. Combined AIC and ADCC rescued γδ T-cell PTM signaling flux and enabled γδ T cells to kill chemorefactory revival colon cancer stem cells. Together, these results demonstrate that multimodal γδ T-cell cytoxicity mechanisms can overcome ITH-specific immunomodulation to kill chemorefractory cancer cells. SIGNIFICANCE: Single-cell phenoscaping of more than 1,000 γδ T-cell and patient-derived organoid cultures shows that cancer cells suppress anticancer γδ T-cell cytotoxicity but γδ T cells can use multimodal killing to overcome immunomodulation.
Oncogenic PIK3CA corrupts growth factor signaling specificity.
Technical limitations have prevented understanding of how growth factor signals are encoded in distinct activity patterns of the phosphoinositide 3-kinase (PI3K)/AKT pathway, and how this is altered by oncogenic pathway mutations. We introduce a kinetic, single-cell framework for precise calculations of PI3K-specific information transfer for different growth factors. This features live-cell imaging of PI3K/AKT activity reporters and multiplexed CyTOF measurements of PI3K/AKT and RAS/ERK signaling markers over time. Using this framework, we found that the PIK3CAH1047R oncogene was not a simple, constitutive activator of the pathway as often presented. Dose-dependent expression of PIK3CAH1047R in human cervical cancer and induced pluripotent stem cells corrupted the fidelity of growth factor-induced information transfer, with preferential amplification of epidermal growth factor receptor (EGFR) signaling responses compared to insulin-like growth factor 1 (IGF1) and insulin receptor signaling. PIK3CAH1047R did not only shift these responses to a higher mean but also enhanced signaling heterogeneity. We conclude that oncogenic PIK3CAH1047R corrupts information transfer in a growth factor-dependent manner and suggest new opportunities for tuning of receptor-specific PI3K pathway outputs for therapeutic benefit.
Functional analysis of cell plasticity using single-cell technologies.
Metazoan organisms are heterocellular systems composed of hundreds of different cell types, which arise from an isogenic genome through differentiation. Cellular 'plasticity' further enables cells to alter their fate in response to exogenous cues and is involved in a variety of processes, such as wound healing, infection, and cancer. Recent advances in cellular model systems, high-dimensional single-cell technologies, and lineage tracing have sparked a renaissance in plasticity research. Here, we discuss the definition of cell plasticity, evaluate state-of-the-art model systems and techniques to study cell-fate dynamics, and explore the application of single-cell technologies to obtain functional insights into cell plasticity in healthy and diseased tissues. The integration of advanced biomimetic model systems, single-cell technologies, and high-throughput perturbation studies is enabling a new era of research into non-genetic plasticity in metazoan systems.
Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment.
Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.
An oncogenic phenoscape of colonic stem cell polarization.
Cancer cells are regulated by oncogenic mutations and microenvironmental signals, yet these processes are often studied separately. To functionally map how cell-intrinsic and cell-extrinsic cues co-regulate cell fate, we performed a systematic single-cell analysis of 1,107 colonic organoid cultures regulated by (1) colorectal cancer (CRC) oncogenic mutations, (2) microenvironmental fibroblasts and macrophages, (3) stromal ligands, and (4) signaling inhibitors. Multiplexed single-cell analysis revealed a stepwise epithelial differentiation phenoscape dictated by combinations of oncogenes and stromal ligands, spanning from fibroblast-induced Clusterin (CLU)+ revival colonic stem cells (revCSCs) to oncogene-driven LRIG1+ hyper-proliferative CSCs (proCSCs). The transition from revCSCs to proCSCs is regulated by decreasing WNT3A and TGF-β-driven YAP signaling and increasing KRASG12D or stromal EGF/Epiregulin-activated MAPK/PI3K flux. We find that APC loss and KRASG12D collaboratively limit access to revCSCs and disrupt stromal-epithelial communication-trapping epithelia in the proCSC fate. These results reveal that oncogenic mutations dominate homeostatic differentiation by obstructing cell-extrinsic regulation of cell-fate plasticity.
Multiplexed single-cell analysis of organoid signaling networks.
Organoids are biomimetic tissue models comprising multiple cell types and cell states. Post-translational modification (PTM) signaling networks control cellular phenotypes and are frequently dysregulated in diseases such as cancer. Although signaling networks vary across cell types, there are limited techniques to study cell type-specific PTMs in heterocellular organoids. Here, we present a multiplexed mass cytometry (MC) protocol for single-cell analysis of PTM signaling and cell states in organoids and organoids co-cultured with fibroblasts and leukocytes. We describe how thiol-reactive organoid barcoding in situ (TOBis) enables 35-plex and 126-plex single-cell comparison of organoid cultures and provide a cytometry by time of flight (CyTOF) signaling analysis pipeline (CyGNAL) for computing cell type-specific PTM signaling networks. The TOBis MC protocol takes ~3 d from organoid fixation to data acquisition and can generate single-cell data for >40 antibodies from millions of cells across 126 organoid cultures in a single MC run.
Deciphering Organoids: High-Dimensional Analysis of Biomimetic Cultures.
Organoids are self-organising stem cell-derived ex vivo cultures widely adopted as biomimetic models of healthy and diseased tissues. Traditional low-dimensional experimental methods such as microscopy and bulk molecular analysis have generated remarkable biological insights from organoids. However, as complex heterocellular systems, organoids are especially well-positioned to take advantage of emerging high-dimensional technologies. In particular, single-cell methods offer considerable opportunities to analyse organoids at unprecedented scale and depth, enabling comprehensive characterisation of cellular processes and spatial organisation underpinning organoid heterogeneity. This review evaluates state-of-the-art analytical methods applied to organoids, discusses the latest advances in single-cell technologies, and speculates on the integration of these two rapidly developing fields.
Cell-type-specific signaling networks in heterocellular organoids.
Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids.
Crosstalk with lung epithelial cells regulates Sfrp2-mediated latency in breast cancer dissemination.
The process of metastasis is complex1. In breast cancer, there are frequently long time intervals between cells leaving the primary tumour and growth of overt metastases2,3. Reasons for disease indolence and subsequent transition back to aggressive growth include interactions with myeloid and fibroblastic cells in the tumour microenvironment and ongoing immune surveillance4-6. However, the signals that cause actively growing cells to enter an indolent state, thereby enabling them to survive for extended periods of time, are not well understood. Here we reveal how the behaviour of indolent breast cancer cells in the lung is determined by their interactions with alveolar epithelial cells, in particular alveolar type 1 cells. This promotes the formation of fibronectin fibrils by indolent cells that drive integrin-dependent pro-survival signals. Combined in vivo RNA sequencing and drop-out screening identified secreted frizzled-related protein 2 (SFRP2) as a key mediator of this interaction. Sfrp2 is induced in breast cancer cells by signals from lung epithelial cells and promotes fibronectin fibril formation and survival, whereas blockade of Sfrp2 expression reduces the burden of indolent disease.
Spatial fibroblast niches define Crohn's fistulae.
Crohn's disease often presents with fistulae, abnormal tunnels that connect the intestine to the skin or other organs. Despite their profound effect on morbidity, the molecular basis of fistula formation remains unclear, largely owing to the challenge of capturing intact fistula tracts and their inherent heterogeneity1-3. Here we construct a subcellular-resolution spatial atlas of 68 intestinal fistulae spanning diverse anatomical locations. We describe fistula-associated epithelial, immune and stromal cell states, revealing abnormal zonation of growth factors and morphogens linked to establishment of tunnelling anatomy. We identify fistula-associated stromal (FAS) fibroblasts, which are assembled in concentric layers: a proliferative, lumen-adjacent zone beneath neutrophil and macrophage-rich granulation tissue, an active lesion core of FAS cells and a quiescent, pro-fibrotic outer zone. We examine the architecture of the extracellular matrix in the fistula tract and demonstrate that FAS populations associate with distinct collagen structures, exhibiting properties ranging from proliferation, migration and extracellular matrix remodelling to dense collagen deposition and fibrosis. We define niches supporting epithelialization of fistula tunnels and a FAS-like population that is detected at the base of ulcers in non-penetrating Crohn's disease. Our study demonstrates that common molecular pathways and cellular niches underpin fistulae across intestinal locations, revealing the cellular protagonists of fistula establishment and persistence. This resource will inform the development of model systems and interventions to mitigate aberrant fibroblast activity while preserving their regenerative properties in Crohn's disease.
Characterizing Behaviors That Influence the Implementation of Digital-Based Interventions in Health Care: Systematic Review.
BACKGROUND: Successful implementation of any digital intervention in a health care setting requires adoption by all stakeholders. Appropriate consideration of behavioral change is a key driver that is often overlooked during implementation. The nonadoption, abandonment, scale-up, spread, and systems (NASSS) behavioral framework offers a broad evaluation of success for digital health solutions, and the theoretical domains framework (TDF) focuses particularly on adopters, identifying determinants of behavior and potential reasons for implementation issues. OBJECTIVE: The aim of this study was to describe and characterize barriers and facilitators to the adoption of digital solutions within health care using behavioral frameworks: the NASSS and TDF. METHODS: A systematic search was performed in 4 databases (ie, Ovid in MEDLINE, Embase, Health Management Information Consortium, and PsycINFO). Included studies reported a behavioral change by health care professionals following digital interventions or the practicality of delivering such interventions. Barriers and facilitators were identified, extracted, and classified using the NASSS framework and TDF. Risk of bias was assessed using the Mixed Methods Appraisal Tool. RESULTS: The initial search result included 2704 unique studies, 12 of which met the inclusion criteria and from which data were extracted. All 12 scored ≥3 out of 5 stars on the Mixed Methods Appraisal Tool risk of bias assessment. Out of the 12 studies, 67% (n=8) were conducted in the United States, and 8% (n=1) each in India, Australia, the Netherlands, and Tanzania. The NASSS framework identified facilitators and barriers in 4 domains: the condition or illness, technology, value proposition, and adopter system. The TDF framework identified 8 relevant domains, including knowledge, skills, and beliefs about capabilities. Key facilitators included intuitive technology design aligned with existing workflows, clear communication of value propositions to adopters, adequate provision of training resources tailored to adopters' knowledge levels, and ensuring organizational readiness for technological change. Conversely, significant barriers involved disruptions to clinical workflow, inadequate adopter training or confidence levels, unclear value propositions leading to disengagement, insufficient consideration of cognitive load impacts, such as alert fatigue, and limited organizational preparedness. Notably, psychological factors such as optimism, intentions, and social influences were underreported. CONCLUSIONS: This study delineated and analyzed various critical behavioral factors impacting the adoption and implementation of digital interventions in health care. Based on these findings, future research must consider the key factors reported and alternative approaches to assess behaviors influencing adoption that are not presented in the current scientific literature. TRIAL REGISTRATION: PROSPERO CRD42022264937; https://www.crd.york.ac.uk/PROSPERO/view/CRD42022264937.