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.

Plastic persisters: revival stem cells in colorectal cancer.

Colorectal cancer (CRC) is traditionally considered to be a genetically driven disease. However, nongenetic plasticity has recently emerged as a major driver of tumour initiation, metastasis, and therapy response in CRC. Central to these processes is a recently discovered cell type, the revival colonic stem cell (revCSC). In contrast to traditional proliferative CSCs (proCSCs), revCSCs prioritise survival over propagation. revCSCs play an essential role in primary tumour formation, metastatic dissemination, and nongenetic chemoresistance. Current evidence suggests that CRC tumours leverage intestinal stem cell plasticity to both proliferate (via proCSCs) when unchallenged and survive (via revCSCs) in response to cell-extrinsic pressures. Although revCSCs likely represent a major source of therapeutic failure in CRC, our increasing knowledge of this important stem cell fate provides novel opportunities for therapeutic intervention.

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-ß-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.

Trellis tree-based analysis reveals stromal regulation of patient-derived organoid drug responses.

Patient-derived organoids (PDOs) can model personalized therapy responses; however, current screening technologies cannot reveal drug response mechanisms or how tumor microenvironment cells alter therapeutic performance. To address this, we developed a highly multiplexed mass cytometry platform to measure post-translational modification (PTM) signaling, DNA damage, cell-cycle activity, and apoptosis in >2,500 colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies at single-cell resolution. To compare patient- and microenvironment-specific drug responses in thousands of single-cell datasets, we developed "Trellis"-a highly scalable, tree-based treatment effect analysis method. Trellis single-cell screening revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is rarer, patient-specific, and aligns with cancer cell PTM signaling. We find that CAFs can regulate PDO plasticity-shifting proliferative colonic stem cells (proCSCs) to slow-cycling revival colonic stem cells (revCSCs) to protect cancer cells from chemotherapy.

Highlights from the 1st European cancer dependency map symposium and workshop.

The systematic identification of tumour vulnerabilities through perturbational experiments on cancer models, including genome editing and drug screens, is playing a crucial role in combating cancer. This collective effort is known as the Cancer Dependency Map (DepMap). The 1st European Cancer Dependency Map Symposium (EuroDepMap), held in Milan last May, featured talks, a roundtable discussion, and a poster session, showcasing the latest discoveries and future challenges related to the DepMap. The symposium aimed to facilitate interactions among participants across Europe, encourage idea exchange with leading experts, and present their work and future projects. Importantly, it sparked discussions on future endeavours, such as screening more complex cancer models and accounting for tumour evolution.

Activation priming and cytokine polyfunctionality modulate the enhanced functionality of low-affinity CD19 CAR T cells.

We recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR) CAT that showed enhanced expansion, cytotoxicity, and antitumor efficacy compared with the high-affinity (FMC63-based) CAR used in tisagenlecleucel, in preclinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced in vivo expansion, and long-term persistence in a phase 1 clinical study. To understand the molecular mechanisms behind these properties of CAT CAR T cells, we performed a systematic in vitro characterization of the transcriptomic (RNA sequencing) and protein (cytometry by time of flight) changes occurring in T cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT CAR T cells exhibit enhanced activation to CD19 stimulation and a distinct transcriptomic and protein profile, with increased activation and cytokine polyfunctionality compared with FMC63 CAR T cells. We demonstrate that the enhanced functionality of low-affinity CAT CAR T cells is a consequence of an antigen-dependent priming induced by residual CD19-expressing B cells present in the manufacture.

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.

Intra- and intercellular signaling pathways associated with drug-induced cardiac pathophysiology.

Cardiac physiology and homeostasis are maintained by the interaction of multiple cell types, via both intra- and intercellular signaling pathways. Perturbations in these signaling pathways induced by oncology therapies can reduce cardiac function, ultimately leading to heart failure. As cancer survival increases, related cardiovascular complications are becoming increasingly prevalent, thus identifying the perturbations and cell signaling drivers of cardiotoxicity is increasingly important. Here, we discuss the homotypic and heterotypic cellular interactions that form the basis of intra- and intercellular cardiac signaling pathways, and how oncological agents disrupt these pathways, leading to heart failure. We also highlight the emerging systems biology techniques that can be applied, enabling a deeper understanding of the intra- and intercellular signaling pathways across multiple cell types associated with cardiovascular toxicity.

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.

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.

Cell-Specific Labeling for Analyzing Bidirectional Signaling by Mass Spectrometry.

Cell-specific proteome labeling enables global proteome-wide analysis of cell signaling in heterotypic co-cultures. Such approaches have provided unique insight in contact-initiated receptor tyrosine kinase signaling, transfer of proteomic material between heterotypic cells, and interactions between normal and oncogenic cells. Here we describe current methods for cell-specific labeling of heterotypic cells with isotopic labeled amino acids (e.g., SILAC and CTAP). We outline the advantages and disadvantages of individual approaches, describe typical experimental scenarios, and discuss where each experimental approach is optimally applied.

The Heterocellular Emergence of Colorectal Cancer.

Tissues contain multiple different cell types and can be considered to be heterocellular systems. Signaling between different cells allows tissues to achieve phenotypes that no cell type can achieve in isolation. Such emergent tissue-level phenotypes can be said to 'supervene upon' heterocellular signaling. It is proposed here that cancer is also an emergent phenotype that supervenes upon heterocellular signaling. Using colorectal cancer (CRC) as an example, I review how heterotypic cells differentially communicate to support emergent malignancy. Studying tumors as integrated heterocellular systems - rather than as solitary expansions of mutated cells - may reveal novel ways to treat cancer.

Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.

Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRAS(G12D)) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS(G12D) signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRAS(G12D) engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS(G12D). Consequently, reciprocal KRAS(G12D) produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS(G12D) alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. VIDEO ABSTRACT.

Microenvironmental Heterogeneity Parallels Breast Cancer Progression: A Histology-Genomic Integration Analysis.

BACKGROUND: The intra-tumor diversity of cancer cells is under intense investigation; however, little is known about the heterogeneity of the tumor microenvironment that is key to cancer progression and evolution. We aimed to assess the degree of microenvironmental heterogeneity in breast cancer and correlate this with genomic and clinical parameters. METHODS AND FINDINGS: We developed a quantitative measure of microenvironmental heterogeneity along three spatial dimensions (3-D) in solid tumors, termed the tumor ecosystem diversity index (EDI), using fully automated histology image analysis coupled with statistical measures commonly used in ecology. This measure was compared with disease-specific survival, key mutations, genome-wide copy number, and expression profiling data in a retrospective study of 510 breast cancer patients as a test set and 516 breast cancer patients as an independent validation set. In high-grade (grade 3) breast cancers, we uncovered a striking link between high microenvironmental heterogeneity measured by EDI and a poor prognosis that cannot be explained by tumor size, genomics, or any other data types. However, this association was not observed in low-grade (grade 1 and 2) breast cancers. The prognostic value of EDI was superior to known prognostic factors and was enhanced with the addition of TP53 mutation status (multivariate analysis test set, p = 9 × 10-4, hazard ratio = 1.47, 95% CI 1.17-1.84; validation set, p = 0.0011, hazard ratio = 1.78, 95% CI 1.26-2.52). Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. Limitations of this study include the number of cell types included in the model, that EDI has prognostic value only in grade 3 tumors, and that our spatial heterogeneity measure was dependent on spatial scale and tumor size. CONCLUSIONS: To our knowledge, this is the first study to couple unbiased measures of microenvironmental heterogeneity with genomic alterations to predict breast cancer clinical outcome. We propose a clinically relevant role of microenvironmental heterogeneity for advanced breast tumors, and highlight that ecological statistics can be translated into medical advances for identifying a new type of biomarker and, furthermore, for understanding the synergistic interplay of microenvironmental heterogeneity with genomic alterations in cancer cells.

Cell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.

We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC(M.tub-KDEL)) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr(M37-KDEL)) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDC(M.tub-KDEL) and Lyr(M37-KDEL) facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell-cell phospho-signaling experiments, we propose DDC(M.tub-KDEL) and Lyr(M37-KDEL) as excellent enzymes for cell-specific labeling with amino acid precursors.

Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody. We first re-investigated the originally selected anti-TACE ectodomain phage-display clones, and isolated a lead 'mouse-human cross-reactive' anti-TACE scFv, clone A9. We reformatted scFv-A9 into an IgG2 framework for comprehensive biochemical and cellular characterization and further demonstrated that A9 is an exosite TACE inhibitor. However, surface plasmon resonance analysis and quenched-fluorescent (QF) peptide assay indicated that IgG reformatting of A9 caused low binding affinity and an 80-fold reduction in TACE ectodomain inhibition, severely limiting its efficacy. To address this, we constructed second generation phage-display randomization libraries focused on the complementarity-determining region 3, and carried out affinity selections shuffling between human and mouse TACE ectodomain as antigen in addition to an off-rate selection to increase the chance of affinity improvement. The bespoke 'three-step' selections enabled a 100-fold affinity enhancement of A9 IgG, and also improved its IC50 in a QF peptide assay to 0.2 nM. In human and mouse cancer cell assays, matured A9 IgG showed significant cell-surface TACE inhibition as a monotherapy or combination therapy with chemotherapeutic agent. Collectively, these data suggest that we successfully developed an exosite inhibitor of TACE with sub-nanomolar affinity, which possesses both murine and human immunoreactive properties that can be used for in vivo application in murine pre-clinical cancer models.

Anti-tumour effects of a specific anti-ADAM17 antibody in an ovarian cancer model in vivo.

ADAM 17 (TNF-α converting enzyme, TACE) is a potential target for cancer therapy, but the small molecule inhibitors reported to date are not specific to this ADAM family member. This membrane-bound metalloproteinase is responsible for ectodomain shedding of pathologically significant substrates including TNF-α and EGFR ligands. The aim of this study was to evaluate the pharmacokinetics, pharmacodynamics and anti-tumour efficacy of the first specific inhibitor, an anti-human ADAM17 IgG antibody, clone D1(A12). We used intraperitoneal xenografts of the human ovarian cancer cell line IGROV1-Luc in Balb/c nude mice, chosen because it was previously reported that growth of these xenografts is inhibited by knock-down of TNF-α. In vitro, 200 nM D1(A12) inhibited shedding of ADAM17 substrates TNF-α, TNFR1-α, TGF-α, amphiregulin (AREG), HB-EGF and IL-6Rα, from IGROV1-Luc cells, (4.7 nM IC(50) for TNF-α shedding). In IGROV1-Luc xenografts in vivo, D1(A12) IgG showed pharmacokinetic properties suitable for efficacy studies, with a single i.p. dose of 10 mg/kg D1(A12) sufficient to maintain IgG plasma and ascites fluid concentrations above 100 nM for more than 7 days. The plasma half life was 8.6 days. Next, an efficacy study was performed, dosing D1(A12) or anti-human TNF-α antibody infliximab at 10 mg/kg q7d, quantifying IGROV1-Luc tumour burden by bioluminescence. D1(A12) IgG showed a significant reduction in tumour growth (p = 0.005), 56% of vehicle control. Surprisingly, D1(A12) did not reduce the concentration of circulating human TNF-α, suggesting that another enzyme may compensate for inhibition of ADAM17 in vivo (but not in vitro). However, D1(A12) did show clear pharmacodynamic effects in the mice, with significant inhibition of shedding from tumour of ADAM17 substrates TNFR1-α, AREG, and TGF-α (4-15-fold reductions, p<0.0001 for all three). Thus, D1(A12) has anti-ADAM17 activity in vivo, inhibits shedding of EGFR ligands and has potential for use in EGF ligand-dependent tumours.

Cross-domain inhibition of TACE ectodomain.

Proteolytic release from the cell surface is an essential activation event for many growth factors and cytokines. TNF-α converting enzyme (TACE) is a membrane-bound metalloprotease responsible for solubilizing many pathologically significant membrane substrates and is an attractive therapeutic target for the treatment of cancer and arthritis. Prior attempts to antagonize cell-surface TACE activity have focused on small-molecule inhibition of the metalloprotease active site. Given the highly conserved nature of metalloprotease active sites, this paradigm has failed to produce a truly specific TACE inhibitor and continues to obstruct the clinical investigation of TACE activity. We report the bespoke development of a specific TACE inhibitory human antibody using "two-step" phage display. This approach combines calculated selection conditions with antibody variable-domain exchange to direct individual antibody variable domains to desired epitopes. The resulting "cross-domain" human antibody is a previously undescribed selective TACE antagonist and provides a unique alternative to small-molecule metalloprotease inhibition.

Thiol isomerases negatively regulate the cellular shedding activity of ADAM17.

ADAM17 (where ADAM is 'a disintegrin and metalloproteinase') can rapidly modulate cell-surface signalling events by the proteolytic release of soluble forms of proligands for cellular receptors. Many regulatory pathways affect the ADAM17 sheddase activity, but the mechanisms for the activation are still not clear. We have utilized a cell-based ADAM17 assay to show that thiol isomerases, specifically PDI (protein disulfide isomerase), could be responsible for maintaining ADAM17 in an inactive form. Down-regulation of thiol isomerases, by changes in the redox environment (for instance as elicited by phorbol ester modulation of mitochondrial reactive oxygen species) markedly enhanced ADAM17 activation. On the basis of ELISA binding studies with novel fragment antibodies against ADAM17 we propose that isomerization of the disulfide bonds in ADAM17, and the subsequent conformational changes, form the basis for the modulation of ADAM17 activity. The shuffling of disulfide bond patterns in ADAMs has been suggested by a number of recent adamalysin crystal structures, with distinct disulfide bond patterns altering the relative orientations of the domains. Such a mechanism is rapid and reversible, and the role of thiol isomerases should be investigated further as a potential factor in the redox regulation of ADAM17.