Organoids are powerful biomimetic tissue models. Despite their widespread adoption, methods to analyse cell-type specific post-translational modification (PTM) signalling networks in organoids are absent. Here we report multivariate single-cell analysis of cell-type specific signalling networks in organoids and organoid co-cultures. Simultaneous measurement of 28 PTMs in >1 million single small intestinal organoid cells by mass cytometry reveals cell-type and cell-state specific signalling networks in stem, Paneth, enteroendocrine, tuft, goblet cells, and enterocytes. Integrating single-cell PTM analysis with Thiol-reactive Organoid Barcoding in situ (TOB is ) enables high-throughput comparison of signalling networks between organoid cultures. Multivariate cell-type specific PTM analysis of colorectal cancer tumour microenvironment organoids reveals that shApc , Kras G12D , and Trp53 R172H cell-autonomously mimic signalling 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 signalling analysis of healthy and cancerous organoids.

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34 þ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte --macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

This multiplexed mass cytometry protocol uses Thiol-reactive Organoid Barcoding in situ (TOBis) and a CyTOF siGNalling AnaLysis pipeline (CyGNAL) to enable 126-plex single-cell analysis of cell-type, cell-state, and posttranslational modification signalling network in organoids.Tweet A multiplexed mass cytometry protocol using Thiol-reactive Organoid Barcoding in situ (TOBis) and a CyTOF siGNalling AnaLysis pipeline (CyGNAL) for 126-plex single-cell analysis of cell-type-specific PTM signalling in organoids (from @QinXiao1990 @FerranC96 and @christophertape).

Hepatocellular Carcinoma (HCC) is increasing in incidence worldwide and requires new approaches to therapy. The combination of anti-angiogenic drug therapy and radiotherapy is one promising new approach. The anti-angiogenic drug vandetanib is a tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and RET proto-oncogene with radio-enhancement potential. To explore the benefit of combined vandetanib and radiotherapy treatment for HCC, we studied outcomes following combined treatment in pre-clinical models. Methods: Vandetanib and radiation treatment were combined in HCC cell lines grown in vitro and in vivo. In addition to 2D migration and clonogenic assays, the combination was studied in 3D spheroids and a syngeneic mouse model of HCC. Results: Vandetanib IC 50 s were measured in 20 cell lines and the drug was found to significantly enhance radiation cell kill and to inhibit both cell migration and invasion in vitro. In vivo, combination therapy significantly reduced cancer growth and improved overall survival, an effect that persisted for the duration of vandetanib treatment. Conclusion: In 2D and 3D studies in vitro and in a syngeneic model in vivo, the combination of vandetanib plus radiotherapy was more efficacious than either treatment alone. This new combination therapy for HCC merits evaluation in clinical trials.

Patient-derived organoids (PDOs) model personalized cancer therapy responses. How-ever, existing bulk PDO screening technologies cannot reveal drug response mechanisms or model how cells of the tumor microenvironment alter therapy performance. To address this, we developed a highly-multiplexed thiol-reactive organoid barcodingin situ(TOBis) mass cytometry platform to perform single-cell post translational modification (PTM) signaling analysis of colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies. To compare patient- and microenvironment-specific treatment effects in thousands of single-cell PTM datasets, we developedTrellis— a highly-scalable, hierarchical tree-based treatment effect analysis method. Trellis analysis of>2,500 single-cell PTM PDO-CAF organoid cultures revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is patient-specific. We found drug-induced apoptosis does not correlate with genotype or clinical staging, but does align with cell-intrinsic PTM signaling in PDOs. We observe that CAFs protect chemosensitive PDOs by shifting cancer cells into a slow-cycling cell-state in a patient-specific manner and show that CAF chemoprotection can be reversed by inhibiting YAP. These results reveal that PTM signaling flux is a major determinant of chemosensitivity and demonstrate CAFs regulate patient-specific drug responses by altering cancer cell-state.

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 in colorectal cancer (CRC), we performed a systematic single-cell analysis of 1,071 colonic organoid cultures regulated by 1) CRC oncogenic mutations, 2) microenvironmental fibroblasts and macrophages, 3) stromal ligands, and 4) signalling inhibitors. Multiplexed single-cell analysis revealed a stepwise epithelial differentiation landscape dictated by combinations of oncogenes and stromal ligands, spanning from fibroblast-induced Clusterin (CLU)+revival colonic stem cells (revCSC) to oncogene-driven LRIG1+hyper-proliferative CSC (proCSC). The transition from revCSC to proCSC is regulated by decreasing WNT3A and TGF-β-driven YAP signalling and increasing KRASG12Dor stromal EGF/Epiregulin-activated MAPK/PI3K flux. We find APC-loss and KRASG12Dcollaboratively limit access to revCSC 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.

Organoids are powerful biomimetic tissue models. Despite their widespread adoption, methods to analyse cell-type specific post-translational modification (PTM) signalling networks in organoids are absent. Here we report multivariate single-cell analysis of cell-type specific signalling networks in organoids and organoid co-cultures. Simultaneous measurement of 28 PTMs in >1 million single small intestinal organoid cells by mass cytometry reveals cell-type and cell-state specific signalling networks in stem, Paneth, enteroendocrine, tuft, goblet cells, and enterocytes. Integrating single-cell PTM analysis with Thiol-reactive Organoid Barcoding in situ (TOBis) enables high-throughput comparison of signalling networks between organoid cultures. Multivariate cell-type specific PTM analysis of colorectal cancer tumour microenvironment organoids reveals that shApc, Kras G12D , and Trp53 R172H cell-autonomously mimic signalling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform highthroughput multivariate cell-type specific signalling analysis of healthy and cancerous organoids.

4600 MicroRNA miR-155 represents a candidate pathogenic factor in chronic lymphocytic leukemia (CLL) and lymphomas (Eis PS, et al. 2005, Fulci V, et al. 2007, Calin GA, et al. 2005, Kluiver J, et al. 2005, Metzler M, et al. 2004, van den Berg A, et al. 2003., Vargova K. et al 2011). In diffuse large B-cell lymphoma (DLBCL) expression of miR-155 was associated with NFkB pathway (Rai et al., 2008). In addition, increased miR-155 levels were associated with more aggressive (Activated B-cell like) DLBCL (Eis et al. 2005). The targets of up-regulated miR-155 apparently include a key hematopoietic transcription factor PU.1. Down-regulation of PU.1 represents an important and critical step for both myeloid as well as lymphoid tumorigenesis. We herein found the increased levels of miR-155 coupled with downregulation of its target PU.1 are frequent events in B cells of a vast majority of CLL patients (N=169). To advance understanding of miR-155 and PU.1 in lymphoid malignancies we studied lymph node biopsies derived from patients with lymphomas. We show that increased miR-155 levels are also found in DLBCL (N=31, P < 0.0001), Hodgkin lymphoma (HL, N=22, P < 0.0001), follicular lymphoma (FL, N=23, P < 0.001), small lymphocytic lymphoma (SLL, N=12, P < 0.01), and marginal zone lymphoma (MZL, N=11, P < 0.01). The miR-155-overexpressing tumors display downregulation of PU.1. In contrast, peripheral T cell lymphoma (T-NHL, N=6) and mantle cell lymphoma (MCL, N=7) expressed miR-155 and PU.1 comparably as control lymph nodes. Our data indicate that expression pattern of miR-155 and its target PU.1 represents a hallmark of some but not all lymphoid malignancies. Interestingly, the miR-155/PU1 levels vary substantially within each diagnosis, therefore, our aim is to further analyze the occurrence of this relationship in different prognostic groups and analyze the clinical outcome as well. (Grants: NT10310-3/2009 & NT11218-6/2010, MPO FR-TI2/509, GAUK251135 82210 & 251070 45410, SVV-2011-262507) Disclosures: No relevant conflicts of interest to declare.