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The contribution of somatic mutations to metastasis of colorectal cancers is currently unknown. To find mutations involved in the colorectal cancer metastatic process, we performed deep mutational analysis of 676 genes in 107 stages II to IV primary colorectal cancer, of which half had metastasized. The mutation prevalence in the ephrin (EPH) family of tyrosine kinase receptors was 10-fold higher in primary tumors of metastatic colorectal than in nonmetastatic cases and preferentially occurred in stage III and IV tumors. Mutational analyses in situ confirmed expression of mutant EPH receptors. To enable functional studies of EPHB1 mutations, we demonstrated that DLD-1 colorectal cancer cells expressing EPHB1 form aggregates upon coculture with ephrin B1 expressing cells. When mutations in the fibronectin type III and kinase domains of EPHB1 were compared with wild-type EPHB1 in DLD-1 colorectal cancer cells, they decreased ephrin B1–induced compartmentalization. These observations provide a mechanistic link between EPHB receptor mutations and metastasis in colorectal cancer. Cancer Res; 77(7); 1730–40. ©2017 AACR.

Carcinoma development in colorectal cancer is driven by genetic alterations in numerous signaling pathways. Alterations in the RAS-ERK1/2 pathway are associated with the shortest overall survival for patients after diagnosis of colorectal cancer metastatic disease, yet how RAS–ERK signaling regulates colorectal cancer metastasis remains unknown. In this study, we used an unbiased screening approach based on selection of highly liver metastatic colorectal cancer cells in vivo to determine genes associated with metastasis. From this, an ERK1/2-controlled metastatic gene set (EMGS) was defined. EMGS was associated with increased recurrence and reduced survival in patients with colorectal cancer tumors. Higher levels of EMGS expression were detected in the colorectal cancer subsets consensus molecular subtype (CMS)1 and CMS4. ANGPT2 and CXCR4, two genes within the EMGS, were subjected to gain-of-function and loss-of-function studies in several colorectal cancer cell lines and then tested in clinical samples. The RAS–ERK1/2 axis controlled expression of the cytokine ANGPT2 and the cytokine receptor CXCR4 in colorectal cancer cells, which facilitated development of liver but not lung metastases, suggesting that ANGPT2 and CXCR4 are important for metastatic outgrowth in the liver. CXCR4 controlled the expression of cytokines IL10 and CXCL1, providing evidence for a causal role of IL10 in supporting liver colonization. In summary, these studies demonstrate that amplification of ERK1/2 signaling in KRAS-mutated colorectal cancer cells affects the cytokine milieu of the tumors, possibly affecting tumor–stroma interactions and favoring liver metastasis formation.Significance:These findings identify amplified ERK1/2 signaling in KRAS-mutated colorectal cancer cells as a driver of tumor–stroma interactions that favor formation of metastases in the liver.

Supplementary Materials and Methods

All somatic mutations found in the study.

SF1: The expression of ANGPT2 and CXCR4 in CRC; SF2: Expression levels of ANGPT2, CXCR4, CXCL12, TEK and VEGFA in SW620_P and SW620_LiM2 cells; SF3: Expression levels of ANGPT2 and CXCR4 upon downregulation and upregulation.; SF4: CXCL1 and IL10 expression is controlled by RAS-ERK signaling pathway; SF5: Correlation of various genes expression and BRAF-signature or CRIS subtypes across CRC tumors; ST1: mRNA expression comparisons; ST2: Hypergeometric test for Signature FC-1.75.up ( 15 genes) - KEGG.

Supplementary Fig. 1. Luciferase reporter assay reveals downregulation of BMP pathway activity upon overexpression of mutant BMPR2 Supplementary Fig. 2. Genes found in the largest number of significant outcomes when comparing mutation rate between metastatic and non-metastatic patients Supplementary Fig. 3. In situ mutation detection in FF colon tumour sections Supplementary Fig. 4. Expression levels of EPHB1 in DLD-1 cell lines overexpressing EPHB1 and its four mutant versions Supplementary Fig. 5. EPHB2 interacts with ephrinB1 ligand used as a positive control for the compartmentalisation experiment Supplementary Fig. 6. Detections of four non-synonymous mutations at the transcript level by Sanger sequencing and real time growth analysis of cell lines used in the compartmentalisation experiment Supplementary Fig. 7. The EPHB1 kinase inhibitor dasatinib abrogates the compartmentalisation phenotype of EPHB1 cells co-cultured with ephrinB1 ligand expressing cells Supplementary Table 1. Probe sequences for in situ mutation detection Supplementary Table 2. Probe performance for in situ mutation detection Supplementary Table 3. LNA primers for reverse transcription in situ Supplementary Table 4. Fluorescently labelled detection probes for RCPs Supplementary Table 5. Genes and corresponding length of the coding regions included in this study Supplementary Table 6. Patient-matched tumour and normal samples Supplementary Table 8. Comparison of pathway mutation rates in metastatic vs non-metastatic samples Supplementary Table 9. Functional annotation of mutated genes unique to metastatic patients Supplementary Table 10. Somatic mutations in Ephrin receptor genes Supplementary Table 11. Mean read depth of Eph receptor tyrosine kinases Supplementary Table 12. Prediction scores for amino acid conservation and effect of point mutations Supplementary Table 13. Mutation counts for Eph receptor mutations in situ Supplementary Table 14. Primer sequences for Sanger sequencing mutant constructs. Supplementary Materials and Methods

Background Colorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1–4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. Methods Using transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n = 577) and mouse (n = 57 across n = 8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. Results We developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. Conclusions When applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package enables researchers to select suitable mouse models of human CRC subtype for their experimental testing.

Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.© 2022. The Author(s), under exclusive licence to Springer Nature Limited.

Intestinal organoids capture essential features of the intestinal epithelium such as crypt folding, cellular compartmentalization and collective movements. Each of these processes and their coordination require patterned forces that are at present unknown. Here we map three-dimensional cellular forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the extracellular matrix and folds through apical constriction, whereas the transit amplifying zone pulls the extracellular matrix and elongates through basal constriction. The size of the stem cell compartment depends on the extracellular-matrix stiffness and endogenous cellular forces. Computational modelling reveals that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. Finally, cells are pulled out of the crypt along a gradient of increasing tension. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal epithelium.

Colorectal cancer (CRC) patient-derived organoids predict responses to chemotherapy. Here we used them to investigate relapse after treatment. Patient-derived organoids expand from highly proliferative LGR5(+) tumor cells; however, we discovered that lack of optimal growth conditions specifies a latent LGR5(+) cell state. This cell population expressed the gene MEX3A, is chemoresistant and regenerated the organoid culture after treatment. In CRC mouse models, Mex3a(+) cells contributed marginally to metastatic outgrowth; however, after chemotherapy, Mex3a(+) cells produced large cell clones that regenerated the disease. Lineage-tracing analysis showed that persister Mex3a(+) cells downregulate the WNT/stem cell gene program immediately after chemotherapy and adopt a transient state reminiscent to that of YAP(+) fetal intestinal progenitors. In contrast, Mex3a-deficient cells differentiated toward a goblet cell-like phenotype and were unable to resist chemotherapy. Our findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from chemotherapy and identify a candidate cell of origin of relapse after treatment in CRC. Batlle and colleagues report that after chemotherapy, Mex3a(+) colorectal cancer cells represent drug-tolerant persister cells that lead to recurrence by downregulating the WNT-Lgr5(+) stem cell program and adopting a transient regenerative state.

Carcinoma development in colorectal cancer is driven by genetic alterations in numerous signaling pathways. Alterations in the RAS-ERK1/2 pathway are associated with the shortest overall survival for patients after diagnosis of colorectal cancer metastatic disease, yet how RAS–ERK signaling regulates colorectal cancer metastasis remains unknown. In this study, we used an unbiased screening approach based on selection of highly liver metastatic colorectal cancer cells in vivo to determine genes associated with metastasis. From this, an ERK1/2-controlled metastatic gene set (EMGS) was defined. EMGS was associated with increased recurrence and reduced survival in patients with colorectal cancer tumors. Higher levels of EMGS expression were detected in the colorectal cancer subsets consensus molecular subtype (CMS)1 and CMS4. ANGPT2 and CXCR4, two genes within the EMGS, were subjected to gain-of-function and loss-of-function studies in several colorectal cancer cell lines and then tested in clinical samples. The RAS–ERK1/2 axis controlled expression of the cytokine ANGPT2 and the cytokine receptor CXCR4 in colorectal cancer cells, which facilitated development of liver but not lung metastases, suggesting that ANGPT2 and CXCR4 are important for metastatic outgrowth in the liver. CXCR4 controlled the expression of cytokines IL10 and CXCL1, providing evidence for a causal role of IL10 in supporting liver colonization. In summary, these studies demonstrate that amplification of ERK1/2 signaling in KRAS-mutated colorectal cancer cells affects the cytokine milieu of the tumors, possibly affecting tumor–stroma interactions and favoring liver metastasis formation. Significance: These findings identify amplified ERK1/2 signaling in KRAS-mutated colorectal cancer cells as a driver of tumor–stroma interactions that favor formation of metastases in the liver.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.

This Review discusses the context-dependent functions of transforming growth factor-beta (TGF beta) with regard to the composition and behaviour of different cell populations in the tumour immune microenvironment, as well as emerging data that demonstrate that TGF beta inhibition can restore cancer immunity. Transforming growth factor-beta (TGF beta) signalling controls multiple cell fate decisions during development and tissue homeostasis; hence, dysregulation of this pathway can drive several diseases, including cancer. Here we discuss the influence that TGF beta exerts on the composition and behaviour of different cell populations present in the tumour immune microenvironment, and the context-dependent functions of this cytokine in suppressing or promoting cancer. During homeostasis, TGF beta controls inflammatory responses triggered by exposure to the outside milieu in barrier tissues. Lack of TGF beta exacerbates inflammation, leading to tissue damage and cellular transformation. In contrast, as tumours progress, they leverage TGF beta to drive an unrestrained wound-healing programme in cancer-associated fibroblasts, as well as to suppress the adaptive immune system and the innate immune system. In consonance with this key role in reprogramming the tumour microenvironment, emerging data demonstrate that TGF beta-inhibitory therapies can restore cancer immunity. Indeed, this approach can synergize with other immunotherapies - including immune checkpoint blockade - to unleash robust antitumour immune responses in preclinical cancer models. Despite initial challenges in clinical translation, these findings have sparked the development of multiple therapeutic strategies that inhibit the TGF beta pathway, many of which are currently in clinical evaluation.

Serrated polyposis syndrome (SPS) is associated with a high risk for colorectal cancer. Intense promoter hypermethylation is a frequent molecular finding in the serrated pathway and may be present in normal mucosa, predisposing to the formation of serrated lesions. To identify novel biomarkers for SPS, fresh-frozen samples of normal mucosa from 50 patients with SPS and 19 healthy individuals were analyzed by using the 850K BeadChip Technology (Infinium). Aberrant methylation levels were correlated with gene expression using a next-generation transcriptome profiling tool. Two validation steps were performed on independent cohorts: first, on formalin-fixed, paraffin-embedded tissue of the normal mucosa; and second, on 24 serrated lesions. The most frequently hypermethylated genes were HLA-F, SLFN12, HLA-DMA, and RARRES3; and the most frequently hypomethylated genes were PIWIL1 and ANK3 ( ß = 10%; P 20%; r > 0.55; P

Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years

The tumor immune microenvironment is a main contributor to cancer progression and a promising therapeutic target for oncology. However, immune microenvironments vary profoundly between patients, and biomarkers for prognosis and treatment response lack precision. A comprehensive compendium of tumor immune cells is required to pinpoint predictive cellular states and their spatial localization. We generated a single-cell tumor immune atlas, jointly analyzing published data sets of >500,000 cells from 217 patients and 13 cancer types, providing the basis for a patient stratification based on immune cell compositions. Projecting immune cells from external tumors onto the atlas facilitated an automated cell annotation system. To enable in situ mapping of immune populations for digital pathology, we applied SPOTlight, combining single-cell and spatial transcriptomics data and identifying colocalization patterns of immune, stromal, and cancer cells in tumor sections. We expect the tumor immune cell atlas, together with our versatile toolbox for precision oncology, to advance currently applied stratification approaches for prognosis and immunotherapy. This publication is part of a project (BCLLATLAS) that has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 810287). This work has received funding from the Ministerio de Ciencia, Innovación y Universidades (SAF2017-89109-P; AEI/FEDER, UE) and the Fundació La Marató de TV3 (201903-30-31-32).We further acknowledge funding from the St. Vincent’s Clinic Foundation (V.T.C.) and the National Health and Medical Research Council Investigator Grant (APP1175781, J.E.P.), the Fundación Asociación Española contra el Cáncer (AECC), FERO (EDM), Ramón Areces Foundation, Cellex Foundation, BBVA (CAIMI), the ISCIII, FIS (PI16/01278), Juan de la Cierva formación fellowship (C.R.-P.) and Sara Borrell fellowship (E.P.-R.). Core funding is from the ISCIII and the Generalitat de Catalunya. We acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programme / Generalitat de Catalunya, the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) through the nstituto de Salud Carlos III and the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement. We also acknowledge the cofinancing by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) with funds from the European Regional Development Fund (ERDF) corresponding to the 2014–2020 Smart Growth Operating Program

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3’UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3’UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A–eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5’-3’ end interaction, mediated by both viral and cellular proteins., Author summary Andes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is unique among other members of the Hantaviridae family of viruses because of its ability to spread from person to person. The molecular mechanisms driving ANDV protein synthesis remain poorly understood. A previous report showed that translation of the Small segment mRNA (SmRNA) of ANDV relied on both the 5’cap and the 3’untranslated region (UTR) of the SmRNA. In this new study, we further characterize the mechanism by which the 5’ and 3’end of the SmRNA interact to assure viral protein synthesis. We establish that the viral nucleocapsid protein N and the cellular protein hMex3A participate in the process. These observations indicated that both viral and cellular proteins regulate viral gene expression during ANDV infection by enabling the viral mRNA to establish a non-covalent 5’-3’end interaction.

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. he authors thank Rene Fabregas for the advice on the in-silico model. The authors also thank Jordi Comelles, María García and Enara Larrañaga for the fruitful discussions. Funding for this project was provided by European Union's Horizon 2020 ERC grant agreements No 647863 (COMIET) and No 884623 (residualCRC), the CERCA Programme/Generalitat de Catalunya (2017-SGR-1079 and 2017-SGR-698), Laserlab-Europe EU-H2020 grant agreement No 871124, the Spanish Ministry of Economy and Competitiveness (Severo Ochoa Program for Centers of Excellence in R&D 2016–2019 and CEX2019-000910-S), the Spanish Ministry of Science (PID2020-119917RB-I00) and also by the Fundació Privada Cellex and the Fundació Mir-Puig. The authors thank Barcelona Institute of Science and Technology (BIST) for the funding of ENGUT project through Ignite Programme. The authors gratefully acknowledge the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) for the funding of G.A. through FI-DGR 2014 and to MINECO/FEDER Ramón y Cajal program (RYC-2015-17935) for the funding of E.G. The results presented here reflect only the views of the authors; the European Commission is not responsible for any use that may be made of the information it contains.

Summary: Upon tissue injury, cytokine expression reprogramming transiently remodels the inflammatory immune microenvironment to activate repair processes and subsequently return to homeostasis. However, chronic inflammation induces permanent changes in cytokine production which exacerbate tissue damage and may even favor tumor development. Here, we address the contribution of post-transcriptional regulation, by the RNA-binding protein CPEB4, to intestinal immune homeostasis and its role in inflammatory bowel diseases (IBD) and colorectal cancer (CRC) development. We found that intestinal damage induces CPEB4 expression in adaptive and innate immune cells, which is required for the translation of cytokine mRNA(s) such as the one encoding interleukin-22. Accordingly, CPEB4 is required for the development of gut-associated lymphoid tissues and to maintain intestinal immune homeostasis, mediating repair and remodeling after acute inflammatory tissue damage and promoting the resolution of intestinal inflammation. CPEB4 is chronically overexpressed in inflammatory cells in patients with IBD and in CRC, favoring tumor development.

© The Author(s) 2019., Metastasis underlies the majority of cancer-related deaths yet remains poorly understood due, in part, to the lack of models in vivo. Here we show that expression of the EMT master inducer Snail in primary adult Drosophila intestinal tumors leads to the dissemination of tumor cells and formation of macrometastases. Snail drives an EMT in tumor cells, which, although retaining some epithelial markers, subsequently break through the basal lamina of the midgut, undergo a collective migration and seed polyclonal metastases. While metastases re-epithelialize over time, we found that early metastases are remarkably mesenchymal, discarding the requirement for a mesenchymal-to-epithelial transition for early stages of metastatic growth. Our results demonstrate the formation of metastases in adult flies, and identify a key role for partial-EMTs in driving it. This model opens the door to investigate the basic mechanisms underlying metastasis, in a powerful in vivo system suited for rapid genetic and drug screens., This study was supported by grants from the Ministerio de Economía y competitividad (Grant Numbers BFU2014–59781-P (to A.C.), BFU2015–73494-JIN (to K.C.), BFU2015–66488-P (to J.C.)). This work was also supported by the Joseph Steiner Foundation (to E.B.) and a Wellcome Trust/Royal Society Sir Henry Dale Award to K.C. (Grant number R/148777–11–1).

Transforming growth factor (TGF)-β is a crucial enforcer of immune homeostasis and tolerance, inhibiting the expansion and function of many components of the immune system. Perturbations in TGF-β signaling underlie inflammatory diseases and promote tumor emergence. TGF-β is also central to immune suppression within the tumor microenvironment, and recent studies have revealed roles in tumor immune evasion and poor responses to cancer immunotherapy. Here, we present an overview of the complex biology of the TGF-β family and its context-dependent nature. Then, focusing on cancer, we discuss the roles of TGF-β signaling in distinct immune cell types and how this knowledge is being leveraged to unleash the immune system against the tumor.

Summary In vivo interrogation of the functional role of genes implicated in colorectal cancer (CRC) is limited by the need for physiological models that mimic the disease. Here, we describe a protocol that provides the steps required for the orthotopic co-implantation of tumoral and stromal cells into the cecum and rectum to investigate the crosstalk between the tumor and its microenvironment. This protocol recapitulates metastases to the lymph nodes, liver, and lungs observed in human CRC. For complete details on the use and execution of this protocol, please refer to Kasashima et al. (2020)., Graphical Abstract, Highlights • Orthotopic implantation in the cecum or rectum is a useful tool to study CRC • These methods recapitulate metastases of CRC to the lymph nodes, liver, and lungs • Co-implantation of tumor and stromal cells results in faster tumor progression, In vivo interrogation of the functional role of genes implicated in colorectal cancer (CRC) is limited by the need for physiological models that mimic the disease. Here, we describe a protocol that provides the steps required for the orthotopic co-implantation of tumoral and stromal cells into the cecum and rectum to investigate the crosstalk between the tumor and its microenvironment. This protocol recapitulates metastases to the lymph nodes, liver, and lungs observed in human CRC.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8

The tumor immune microenvironment is a main contributor to cancer progression and a promising therapeutic target for oncology. However, immune microenvironments vary profoundly between patients and biomarkers for prognosis and treatment response lack precision. A comprehensive compendium of tumor immune cells is required to pinpoint predictive cellular states and their spatial localization. We generated a single-cell tumor immune atlas, jointly analyzing >500,000 cells from 217 patients and 13 cancer types, providing the basis for a patient stratification based on immune cell compositions. Projecting immune cells from external tumors onto the atlas facilitated an automated cell annotation system for a harmonized interpretation. To enable in situ mapping of immune populations for digital pathology, we applied SPOTlight, combining single-cell and spatial transcriptomics data and identifying striking spatial immune cell patterns in tumor sections. We expect the tumor immune cell atlas, together with our versatile toolbox for precision oncology, to advance currently applied stratification approaches for prognosis and immuno-therapy.

Intestinal organoids capture essential features of the intestinal epithelium such as folding of the crypt, spatial compartmentalization of different cell types, and cellular movements from crypt to villus-like domains. Each of these processes and their coordination in time and space requires patterned physical forces that are currently unknown. Here we map the three-dimensional cell-ECM and cell-cell forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the ECM and folds through apical constriction, whereas the transit amplifying zone pulls the ECM and elongates through basal constriction. Tension measurements establish that the transit amplifying zone isolates mechanically the stem cell compartment and the villus-like domain. A 3D vertex model shows that the shape and force distribution of the crypt can be largely explained by cell surface tensions following the measured apical and basal actomyosin density. Finally, we show that cells are pulled out of the crypt along a gradient of increasing tension, rather than pushed by a compressive stress downstream of mitotic pressure as previously assumed. Our study unveils how patterned forces enable folding and collective migration in the intestinal crypt.

Summary Here, we describe a protocol to detect and visualize protein synthesis by click-chemistry-based immunofluorescence in patient-derived organoids (PDOs) in vitro. The protocol uses O-propargyl puromycin (OPP), an analog of puromycin that enters the acceptor site of ribosomes and is incorporated into nascent polypeptides. OPP can be detected by a click chemistry reaction and can be combined with conventional antibody staining. We describe procedures for imaging intact organoids in 3D format or imaging sections of organoids from paraffin blocks. For complete details on the use and execution of this protocol, please refer to Morral, Stanisavljevic et al. (2020)., Graphical Abstract, Highlights • Protocol allows for protein synthesis visualization in CRC organoids by OPP incorporation • Protein synthesis can be observed within intact organoids in 3D, or in paraffin sections • OPP detection can be combined with antibody staining, Here, we describe a protocol to detect and visualize protein synthesis by click-chemistry-based immunofluorescence in patient-derived organoids (PDOs) in vitro. The protocol uses O-propargyl puromycin (OPP), an analog of puromycin that enters the acceptor site of ribosomes and is incorporated into nascent polypeptides. OPP can be detected by a click chemistry reaction and can be combined with conventional antibody staining. We describe procedures for imaging intact organoids in 3D format or imaging sections of organoids from paraffin blocks.

Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas. This project has been made possible in part by grant no. 2018-182827 from the Chan Zuckerberg Initiative DAF, an advised fund of the Silicon Valley Community Foundation. H.H. is a Miguel Servet (CP14/00229) researcher funded by the Spanish Institute of Health Carlos III (ISCIII). C.M. is supported by an AECC postdoctoral fellowship. This work has received funding from the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement no. H2020-MSCA-ITN-2015-675752 (Singek), and the Ministerio de Ciencia, Innovación y Universidades (SAF2017-89109-P; AEI/FEDER, UE). S. was supported by the German Research Foundation’s (DFG’s) (GR4980) Behrens-Weise-Foundation. C.Z. was supported by the European Molecular Biology Organization through the long-term fellowship ALTF 673-2017. The snRNA-seq data were generated with support from the National Institute of Allergy and Infectious Diseases (grant no. U24AI118672), I.N. was supported by JST CREST (grant no. JPMJCR16G3) , Japan. A.J., L.E.W., J.W.B. and W.E. were supported by funding from the DFG (EN 1093/2-1 and SFB1243 TP A14). This publication is part of a project (BCLLATLAS) that received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 810287). Core funding was from the ISCIII and the Generalitat de Catalunya

The cancer stem cell (CSC) concept was proposed four decades ago, and states that tumor growth, analogous to the renewal of healthy tissues, is fueled by small numbers of dedicated stem cells. It has gradually become clear that many tumors harbor CSCs in dedicated niches, and yet their identification and eradication has not been as obvious as was initially hoped. Recently developed lineage-tracing and cell-ablation strategies have provided insights into CSC plasticity, quiescence, renewal, and therapeutic response. Here we discuss new developments in the CSC field in relationship to changing insights into how normal stem cells maintain healthy tissues. Expectations in the field have become more realistic, and now, the first successes of therapies based on the CSC concept are emerging.

The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient‐derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage‐tracing cassettes knocked in the LGR5 locus. Analysis of LGR5‐EGFP + cells isolated from organoid‐derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage‐tracing experiments showed that LGR5+ CRC cells self‐renew and generate progeny over long time periods that undergo differentiation toward mucosecreting‐ and absorptive‐like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.