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The peritoneum is a common site of dissemination in patients with colorectal cancer. In order to identify high-risk patients and improve therapeutic strategies, a better understanding of the peritoneal dissemination process and the reasons behind the high heterogeneity that is observed between patients is required. We aimed to create a murine model to further elucidate the process of peritoneal dissemination and to provide an experimental platform for further studies. We developed an in vivo model to assess patterns of peritoneal dissemination of 15 colorectal cancer cell lines. Immune deficient mice were intraperitoneally injected with 10,000 human colorectal cancer cells. Ten weeks after injection, or earlier in case of severe discomfort, the mice were sacrificed followed by dissection including assessment of the outgrowth and localization of peritoneal metastases. Furthermore, using a color-based clonal tracing method, the clonal dynamics of peritoneal nodules were observed. The different cell lines showed great variation in the extent of peritoneal outgrowth, ranging from no outgrowth to localized or widespread outgrowth of cells. An association between KRAS pathway activation and the formation of peritoneal metastases was identified. Also, cell line specific tumor location preferences were observed, with similar patterns of outgrowth in anatomically related areas. Furthermore, different patterns regarding clonal dynamics were found, varying from monoclonal or polyclonal outgrowth to extensively dispersed polyclonal lesions. The established murine model recapitulates heterogeneity as observed in human peritoneal metastases, which makes it a suitable platform for future (intervention) studies. The authors developed an in vivo model to assess patterns of peritoneal dissemination of colorectal cancer cell lines that recapitulates the high heterogeneity observed between patients. Great variation in the extent of peritoneal outgrowth, localization and clonal dynamics between cell lines was observed and a putative association with KRAS pathway activation was identified.

The intestinal epithelial lining is one of the most rapidly renewing cell populations in the body. As a result, the gut has been an attractive model to resolve key mechanisms in epithelial homeostasis. In particular the role of intestinal stem cells (ISCs) in the renewal process has been intensely studied. Interestingly, as opposed to the traditional stem cell theory, the ISC is not a static population but displays significant plasticity and in situations of tissue regeneration more differentiated cells can revert back to a stem cell state upon exposure to extracellular signals. Importantly, normal intestinal homeostasis provides important insight into mechanisms that drive colorectal cancer (CRC) development and growth. Specifically, the dynamics of cancer stem cells bear important resemblance to ISC functionality. In this review we present an overview of the current knowledge on ISCs in homeostasis and their role in malignant transformation. Also, we discuss the existence of stem cells in intestinal adenomas and CRC and how these cells contribute to (pre-)malignant growth. Furthermore, we will focus on new paradigms in the field of dynamical cellular hierarchies in CRC and the intimate relationship between tumor cells and their niche.

Cancer is a disease in which cells accumulate genetic aberrations that are believed to confer a clonal advantage over cells in the surrounding tissue. However, the quantitative benefit of frequently occurring mutations during tumor development remains unknown. We quantified the competitive advantage of Ape loss, Kras activation, and P53 mutations in the mouse intestine. Our findings indicate that the fate conferred by these mutations is not deterministic, and many mutated stem cells are replaced by wild-type stem cells after biased, but still stochastic events. Furthermore, P53 mutations display a condition-dependent advantage, and especially in colitis-affected intestines, clones harboring mutations in this gene are favored. Our work confirms the previously theoretical notion that the tissue architecture of the intestine suppresses the accumulation of mutated lineages.

Analysis of gene-expression profiles led to the identification of three molecularly distinct subtypes of colon cancer. One of these subtypes is new, and its identification is of clinical interest because it tends to have a particularly unfavorable prognosis and is refractory to existing targeted therapies. Colon cancer is a clinically diverse disease. This heterogeneity makes it difficult to determine which patients will benefit most from adjuvant therapy and impedes the development of new targeted agents 1 . More insight into the biological diversity of colon cancers, especially in relation to clinical features, is therefore needed. We demonstrate, using an unsupervised classification strategy involving over 1,100 individuals with colon cancer, that three main molecularly distinct subtypes can be recognized. Two subtypes have been previously identified and are well characterized (chromosomal-instable and microsatellite-instable cancers) 2 . The third subtype is largely microsatellite stable and contains relatively more CpG island methylator phenotype–positive carcinomas but cannot be identified on the basis of characteristic mutations. We provide evidence that this subtype relates to sessile-serrated adenomas, which show highly similar gene expression profiles, including upregulation of genes involved in matrix remodeling and epithelial-mesenchymal transition. The identification of this subtype is crucial, as it has a very unfavorable prognosis and, moreover, is refractory to epidermal growth factor receptor–targeted therapy.

Solid malignancies have been speculated to depend on cancer stem cells (CSCs) for expansion and relapse after therapy. Here we report on quantitative analyses of lineage tracing data from primary colon cancer xenograft tissue to assess CSC functionality in a human solid malignancy. The temporally obtained clone size distribution data support a model in which stem cell function in established cancers is not intrinsically, but is entirely spatiotemporally orchestrated. Functional stem cells that drive tumour expansion predominantly reside at the tumour edge, close to cancer-associated fibroblasts. Hence, stem cell properties change in time depending on the cell location. Furthermore, although chemotherapy enriches for cells with a CSC phenotype, in this context functional stem cell properties are also fully defined by the microenvironment. To conclude, we identified osteopontin as a key cancer-associated fibroblast-produced factor that drives in situ clonogenicity in colon cancer. Lenos et al. report that the spatiotemporal regulation of stem cell functionality is not intrinsically determined but environmentally defined during tumour growth and drug response in colon cancer.

Cancer stem cell activity may not merely be an intrinsic characteristic of a subset of cancer cells and could be regulated by environmental cues. High Wnt activity in human colorectal cancer designates a tumour-initiating population and is orchestrated by the microenvironment. Despite the presence of mutations in APC or β-catenin, which are believed to activate the Wnt signalling cascade constitutively, most colorectal cancers show cellular heterogeneity when β-catenin localization is analysed, indicating a more complex regulation of Wnt signalling. We explored this heterogeneity with a Wnt reporter construct and observed that high Wnt activity functionally designates the colon cancer stem cell (CSC) population. In adenocarcinomas, high activity of the Wnt pathway is observed preferentially in tumour cells located close to stromal myofibroblasts, indicating that Wnt activity and cancer stemness may be regulated by extrinsic cues. In agreement with this notion, myofibroblast-secreted factors, specifically hepatocyte growth factor, activate β-catenin-dependent transcription and subsequently CSC clonogenicity. More significantly, myofibroblast-secreted factors also restore the CSC phenotype in more differentiated tumour cells both in vitro and in vivo . We therefore propose that stemness of colon cancer cells is in part orchestrated by the microenvironment and is a much more dynamic quality than previously expected that can be defined by high Wnt activity.

Cancer evolution is predominantly studied by focusing on differences in the genetic characteristics of malignant cells within tumors. However, the spatiotemporal dynamics of clonal outgrowth that underlie evolutionary trajectories remain largely unresolved. Here, we sought to unravel the clonal dynamics of colorectal cancer (CRC) expansion in space and time by using a color-based clonal tracing method. This method involves lentiviral red-green-blue (RGB) marking of cell populations, which enabled us to track individual cells and their clonal outgrowth during tumor initiation and growth in a xenograft model. We found that clonal expansion largely depends on the location of a clone, as small clones reside in the center and large clones mostly drive tumor growth at the border. These dynamics are recapitulated in a computational model, which confirms that the clone position within a tumor rather than cell-intrinsic features, is crucial for clonal outgrowth. We also found that no significant clonal loss occurs during tumor growth and clonal dispersal is limited in most models. Our results imply that, in addition to molecular features of clones such as (epi-)genetic differences between cells, clone location and the geometry of tumor growth are crucial for clonal expansion. Our findings suggest that either microenvironmental signals on the tumor border or differences in physical properties within the tumor, are major contributors to explain heterogeneous clonal expansion. Thus, this study provides further insights into the dynamics of solid tumor growth and progression, as well as the origins of tumor cell heterogeneity in a relevant model system.

Intestinal tumour formation is generally thought to occur following mutational events in the stem cell pool. However, active NF-κB signalling additionally facilitates malignant transformation of differentiated cells. We hypothesized that genes shared between NF-κB and intestinal stem cell (ISCs) signatures might identify common pathways that are required for malignant growth. Here, we find that the NF-κB target Bcl-2 , an anti-apoptotic gene, is specifically expressed in ISCs in both mice and humans. Bcl-2 is dispensable in homeostasis and, although involved in protecting ISCs from radiation-induced damage, it is non-essential in tissue regeneration. Bcl-2 is upregulated in adenomas, and its loss or inhibition impairs outgrowth of oncogenic clones, because Bcl-2 alleviates apoptotic priming in epithelial cells following Apc loss. Furthermore, Bcl-2 expression in differentiated epithelial cells renders these cells amenable to clonogenic outgrowth. Collectively, our results indicate that Bcl-2 is required for efficient intestinal transformation following Apc -loss and constitutes a potential chemoprevention target. The anti-apoptotic protein Bcl-2 is selectively expressed in intestinal stem cells (ISCs). Here, the authors show that, in intestinal stem cells, Bcl-2 alleviates apoptotic priming induced by the loss of the tumour suppressor Apc in ISCs and that the absence of Bcl-2 or pharmacological blockade of Bcl-2 can inhibit the intestinal tumorigenesis driven by the Apc-loss.

To design and validate a practical questionnaire for clinicians, to identify barriers and facilitators for evidence-based practice (EBP), that is, the use of research evidence in patient care. The inventory is ultimately intended for departments to assess local conditions for EBP, to aim and evaluate efforts at improving or maximizing EBP. We derived candidate items from existing EBP scales, psychology, and behavioral economics. In an online Delphi study, 537 international expert clinicians, researchers, teachers, and policymakers interested in EBP identified items with sufficient face and content validity. We piloted and validated the resulting draft inventory among 127 clinicians from various specialties and career stages. The Delphi study started with 114 items and resulted in a draft inventory with 29 items in five dimensions. During the pilot, the inventory was easy to complete within 15 minutes and the items showed sufficient response variation. In four of five dimensions, test–retest reliability was substantial to almost perfect and the power to discriminate between groups with different expertise was adequate, whereas internal consistency showed that the items generally measured the same construct. On the basis of internal consistency and factor analysis, we excluded three items. The final EBP inventory consists of 26 items in five dimensions: decision making, subjective norm, attitude, perceived behavior control, and intention and behavior. The EBP inventory was developed with support of EBP experts and validated among various academic clinicians. It shows adequate face and content validity, internal consistency, test–retest reliability, discriminative power, and completion will take <15 minutes. We recommend further evaluation of its value in field trials.

People living with HIV (PLHIV) are characterized by functional reprogramming of innate immune cells even after long-term antiretroviral therapy (ART). In order to assess technical feasibility of omics technologies for application to larger cohorts, we compared multiple omics data layers. Bulk and single-cell transcriptomics, flow cytometry, proteomics, chromatin landscape analysis by ATAC-seq as well as drug stimulation were performed in a small number of blood samples derived from PLHIV and healthy controls from the 200-HIV cohort study. Single-cell RNA-seq analysis revealed that most immune cells in peripheral blood of PLHIV are altered in their transcriptomes and that a specific functional monocyte state previously described in acute HIV infection is still existing in PLHIV while other monocyte cell states are only occurring acute infection. Further, a reverse transcriptome approach on a rather small number of PLHIV was sufficient to identify drug candidates for reversing the transcriptional phenotype of monocytes in PLHIV. These scientific findings and technological advancements for clinical application of single-cell transcriptomics form the basis for the larger 2000-HIV multicenter cohort study on PLHIV, for which a combination of bulk and single-cell transcriptomics will be included as the leading technology to determine disease endotypes in PLHIV and to predict disease trajectories and outcomes.