Search

The sebaceous gland (SG) is a vital appendage of the epidermis, and its normal homeostasis and function is crucial for effective maintenance of the skin barrier. Notch signaling is a well-known regulator of epidermal differentiation, and has also been shown to be involved in postnatal maintenance of SGs. However, the precise role of Notch signaling in regulating SG differentiation in the adult homeostatic skin remains unclear. While there is evidence to suggest that Notch1 is the primary Notch receptor involved in regulating the differentiation process, the ligand remains unknown. Using monoclonal therapeutic antibodies designed to specifically inhibit of each of the Notch ligands or receptors, we have identified the Jag2/Notch1 signaling axis as the primary regulator of sebocyte differentiation in mouse homeostatic skin. Mature sebocytes are lost upon specific inhibition of the Jag2 ligand or Notch1 receptor, resulting in the accumulation of proliferative stem/progenitor cells in the SG. Strikingly, this phenotype is reversible, as these stem/progenitor cells re-enter differentiation when the inhibition of Notch activity is lifted. Thus, Notch activity promotes correct sebocyte differentiation, and is required to restrict progenitor proliferation.

BackgroundThe occurrence of peritoneal metastasis (PM) in patients with colorectal cancer (CRC) has a dismal prognosis. There is often limited response to systemic- and immunotherapy, even in microsatellite unstable (MSI) CRC. To overcome therapy resistance, it is critical to understand local immune environment in the peritoneal cavity, and to develop models to study anti-tumor immune responses. Here, we defined the peritoneal immune system (PerIS) in PM-CRC patients and evaluate the pre-clinical potential of a humanized immune system (HIS) mouse model for PM-CRC.MethodsWe studied the human PerIS in PM-CRC patients (n=20; MSS 19/20; 95%) and in healthy controls (n=3). HIS mice (NODscid gamma background; n=18) were generated, followed by intraperitoneal injection of either saline (HIS control; n=3) or human MSS/MSI CRC cell lines HUTU80, MDST8 and HCT116 (HIS-PM, n=15). Immune cells in peritoneal fluid and peritoneal tumors were analyzed using cytometry by time of flight (CyTOF).ResultsThe human and HIS mouse homeostatic PerIS was equally populated by NK cells and CD4+- and CD8+ T cells, however differences were observed in macrophage and B cell abundance. In HIS mice, successful peritoneal engraftment of both MSI and MSS tumors was observed (15/15; 100%). Both in human PM-CRC and in the HIS mouse PM-CRC model, we observed that MSS PM-CRC triggered a CD4+ Treg response in the PerIS, while MSI PM-CRC drives CD8+ TEMs responses.ConclusionIn conclusion, T cell responses in PM-CRC in HIS mice mirror those in human PM-CRC, making this model suitable to study antitumor T cell responses in PM-CRC.

Abstract Clonal growth and competition underlie processes of key relevance in etiology, progression and therapy response across all cancers. Here, we demonstrate a novel experimental approach, based on multi-color, fluorescent tagging of cell nuclei, in combination with picoliter droplet deposition, to study the clonal dynamics in two- and three-dimensional cell cultures. The method allows for the simultaneous visualization and analysis of multiple clones in individual multi-clonal colonies, providing a powerful tool for studying clonal dynamics and identifying clonal populations with distinct characteristics. Results of our experiments validate the utility of the method in studying clonal dynamics in vitro, and reveal differences in key aspects of clonal behavior of different cancer cell lines in monoculture conditions, as well as in co-cultures with stromal fibroblasts.

Abstract Cell‐free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short‐read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor‐derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated (R = 0.98) with the tumor fraction calculated from short‐read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment‐end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor‐derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost‐effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations.

Abstract Background Existing methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection. Results We survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81. Conclusions The mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.

Summary: Long non-coding RNAs (lncRNAs) are emerging as key regulators in the initiation, growth, and progression of cancer. High-throughput CRISPR-based techniques systematically assess the function of genes or regulatory elements present in the human genome. Here, we present a protocol for identifying essential lncRNAs in cancer using CRISPRi-based dropout screens. We describe steps to select target sites, design guide RNAs, and generate CRISPRi cell lines. We then detail the execution and analysis of CRISPRi-based dropout screens. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal infiltration (Guinney et al., Nat Med 21:1350–6, 2015; Linnekamp et al., Cell Death Differ 25:616–33, 2018), whereas clinically it is characterized by lower responses to adjuvant therapy, higher incidence of metastatic spreading and hence dismal prognosis (Buikhuisen et al., Oncogenesis 9:66, 2020). Methods To understand the biology of the mesenchymal subtype and unveil specific vulnerabilities, a large CRISPR-Cas9 drop-out screen was performed on 14 subtyped CRC cell lines to uncover essential kinases in all CMSs. Dependency of CMS4 cells on p21-activated kinase 2 (PAK2) was validated in independent 2D and 3D in vitro cultures and in vivo models assessing primary and metastatic outgrowth in liver and peritoneum. TIRF microscopy was used to uncover actin cytoskeleton dynamics and focal adhesion localization upon PAK2 loss. Subsequent functional assays were performed to determine altered growth and invasion patterns. Results PAK2 was identified as a key kinase uniquely required for growth of the mesenchymal subtype CMS4, both in vitro and in vivo. PAK2 plays an important role in cellular attachment and cytoskeletal rearrangements (Coniglio et al., Mol Cell Biol 28:4162–72, 2008; Grebenova et al., Sci Rep 9:17171, 2019). In agreement, deletion or inhibition of PAK2 impaired actin cytoskeleton dynamics in CMS4 cells and, as a consequence, significantly reduced invasive capacity, while it was dispensable for CMS2 cells. Clinical relevance of these findings was supported by the observation that deletion of PAK2 from CMS4 cells prevented metastatic spreading in vivo. Moreover, growth in a model for peritoneal metastasis was hampered when CMS4 tumor cells were deficient for PAK2. Conclusion Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.

Abstract Peritoneal metastases are a common form of tumor cell dissemination in gastrointestinal malignancies. Peritoneal metastatic disease (PMD) is associated with severe morbidity and resistance to currently employed therapies. Given the distinct route of dissemination compared with distant organ metastases, and the unique microenvironment of the peritoneal cavity, specific tumor cell characteristics are needed for the development of PMD. In this review, we provide an overview of the known histopathological, genomic, and transcriptomic features of PMD. We find that cancers representing the mesenchymal subtype are strongly associated with PMD in various malignancies. Furthermore, we discuss the peritoneal niche in which the metastatic cancer cells reside, including the critical role of the peritoneal immune system. Altogether, we show that PMD should be regarded as a distinct disease entity, that requires tailored treatment strategies.

Abstract The majority of colorectal cancers (CRCs) present with early mutations in tumor suppressor gene APC. APC mutations result in oncogenic activation of the Wnt pathway, which is associated with hyperproliferation, cytoskeletal remodeling, and a global increase in mRNA translation. To compensate for the increased biosynthetic demand, cancer cells critically depend on protein chaperones to maintain proteostasis, although their function in CRC remains largely unexplored. In order to investigate the role of molecular chaperones in driving CRC initiation, we captured the transcriptomic profiles of murine wild type and Apc‐mutant organoids during active transformation. We discovered a strong transcriptional upregulation of Hspb1, which encodes small heat shock protein 25 (HSP25). We reveal an indispensable role for HSP25 in facilitating Apc‐driven transformation, using both in vitro organoid cultures and mouse models, and demonstrate that chemical inhibition of HSP25 using brivudine reduces the development of premalignant adenomas. These findings uncover a hitherto unknown vulnerability in intestinal transformation that could be exploited for the development of chemopreventive strategies in high‐risk individuals.

Colorectal cancer can lead to the development of peritoneal metastases, which are associated with worse disease outcome. Here, the authors characterize peritoneal metastases from 52 patients using RNA-seq and mutational sequencing and show a distinct molecular subtype.

Abstract Background Familial adenomatous polyposis (FAP) is a rare autosomal dominant disease characterized by germline mutations in the Adenomatous Polyposis Coli (APC) gene, resulting in the development of numerous colorectal adenomas. As these patients have a high risk of developing colorectal cancer (CRC), guidelines suggest prophylactic colectomy during early adulthood, however, adenoma development is still observed in the remaining intestinal tract. Therefore, FAP patients would benefit from chemoprevention strategies reducing the development of adenomas. Recent work in mice reveals a chemopreventive effect of lithium on the development of adenomas by inhibiting the expansion of Apc mutated intestinal stem cells (ISCs) within the crypts of normal intestinal mucosa. Here, we aim to investigate the effect of lithium on the spread of APC mutant cells within the human intestinal epithelium. Methods This prospective phase II single arm trial has a duration of 18 months. FAP patients (18–35 years) with a genetically confirmed APC mutation who did not undergo colectomy will be treated with lithium carbonate orally achieving a serum level of 0.2–0.4 mmol/l between month 6 and 12. Colonoscopy with biopsies of normal intestinal mucosa will be performed at baseline and every six months. The primary endpoint is the effect of lithium on the spread of APC mutant cells within intestinal crypts over time by using APC specific marker NOTUM in situ hybridization. Secondary endpoints include change in adenoma burden, patient reported side effects and safety-outcomes. Total sample size is 12 patients and recruitment will take place in the Amsterdam UMC, location AMC in the Netherlands. Discussion The outcome of this study will function as a proof-of-concept for the development of novel chemoprevention approaches that interfere with the competition between normal and mutant ISCs. Trial registration: ClinicalTrials.gov ( https://clinicaltrials.gov/ ): NCT05402891 (June 1, 2022) and the EU Clinical Trials Register: EuraCT 2022-000240-30 (January 1, 2022).

Previously, colorectal cancer (CRC) has been classified into four distinct molecular subtypes based on transcriptome data. These consensus molecular subtypes (CMSs) have implications for our understanding of tumor heterogeneity and the prognosis of patients. So far, this classification has been based on the use of messenger RNAs (mRNAs), although microRNAs (miRNAs) have also been shown to play a role in tumor heterogeneity and biological differences between CMSs. In contrast to mRNAs, miRNAs have a smaller size and increased stability, facilitating their detection. Therefore, we built a miRNA‐based CMS classifier by converting the existing mRNA‐based CMS classification using machine learning (training dataset of n = 271). The performance of this miRNA‐assigned CMS classifier (CMS‐miRaCl) was evaluated in several datasets, achieving an overall accuracy of ~ 0.72 (0.6329–0.7987) in the largest dataset (n = 158). To gain insight into the biological relevance of CMS‐miRaCl, we evaluated the most important features in the classifier. We found that miRNAs previously reported to be relevant in microsatellite‐instable CRCs or Wnt signaling were important features for CMS‐miRaCl. Following further studies to validate its robustness, this miRNA‐based alternative might simplify the implementation of CMS classification in clinical workflows.

Abstract Background Recently it has been recognized that stromal markers could be used as a clinically relevant biomarker for therapy response and prognosis. Here, we report on a serum marker for stromal activation, A Disintegrin and Metalloprotease 12 (ADAM12) in colorectal cancer (CRC). Methods Using gene expression databases we investigated ADAM12 expression in CRC and delineated the source of ADAM12 expression. The clinical value of ADAM12 was retrospectively assessed in the CAIRO2 trial in metastatic CRC with 235 patients (31% of total cohort), and an independent rectal cancer cohort (n = 20). Results ADAM12 is expressed by activated CRC associated fibroblasts. In the CAIRO2 trial cohort, ADAM12 serum levels were prognostic (ADAM12 low versus ADAM12 high; median OS 25.3 vs. 17.1 months, HR 1.48 [95% CI 1.11–1.96], P = 0.007). The prognostic potential was specifically high for metastatic rectal cancer (HR 1.78 [95% CI 1.06–3.00], P = 0.030) and mesenchymal subtype tumors (HR 2.12 [95% CI 1.25–3.60], P = 0.004). ADAM12 also showed potential for predicting recurrence in an exploratory analysis of non-metastatic rectal cancers. Conclusions Here we describe a non-invasive marker for activated stroma in CRC which associates with poor outcome, especially for primary cancers located in the rectum.

Intratumour heterogeneity (ITH) is associated with worse prognosis in cancer, and efficient frameworks to measure it are needed. Here the authors develop a method to estimate copy number heterogeneity, and propose that it is driven by chromosomal instability and can predict pan-cancer survival.

Abstract Intratumor heterogeneity (ITH) is an omnipresent property of cancers and predicts poor survival in most types of cancer. The propensity to metastasize and the regrowth of tumors after therapy are both associated with ITH. Quantification of the level of ITH in a malignancy is hence of great interest, and accurate inference of ITH could guide clinical decision making. However, ITH is an emergent property of billions of cells and requires mathematical modeling for inference from a limited number of measurements. Over the last decade, numerous mathematical and computational models have been introduced to infer ITH from variant‐allele frequencies, copy number variations, or from data of experimental model systems. These quantitative modeling efforts have advanced the understanding of tumor evolution, underlined poor prognosis associated with ITH, and elucidated the importance of functional heterogeneity, that is, cancer stem cells. Yet, a comprehensive overview of the different mathematical models, their underlying assumptions, their limitations, and their strengths is missing. In this Perspective, we highlight the achievements of mathematical modeling and present a framework which allows better understanding of the mathematical models themselves.

Abstract Background Cancer results from the accumulation of mutations leading to the acquisition of cancer promoting characteristics such as increased proliferation and resistance to cell death. In colorectal cancer, an early mutation leading to such features usually occurs in the APC or CTNNB1 genes, thereby activating Wnt signalling. However, substantial phenotypic differences between cancers originating within the same organ, such as molecular subtypes, are not fully reflected by differences in mutations. Indeed, the phenotype seems to result from a complex interplay between the cell-intrinsic features and the acquired mutations, which is difficult to disentangle when established tumours are studied. Methods We use a 3D in vitro organoid model to study the early phase of colorectal cancer development. From three different murine intestinal locations we grow organoids. These are transformed to resemble adenomas after Wnt activation through lentiviral transduction with a stable form of β-Catenin. The gene expression before and after Wnt activation is compared within each intestinal origin and across the three locations using RNA sequencing. To validate and generalize our findings, we use gene expression data from patients. Results In reaction to Wnt activation we observe downregulation of location specific genes and differentiation markers. A similar effect is seen in patient data, where genes with significant differential expression between the normal left and right colon are downregulated in the cancer samples. Furthermore, the signature of Wnt target genes differs between the three intestinal locations in the organoids. The location specific Wnt signatures are dominated by genes which have been lowly expressed in the tissue of origin, and are the targets of transcription factors that are activated following enhanced Wnt signalling. Conclusion We observed that the region-specific cell identity has a substantial effect on the reaction to Wnt activation in a simple intestinal adenoma model. These findings provide a way forward in resolving the distinct biology between left- and right-sided human colon cancers with potential clinical relevance.

Abstract Histological ‘phenotypic subtypes’ that classify patients into four groups (immune, canonical, latent and stromal) have previously been demonstrated to stratify survival in a stage I–III colorectal cancer (CRC) pilot cohort. However, clinical utility has not yet been validated. Therefore, this study assessed prognostic value of these subtypes in additional patient cohorts along with associations with risk of recurrence and response to chemotherapy. Two independent stage I–III CRC patient cohorts (internal and external cohort) were utilised to investigate phenotypic subtypes. The primary endpoint was disease‐free survival (DFS) and the secondary endpoint was recurrence risk (RR). Stage II–III patients, from the SCOT adjuvant chemotherapy trial, were utilised to further validate prognostic value and for exploratory analysis assessing associations with adjuvant chemotherapy. In an 893‐patient internal cohort, phenotypic subtype independently associated with DFS (p = 0.025) and this was attenuated in stage III patients (p = 0.020). Phenotypic subtype also independently associated with RR (p

Drug synergies impact the efficacy of combination therapies but are difficult to identify. Here Narayan et al. describe the drug atlas, a method to predict effective drug combinations from common exclusive drug effects providing a resource for exploring and understanding effective drug combinations.

Summary: Intestinal organoid cultures are a powerful tool to study epithelial cells in vitro, as they are able to proliferate and differentiate into all cell lineages observed in vivo. Co-culturing organoids with distinct genetic backgrounds provides an excellent approach to study contact dependent and independent interactions between healthy and mutant epithelial intestinal cells. Here, we provide 2D and 3D approaches to mouse organoid co-cultures using fluorescently labeled organoids and demonstrate the analysis of these co-cultures using flow cytometry and microscopy-based approaches.For complete details on the use and execution of this profile, please refer to van Neerven et al., 2021.

Summary: Effective treatments for pancreatic ductal adenocarcinoma (PDAC) are lacking, and targeted agents have demonstrated limited efficacy. It has been speculated that a rare population of cancer stem cells (CSCs) drives growth, therapy resistance, and rapid metastatic progression in PDAC. These CSCs demonstrate high clonogenicity in vitro and tumorigenic potential in vivo. However, their relevance in established PDAC tissue has not been determined. Here, we use marker-independent stochastic clonal labeling, combined with quantitative modeling of tumor expansion, to uncover PDAC tissue growth dynamics. We find that in contrast to the CSC model, all PDAC cells display clonogenic potential in situ. Furthermore, the proximity to activated cancer-associated fibroblasts determines tumor cell clonogenicity. This means that the microenvironment is dominant in defining the clonogenic activity of PDAC cells. Indeed, manipulating the stroma by Hedgehog pathway inhibition alters the tumor growth mode, revealing that tumor-stroma crosstalk shapes tumor growth dynamics and clonal architecture.

The exact immunopathophysiology of community-acquired pneumonia (CAP) caused by SARS-CoV-2 (COVID-19) remains clouded by a general lack of relevant disease controls. The scarcity of single-cell investigations in the broader population of patients with CAP renders it difficult to distinguish immune features unique to COVID-19 from the common characteristics of a dysregulated host response to pneumonia. We performed integrated single-cell transcriptomic and proteomic analyses in peripheral blood mononuclear cells from a matched cohort of eight patients with COVID-19, eight patients with CAP caused by Influenza A or other pathogens, and four non-infectious control subjects. Using this balanced, multi-omics approach, we describe shared and diverging transcriptional and phenotypic patterns—including increased levels of type I interferon-stimulated natural killer cells in COVID-19, cytotoxic CD8 T EMRA cells in both COVID-19 and influenza, and distinctive monocyte compositions between all groups—and thereby expand our understanding of the peripheral immune response in different etiologies of pneumonia.

We recently introduced a method to derive intra-tumor heterogeneity (ITH) from a single copy number measurement. This method stratifies patients for survival and could potentially help to identify low and high-risk patients with clinical relevance.

Abstract 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.

Organs-on-chips are in vitro models in which human tissues are cultured in microfluidic compartments with a controlled, dynamic micro-environment. Specific organs-on-chips are being developed to mimic human tumors, but the validation of such ‘cancer-on-chip’ models for use in drug development is hampered by the complexity and variability of human tumors. An important step towards validation of cancer-on-chip technology could be to first mimic cancer xenograft models, which share multiple characteristics with human cancers but are significantly less complex. Here we review the relevant biological characteristics of a xenograft tumor and show that organ-on-chip technology is capable of mimicking many of these aspects. Actual comparisons between on-chip tumor growth and xenografts are promising but also demonstrate that further development and empirical validation is still needed. Validation of cancer-on-chip models to xenografts would not only represent an important milestone towards acceptance of cancer-on-chip technology, but could also improve drug discovery, personalized cancer medicine, and reduce animal testing.

The cancer xenograft model in which human cancer cells are implanted in a mouse is one of the most used preclinical models to test the efficacy of novel cancer drugs. However, the model is imperfect; animal models are ethically burdened, and the imperfect efficacy predictions contribute to high clinical attrition of novel drugs. If microfluidic cancer-on-chip models could recapitulate key elements of the xenograft model, then these models could substitute the xenograft model and subsequently surpass the xenograft model by reducing variation, increasing sensitivity and scale, and adding human factors. Here, we exposed HCT116 colorectal cancer spheroids to dynamic, in vivo-like, concentrations of oxaliplatin, including a 5 day drug-free period, on-chip. Growth inhibition on-chip was comparable to existing xenograft studies. Furthermore, immunohistochemistry showed a similar response in proliferation and apoptosis markers. While small volume changes in xenografts are hard to detect, in the chip-system, we could observe a temporary growth delay. Lastly, histopathology and a pharmacodynamic model showed that the cancer spheroid-on-chip was representative of the proliferating outer part of a HCT116 xenograft, thereby capturing the major driver of the drug response of the xenograft. Hence, the cancer-on-chip model recapitulated the response of HCT116 xenografts to oxaliplatin and provided additional drug efficacy information.

Background and study aims Microsatellite instability accelerates colorectal cancer development in patients with Lynch syndrome (LS). Previous research showed that virtual chromoendoscopy increases detection of adenomas during colonoscopy surveillance of patients with LS. Because previous research revealed that Lynch patients have an increased vascular network in the oral mucosa, we hypothesized that increased vascularization of LS-associated adenomas is the cause of better detection with virtual chromoendoscopy. Patients and methods In this pilot study, patients with LS having a proven germline mutation were selected from two tertiary referral hospitals and non-LS patients from an outpatient colonoscopy center. Adenomas from patients with LS were exactly matched in size and histology with adenomas from non-LS patients. Initial adenoma diagnosis was confirmed by a specialist pathologist. All adenomas were stained with CD31 and adenomatous tissue was annotated by the specialist pathologist. Image analysis of CD31-positive microvessel density was conducted using FIJI software. Results Colonoscopy of 63 patients with LS and 24 non-LS patients provided 40 adenomas that could be exactly matched in size and histology. In image-analysis, the CD31-positive microvessel density (2.49 % vs. 2.47 %, P = 0.96), the average size of CD31-positive structures (514 μm2 vs. 523 μm2, P = 0.26) nor the amount of vascular structures per mm2 (183 vs. 176, P = 0.50) differed between adenomas of LS patients and non-Lynch patients. Conclusion The outcomes of this pilot case-control study did not provide further insights into the mechanism of increased adenoma detection in LS patients using virtual chromoendoscopy techniques.

By using marker-free lineage tracing in combination with quantitative analysis, we recently revealed cancer stem cell functionality in established human colon cancer is not intrinsically defined, but fully spatiotemporally regulated.

Abstract The heterogeneous nature of colorectal cancer (CRC) complicates prognosis and is suggested to be a determining factor in the efficacy of adjuvant therapy for individual patients. Based on gene expression profiling, CRC is currently classified into four consensus molecular subtypes (CMSs), characterized by specific biological programs, thus suggesting the existence of unifying developmental drivers for each CMS. Using human organoid cultures, we investigated the role of such developmental drivers at the premalignant stage of distinct CRC subtypes and found that TGFβ plays an important role in the development of the mesenchymal CMS4, which is of special interest due to its association with dismal prognosis. We show that in tubular adenomas (TAs), which progress to classical CRCs, the dominating response to TGFβ is death by apoptosis. By contrast, induction of a mesenchymal phenotype upon TGFβ treatment prevails in a genetically engineered organoid culture carrying a BRAFV600E mutation, constituting a model system for sessile serrated adenomas (SSAs). Our data indicate that TGFβ signaling is already active in SSA precursor lesions and that TGFβ is a critical cue for directing SSAs to the mesenchymal, poor‐prognosis CMS4 of CRC.

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.

Colon cancer stem cells (colon-CSCs) are more resistant to conventional chemotherapy than differentiated cancer cells. This subset of therapy refractory cells is therefore believed to play an important role in post-therapeutic tumor relapse. In order to improve the rate of sustained response to conventional chemotherapy, development of approaches is warranted that specifically sensitize colon-CSCs to treatment. Here, we report that ER-stress-induced activation of the unfolded protein response (UPR) forces colon-CSCs to differentiate, resulting in their enhanced sensitivity to chemotherapy in vitro and in vivo. Our data suggest that agents that induce activation of the UPR may be used to specifically increase sensitivity of colon-CSCs to the effects of conventional chemotherapy.

CD44 marks stem cell-like cells in a number of tumour types, including colorectal cancer (CRC), while aberrant CD44 expression conveys increased tumourigenic, invasive, and metastatic potential. Previous data indicate that CD44 is a direct target of p53-mediated transcriptional repression in breast cancer. Since inactivating p53 mutations are frequent genetic events in CRC these could unleash expression of CD44. In the present study, we therefore explored the relation between p53 mutational status and CD44 expression in a cohort of 90 localized primary CRCs and studied the effect of radiation-induced p53 activation on CD44 expression. Interestingly, we observed that, in contrast to breast cancer, loss of function p53 mutations were not associated with elevated CD44 expression in colon cancer. Moreover, DNA-damage induced p53 activation did not result in repression of CD44 expression, neither in colon cancer cells nor in normal intestinal epithelial cells. Our data demonstrate that CD44 expression in normal and malignant intestinal epithelial cells is not regulated by p53, implying that regulation of this potentially important therapeutic target is tissue and cancer-type specific.

The cancer stem cell (CSC) concept is a highly debated topic in cancer research. While experimental evidence in favor of the cancer stem cell theory is apparently abundant, the results are often criticized as being difficult to interpret. An important reason for this is that most experimental data that support this model rely on transplantation studies. In this study we use a novel cellular Potts model to elucidate the dynamics of established malignancies that are driven by a small subset of CSCs. Our results demonstrate that epigenetic mutations that occur during mitosis display highly altered dynamics in CSC-driven malignancies compared to a classical, non-hierarchical model of growth. In particular, the heterogeneity observed in CSC-driven tumors is considerably higher. We speculate that this feature could be used in combination with epigenetic (methylation) sequencing studies of human malignancies to prove or refute the CSC hypothesis in established tumors without the need for transplantation. Moreover our tumor growth simulations indicate that CSC-driven tumors display evolutionary features that can be considered beneficial during tumor progression. Besides an increased heterogeneity they also exhibit properties that allow the escape of clones from local fitness peaks. This leads to more aggressive phenotypes in the long run and makes the neoplasm more adaptable to stringent selective forces such as cancer treatment. Indeed when therapy is applied the clone landscape of the regrown tumor is more aggressive with respect to the primary tumor, whereas the classical model demonstrated similar patterns before and after therapy. Understanding these often counter-intuitive fundamental properties of (non-)hierarchically organized malignancies is a crucial step in validating the CSC concept as well as providing insight into the therapeutical consequences of this model.