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Generation and initial assignment of CRC single-cell RNA sequencing data. A Clinical data for the eight patients under investigation in this study. For mutational data, see also Supplementary table 6. Loc (Localisation): C: cecum; S: sigmoid colon; T: transverse colon; A: ascending colon; R: rectum. Prog (Predicted Progression): S: via serrated precursor; I: inflammatory/colitis-associated; C: canonical. Tissues used for single-cell RNA sequencing: N: Normal; T: Tumor; O: Organoid. B UMAPs of epithelial, immune and stromal cells, color-coded for patients. C UMAPs of epithelial, immune and stromal cells, color-coded by tissue of origin.
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In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to identify differences in cellular composition between normal colon and colorectal cancer, and to define signals controlling cancer cell development.
We used single cell RNA a...
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... RNA sequencing of CRC To capture the cellular diversity in CRC and track changes from normalcy to disease, we performed single-cell transcriptome analysis of eight previously untreated CRC patients (Fig. 1A). We utilized tissue samples that included the invasive tumor front and matched normal tissues ( Supplementary Fig. 1). Tumors under investigation encompass stages pTis (Tumor in situ) to pT4, that is, from cancer confined within the lamina propria to invasive through the visceral peritoneum, with or without metastasis, and with various ...
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... RNA sequencing of CRC To capture the cellular diversity in CRC and track changes from normalcy to disease, we performed single-cell transcriptome analysis of eight previously untreated CRC patients (Fig. 1A). We utilized tissue samples that included the invasive tumor front and matched normal tissues ( Supplementary Fig. 1). Tumors under investigation encompass stages pTis (Tumor in situ) to pT4, that is, from cancer confined within the lamina propria to invasive through the visceral peritoneum, with or without metastasis, and with various locations along the cephalocaudal axis of the colon. ...
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... observed that single-cell transcriptomes derived from all patients intermingled within the epithelial, immune, and stromal compartments (Fig. 1B). When distinguishing normal versus tumor samples, distributions of single-cell profiles largely overlapped, although several regions within each plot were preferentially inhabited by transcriptomes derived from either normal or tumor tissues (Fig. 1C). This indicates that our data are generally free from patient-or sample-specific ...
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... derived from all patients intermingled within the epithelial, immune, and stromal compartments (Fig. 1B). When distinguishing normal versus tumor samples, distributions of single-cell profiles largely overlapped, although several regions within each plot were preferentially inhabited by transcriptomes derived from either normal or tumor tissues (Fig. 1C). This indicates that our data are generally free from patient-or sample-specific batch effects confounding the cell type distributions, but that general differences occur between normal and tumor transcriptome ...
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... tumor samples in our analysis contained a small proportion of normal epithelial tissue (see Supplementary Fig. 1). To ascertain the origin of transcriptomes assigned to differentiated epithelial cell clusters, we calculated probabilities for single-cell transcriptomes to be derived from the tumor, taking into account RNA reads covering somatic mutations. ...
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... ascertain the origin of transcriptomes assigned to differentiated epithelial cell clusters, we calculated probabilities for single-cell transcriptomes to be derived from the tumor, taking into account RNA reads covering somatic mutations. While this approach successfully assigned a small fraction of transcriptomes as deriving from normal or tumor cells, respectively, a large majority of single-cell profiles remained unassigned (Supplementary Fig. 10). We conclude that single-cell transcriptomes acquired by our droplet-based sequencing platform contain insufficient information ...
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Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide. It is also the second most common cause of cancer‑associated mortality; it accounted for about 9.2% of all cancer deaths in 2018, most of which were due to resistance to therapy. The main treatment for CRC is surgery, generally associated with chemotherapy, radiation ther...
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... For the metabolic labeling of RNA in single cells, we made two major improvements: First, we made the method accessible for high-throughput microfluidics by performing the chemical conversion of 4sU in methanol fixed cells. Since microfluidic platforms such as 10X Chromium are becoming increasingly popular, the combination of both will pave the way for more studies of transcriptome dynamics, for example for studying the effects of drugs or novel compounds on cells and organoids (Uhlitz et al., 2020). For the first time, we applied this method in an in vivo system to study the fate of maternally provided transcripts by injecting 4sU into one-cell stage zebrafish embryos. ...
Die Lokalisierung von mRNA ist ein wichtiger regulativer Mechanismus in polarisierten Zellen und in frühen Embryonalstadien. Dort sind räumliche Muster maternaler mRNA für die korrekte Entwicklung der Körperachsen und die Spezifizierung der Keimzellen verantwortlich. Systematische Analysen dieser Prozesse wurden jedoch bisher limitiert durch einen Mangel an räumlicher und zeitlicher Auflösung von Einzelzell- Sequenzierungsdaten. Wir analysierten die Dynamik des räumlichen und zeitlichen Transkriptoms während frühen Embryonalstadien von Zebrafischen. Wir verbesserten Empfindlichkeit und Auflösung von tomo-seq und erfassten damit systematisch räumlich aufgelöste Transkriptome entlang der animal-vegetalen-Achse Embryonen im Einzell-Stadium und fanden 97 vegetal lokalisierte Gene. Außerdem etablierten wir eine Hochdurchsatz kompatible Variante der RNA-Markierungsmethode scSLAM-seq. Wir wendeten diese in Embryonen während der Gastrulation. Von den vegetal lokalisierten Genen waren 22 angereichert in Keimzellen, was eine funktionelle Rolle bei der Spezifizierung von Keimzellen nahelegt. Mit tomo-seq untersuchten wir die evolutionäre Konservierung der RNA-Lokalisierung zwischen Zebrafischen und gereiften Oozyten zweier Xenopus-Arten. Wir verglichen die lokalisierten Gene, suchten nach konservierten 3'UTR-Motiven, und fanden zum Teil überlappende Motive, was auf eine mögliche mechanistische Konservierung der Lokalisierungsmechanismen hinweist. Wir untersuchten auch RNA-Editierung von Adenin zu Inosin während der Embryonalentwicklung und in den Organen erwachsener Fische. In im Gehirn exprimierten Transkripten fanden wir 117 Editierstellen, die hauptsächlich für Ionentransporter kodieren und zum Teil zum Menschen konserviert sind. Die höchsten Editierraten konnten wir in Eierstöcken, Hoden und frühen Embryonen nachweisen, was auf eine mögliche Rolle bei der Regulierung der RNA-Stabilität hindeutet.
... The tumour microenvironment in CRC consists of multiple stromal and immune cell types, including cancer-associated fibroblasts, pericytes, monocytes, macrophages and a subset of T cells [88]. The numbers of CD66b-positive neutrophils are increased in CRC in comparison to adjacent mucosa, and a high density of intratumoural neutrophils is an independent factor for poor prognosis of patients with CRC. ...
In global cancer statistics, colorectal carcinoma (CRC) ranks third by incidence and second by mortality, causing 10.0% of new cancer cases and 9.4% of oncological deaths worldwide. Despite the development of screening programs and preventive measures, there are still high numbers of advanced cases. Multiple problems compromise the treatment of metastatic colorectal cancer, one of these being cancer stem cells—a minor fraction of pluripotent, self-renewing malignant cells capable of maintaining steady, low proliferation and exhibiting an intriguing arsenal of treatment resistance mechanisms. Currently, there is an increasing body of evidence for intricate associations between inflammation, epithelial–mesenchymal transition and cancer stem cells. In this review, we focus on inflammation and its role in CRC stemness development through epithelial–mesenchymal transition.
... Paracrine or microenvironment-derived EGFR stimuli can provide essential amplification of oncogenic MAPK signalling to promote tumour growth, as we also deduced from multiple PDX models of CRC cancer (Fig. 7). Accordingly, transcriptome analysis of human CRCs identified increased levels of paracrine EGFR stimuli in tumour tissue 60 . Autocrine production of EGFR-activating ligands has been demonstrated to predict the response to EGFR inhibition [61][62][63] . ...
Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC. Ponsioen et al. use a FRET‐based ERK biosensor EKAREN5 in patient‐derived organoids to show that EGFR activity amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways.
... In all the cohorts of CRC patients studied, S3 CRCs display a better survival than the two other groups [14]. A more recent study based on the analysis of >50,000 single cells from CRCs and matched normal tissues provided evidence that CRC development is associated in all cases analyzed with changes at the level of epithelial, immune and stromal cell compartments [15]. Interestingly, in the epithelium, five different tumor-specific stem and progenitor-like cell populations were identified [15]. ...
... A more recent study based on the analysis of >50,000 single cells from CRCs and matched normal tissues provided evidence that CRC development is associated in all cases analyzed with changes at the level of epithelial, immune and stromal cell compartments [15]. Interestingly, in the epithelium, five different tumor-specific stem and progenitor-like cell populations were identified [15]. This single-cell analysis showed also that epithelial tumor cells and cancer-associated fibroblasts are fundamental and essential for the assignment of each CRC to a given CMS subtype [15]. ...
... Interestingly, in the epithelium, five different tumor-specific stem and progenitor-like cell populations were identified [15]. This single-cell analysis showed also that epithelial tumor cells and cancer-associated fibroblasts are fundamental and essential for the assignment of each CRC to a given CMS subtype [15]. ...
Genome sequencing studies have characterized the genetic alterations of different tumor types, highlighting the diversity of the molecular processes driving tumor development. Comprehensive sequencing studies have defined molecular subtypes of colorectal cancers (CRCs) through the identification of genetic events associated with microsatellite stability (MSS), microsatellite-instability-high (MSI-H), and hypermutation. Most of these studies characterized primary tumors. Only recent studies have addressed the characterization of the genetic and clinical heterogeneity of metastatic CRC. Metastatic CRC genomes were found to be not fundamentally different from primary CRCs in terms of the mutational landscape or of genes that drive tumorigenesis, and a genomic heterogeneity associated with tumor location of primary tumors helps to define different clinical behaviors of metastatic CRCs. Although CRC metastatic spreading was traditionally seen as a late-occurring event, growing evidence suggests that this process can begin early during tumor development and the clonal architecture of these tumors is consistently influenced by cancer treatment. Although the survival rate of patients with metastatic CRC patients improved in the last years, the response to current treatments and prognosis of many of these patients remain still poor, indicating the need to discover new improvements for therapeutic vulnerabilities and to formulate a rational prospective of personalized therapies.
