bims-instec Biomed News
on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration
Issue of 2022‒08‒21
eight papers selected by
Maria-Virginia Giolito

  1. J Vis Exp. 2022 Jul 27.
      The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.
  2. Gastroenterology. 2022 Aug 10. pii: S0016-5085(22)00912-X. [Epub ahead of print]
      BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown.METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by scRNA-seq analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCT, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells was evaluated using EdU pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation and 5-FU-induced mucosal injury.
    RESULTS: ScRNA-seq analysis illuminated the presence of non-dividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. TGF-β signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth following 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating.
    CONCLUSIONS: Our results highlighted the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.
    Keywords:  Intestinal stem cells; Organoids; Slow-cycling stem cell; TGF-β signaling
  3. Front Oncol. 2022 ;12 954329
      Metabolic reprogramming plays a critical role in colorectal cancer (CRC). It contributes to CRC by shaping metabolic phenotypes and causing uncontrolled proliferation of CRC cells. Glucose metabolic reprogramming is common in carcinogenesis and cancer progression. Growing evidence has implicated the modifying effects of non-coding RNAs (ncRNAs) in glucose metabolic reprogramming and chemoresistance in CRC. In this review, we have summarized currently published studies investigating the role of ncRNAs in glucose metabolic alterations and chemoresistance in CRC. Elucidating the interplay between ncRNAs and glucose metabolic reprogramming provides insight into exploring novel biomarkers for the diagnosis and prognosis prediction of CRC.
    Keywords:  Circular RNA; colorectal cancer; glucose metabolism; long noncoding RNA; metabolic reprogramming; microRNA
  4. Genome Med. 2022 Aug 16. 14(1): 93
      BACKGROUND: Colorectal cancer (CRC) ranks as the second-leading cause of cancer-related death worldwide with metastases being the main cause of cancer-related death. Here, we investigated the genomic and transcriptomic alterations in matching adjacent normal tissues, primary tumors, and metastatic tumors of CRC patients.METHODS: We performed whole genome sequencing (WGS), multi-region whole exome sequencing (WES), simultaneous single-cell RNA-Seq, and single-cell targeted cDNA Sanger sequencing on matching adjacent normal tissues, primary tumors, and metastatic tumors from 12 metastatic colorectal cancer patients (n=84 for genomes, n=81 for exomes, n=9120 for single cells). Patient-derived tumor organoids were used to estimate the anti-tumor effects of a PPAR inhibitor, and self-renewal and differentiation ability of stem cell-like tumor cells.
    RESULTS: We found that the PPAR signaling pathway was prevalently and aberrantly activated in CRC tumors. Blocking of PPAR pathway both suppressed the growth and promoted the apoptosis of CRC organoids in vitro, indicating that aberrant activation of the PPAR signaling pathway plays a critical role in CRC tumorigenesis. Using matched samples from the same patient, distinct origins of the metastasized tumors between lymph node and liver were revealed, which was further verified by both copy number variation and mitochondrial mutation profiles at single-cell resolution. By combining single-cell RNA-Seq and single-cell point mutation identification by targeted cDNA Sanger sequencing, we revealed important phenotypic differences between cancer cells with and without critical point mutations (KRAS and TP53) in the same patient in vivo at single-cell resolution.
    CONCLUSIONS: Our data provides deep insights into how driver mutations interfere with the transcriptomic state of cancer cells in vivo at a single-cell resolution. Our findings offer novel knowledge on metastatic mechanisms as well as potential markers and therapeutic targets for CRC diagnosis and therapy. The high-precision single-cell RNA-seq dataset of matched adjacent normal tissues, primary tumors, and metastases from CRCs may serve as a rich resource for further studies.
    Keywords:  Genotype-phenotype relationship; Lineage tracing; Metastatic colorectal cancer; Mitochondrial mutations; PPAR signaling pathway; Patient-derived organoids; Single-cell transcriptome profiling
  5. Stem Cell Reports. 2022 Aug 10. pii: S2213-6711(22)00376-9. [Epub ahead of print]
      In vitro tissue models hold great promise for modeling diseases and drug responses. Here, we used emulsion microfluidics to form micro-organospheres (MOSs), which are droplet-encapsulated miniature three-dimensional (3D) tissue models that can be established rapidly from patient tissues or cells. MOSs retain key biological features and responses to chemo-, targeted, and radiation therapies compared with organoids. The small size and large surface-to-volume ratio of MOSs enable various applications including quantitative assessment of nutrient dependence, pathogen-host interaction for anti-viral drug screening, and a rapid potency assay for chimeric antigen receptor (CAR)-T therapy. An automated MOS imaging pipeline combined with machine learning overcomes plating variation, distinguishes tumorspheres from stroma, differentiates cytostatic versus cytotoxic drug effects, and captures resistant clones and heterogeneity in drug response. This pipeline is capable of robust assessments of drug response at individual-tumorsphere resolution and provides a rapid and high-throughput therapeutic profiling platform for precision medicine.
    Keywords:  CAR-T; SARS-COV-2; cytostatic; cytotoxic; deep learning; demulsification; drug resistant; micro-organospheres; organoid; patient derived organoid
  6. Nat Commun. 2022 Aug 18. 13(1): 4864
      Organoids provide an accessible in vitro system to mimic the dynamics of tissue regeneration and development. However, long-term live-imaging of organoids remains challenging. Here we present an experimental and image-processing framework capable of turning long-term light-sheet imaging of intestinal organoids into digital organoids. The framework combines specific imaging optimization combined with data processing via deep learning techniques to segment single organoids, their lumen, cells and nuclei in 3D over long periods of time. By linking lineage trees with corresponding 3D segmentation meshes for each organoid, the extracted information is visualized using a web-based "Digital Organoid Viewer" tool allowing combined understanding of the multivariate and multiscale data. We also show backtracking of cells of interest, providing detailed information about their history within entire organoid contexts. Furthermore, we show cytokinesis failure of regenerative cells and that these cells never reside in the intestinal crypt, hinting at a tissue scale control on cellular fidelity.
  7. Gastroenterol Rep (Oxf). 2022 ;10 goac035
      Recent studies have mapped key genetic changes in colorectal cancer (CRC) that impact important pathways contributing to the multistep models for CRC initiation and development. In parallel with genetic changes, normal and cancer tissues harbor epigenetic alterations impacting regulation of critical genes that have been shown to play profound roles in the tumor initiation. Cumulatively, these molecular changes are only loosely associated with heterogenous transcriptional programs, reflecting the heterogeneity in the various CRC molecular subtypes and the paths to CRC development. Studies from mapping molecular alterations in early CRC lesions and use of experimental models suggest that the intricate dependencies of various genetic and epigenetic hits shape the early development of CRC via different pathways and its manifestation into various CRC subtypes. We highlight the dependency of epigenetic and genetic changes in driving CRC development and discuss factors affecting epigenetic alterations over time and, by extension, risk for cancer.
    Keywords:  DNA methylation; aging; classification; colorectal cancer; consensus molecular subtypes; epigenetics; subtyping
  8. Int J Biol Sci. 2022 ;18(13): 5185-5206
      Intestinal stem cells (ISCs) play an important role in maintaining intestinal homeostasis via promoting a healthy gut barrier. Within the stem cell niche, gut microbiota linking the crosstalk of dietary influence and host response has been identified as a key regulator of ISCs. Emerging insights from recent research reveal that ISC and gut microbiota interplay regulates epithelial self-renewal. This article reviews the recent knowledge on the key role of ISC in their local environment (stem cell niche) associating with gut microbiota and their metabolites as well as the signaling pathways. The current progress of intestinal organoid culture is further summarized. Subsequently, the key challenges and future directions are discussed.
    Keywords:  Gut microbiota; Intestinal barrier; Intestinal homeostasis; Intestinal stem cell; Signaling pathway