bims-instec Biomed News
on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration
Issue of 2022–09–04
twelve papers selected by
Maria-Virginia Giolito, IRFAC/UMR-S1113 INSERM
  1. Intestinal cellular heterogeneity and disease development revealed by single-cell technology.
  2. Fibroblasts in intestinal homeostasis, damage, and repair.
  3. Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids.
  4. The neuronal protein Neuroligin 1 promotes colorectal cancer progression by modulating the APC/β-catenin pathway.
  5. Limitations to understanding intestinal stem cell activity via Cre-Lox-based lineage tracing.
  6. Organoids capture tissue-specific innate lymphoid cell development in mice and humans.
  7. PD-L1/PD-L1 signaling promotes colorectal cancer cell migration ability through RAS/MEK/ERK.
  8. Deciphering colorectal cancer immune microenvironment transcriptional landscape on single cell resolution - A role for immunotherapy.
  9. Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation.
  10. 3D matrix promotes cell dedifferentiation into colorectal cancer stem cells via integrin/cytoskeleton/glycolysis signaling.
  11. Preventive cancer stem cell-based vaccination modulates tumor development in syngeneic colon adenocarcinoma murine model.
  12. Culture Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids.
  1. Cell Regen. 2022 Sep 01. 11(1): 26
    Intestinal cellular heterogeneity and disease development revealed by single-cell technology.
    Yalong Wang, Wanlu Song, Shicheng Yu, Yuan Liu, Ye-Guang Chen.
      The intestinal epithelium is responsible for food digestion and nutrient absorption and plays a critical role in hormone secretion, microorganism defense, and immune response. These functions depend on the integral single-layered intestinal epithelium, which shows diversified cell constitution and rapid self-renewal and presents powerful regeneration plasticity after injury. Derailment of homeostasis of the intestine epithelium leads to the development of diseases, most commonly including enteritis and colorectal cancer. Therefore, it is important to understand the cellular characterization of the intestinal epithelium at the molecular level and the mechanisms underlying its homeostatic maintenance. Single-cell technologies allow us to gain molecular insights at the single-cell level. In this review, we summarize the single-cell RNA sequencing applications to understand intestinal cell characteristics, spatiotemporal evolution, and intestinal disease development.
    Keywords:  Colorectal cancer; Enteritis; Gut; Heterogeneity; scRNA-seq
    DOI:  https://doi.org/10.1186/s13619-022-00127-6
  2. Front Immunol. 2022 ;13 924866
    Fibroblasts in intestinal homeostasis, damage, and repair.
    Niki Chalkidi, Christina Paraskeva, Vasiliki Koliaraki.
      The mammalian intestine is a self-renewing tissue that ensures nutrient absorption while acting as a barrier against environmental insults. This is achieved by mature intestinal epithelial cells, the renewing capacity of intestinal stem cells at the base of the crypts, the development of immune tolerance, and the regulatory functions of stromal cells. Upon intestinal injury or inflammation, this tightly regulated mucosal homeostasis is disrupted and is followed by a series of events that lead to tissue repair and the restoration of organ function. It is now well established that fibroblasts play significant roles both in the maintenance of epithelial and immune homeostasis in the intestine and the response to tissue damage mainly through the secretion of a variety of soluble mediators and ligands and the remodeling of the extracellular matrix. In addition, recent advances in single-cell transcriptomics have revealed an unexpected heterogeneity of fibroblasts that comprise distinct cell subsets in normal and inflammatory conditions, indicative of diverse functions. However, there is still little consensus on the number, terminology, and functional properties of these subsets. Moreover, it is still unclear how individual fibroblast subsets can regulate intestinal repair processes and what is their impact on the pathogenesis of inflammatory bowel disease. In this mini-review, we aim to provide a concise overview of recent advances in the field, that we believe will help clarify current concepts on fibroblast heterogeneity and functions and advance our understanding of the contribution of fibroblasts in intestinal damage and repair.
    Keywords:  epithelial homeostasis; heterogeneity; immune responses; mesenchymal cells; regeneration; tissue injury
    DOI:  https://doi.org/10.3389/fimmu.2022.924866
  3. STAR Protoc. 2022 Sep 16. 3(3): 101639
    Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids.
    Adriana Martinez-Silgado, Fjodor A Yousef Yengej, Jens Puschhof, Veerle Geurts, Charelle Boot, Maarten H Geurts, Maarten B Rookmaaker, Marianne C Verhaar, Joep Beumer, Hans Clevers.
      Intestinal organoids are three-dimensional cultures that resemble key aspects of the epithelium of origin. Here, we describe how to differentiate human small intestinal organoids by combining growth media variations and genetic engineering. We detail the differentiation of human intestinal organoids in the presence and absence of BMP agonists to recapitulate a broader scope of functional cell states found in vivo. Using transient overexpression of the transcription factor Neurogenin-3, we describe the enhancement of differentiation toward rare enteroendocrine cells. For complete details on the use and execution of this protocol, please refer to Beumer et al. (2022).
    Keywords:  CRISPR; Cell differentiation; Organoids
    DOI:  https://doi.org/10.1016/j.xpro.2022.101639
  4. J Exp Clin Cancer Res. 2022 Sep 02. 41(1): 266
    The neuronal protein Neuroligin 1 promotes colorectal cancer progression by modulating the APC/β-catenin pathway.
    Margherita Pergolizzi, Laura Bizzozero, Federica Maione, Elena Maldi, Claudio Isella, Marco Macagno, Elisa Mariella, Alberto Bardelli, Enzo Medico, Caterina Marchiò, Guido Serini, Federica Di Nicolantonio, Federico Bussolino, Marco Arese.
       BACKGROUND: Colorectal cancer (CRC) remains largely incurable when diagnosed at the metastatic stage. Despite some advances in precision medicine for this disease in recent years, new molecular targets, as well as prognostic/predictive markers, are highly needed. Neuroligin 1 (NLGN1) is a transmembrane protein that interacts at the synapse with the tumor suppressor adenomatous polyposis Coli (APC), which is heavily involved in the pathogenesis of CRC and is a key player in the WNT/β-catenin pathway.
    METHODS: After performing expression studies of NLGN1 on human CRC samples, in this paper we used in vitro and in vivo approaches to study CRC cells extravasation and metastasis formation capabilities. At the molecular level, the functional link between APC and NLGN1 in the cancer context was studied.
    RESULTS: Here we show that NLGN1 is expressed in human colorectal tumors, including clusters of aggressive migrating (budding) single tumor cells and vascular emboli. We found that NLGN1 promotes CRC cells crossing of an endothelial monolayer (i.e. Trans-Endothelial Migration or TEM) in vitro, as well as cell extravasation/lung invasion and differential organ metastatization in two mouse models. Mechanistically, NLGN1 promotes APC localization to the cell membrane and co-immunoprecipitates with some isoforms of this protein stimulates β-catenin translocation to the nucleus, upregulates mesenchymal markers and WNT target genes and induces an "EMT phenotype" in CRC cell lines CONCLUSIONS: In conclusion, we have uncovered a novel modulator of CRC aggressiveness which impacts on a critical pathogenetic pathway of this disease, and may represent a novel therapeutic target, with the added benefit of carrying over substantial knowledge from the neurobiology field.
    Keywords:  APC; Neuroligin 1; WNT pathway; intravasation/extravasation; metastasis; tumor budding
    DOI:  https://doi.org/10.1186/s13046-022-02465-4
  5. Cell Mol Gastroenterol Hepatol. 2022 Aug 25. pii: S2352-345X(22)00185-0. [Epub ahead of print]
    Limitations to understanding intestinal stem cell activity via Cre-Lox-based lineage tracing.
    N M Johnson, J Na, K E Monaghan, A T Tang, Y Tian, N A Leu, N Li, M L Kahn, C J Lengner.
      
    DOI:  https://doi.org/10.1016/j.jcmgh.2022.08.007
  6. Cell Rep. 2022 Aug 30. pii: S2211-1247(22)01101-9. [Epub ahead of print]40(9): 111281
    Organoids capture tissue-specific innate lymphoid cell development in mice and humans.
    Geraldine M Jowett, Emily Read, Luke B Roberts, Diana Coman, Marta Vilà González, Tomasz Zabinski, Umar Niazi, Rita Reis, Tung-Jui Trieu, Davide Danovi, Eileen Gentleman, Ludovic Vallier, Michael A Curtis, Graham M Lord, Joana F Neves.
      Organoid-based models of murine and human innate lymphoid cell precursor (ILCP) maturation are presented. First, murine intestinal and pulmonary organoids are harnessed to demonstrate that the epithelial niche is sufficient to drive tissue-specific maturation of all innate lymphoid cell (ILC) groups in parallel, without requiring subset-specific cytokine supplementation. Then, more complex human induced pluripotent stem cell (hiPSC)-based gut and lung organoid models are used to demonstrate that human epithelial cells recapitulate maturation of ILC from a stringent systemic human ILCP population, but only when the organoid-associated stromal cells are depleted. These systems offer versatile and reductionist models to dissect the impact of environmental and mucosal niche cues on ILC maturation. In the future, these could provide insight into how ILC activity and development might become dysregulated in chronic inflammatory diseases.
    Keywords:  CP: Developmental biology; CP: Immunology; epithelial organoids; innate lymphoid cell development; mucosal co-cultures; stromal cells
    DOI:  https://doi.org/10.1016/j.celrep.2022.111281
  7. Clin Exp Pharmacol Physiol. 2022 Sep 01.
    PD-L1/PD-L1 signaling promotes colorectal cancer cell migration ability through RAS/MEK/ERK.
    Yihui Cao, Lian Fang, Weiye Liang, Ming-Kai Liu, Jia Zuo, Ying-Long Peng, Jia-Jie Shan, Rui-Xia Sun, Jie Zhao, Jian Wang.
      PD-L1 is widely known as an immune checkpoint, and immunotherapy through the inhibition of checkpoint molecules has become an important component in the successful treatment of tumours via PD-1/PD-L1 signaling pathways. However, its biological functions and expression profile in colorectal cancer (CRC) are elusive. We previously found that PD-L1 can bind to PD-L1 and cause cell detachment. However, the detailed molecular mechanisms of how PD-L1 binds to PD-L1 and how it transmits signals to the cell remain unclear. In this study, we disclosed that PD-L1 expression was dramatically upregulated in CRC compared to normal tissues. Ectopic expression of PD-L1 inhibits cell adhesive capacity and promotes cell migration in CRC cell lines, while silencing PD-L1 had the opposite effects and suppressed invasion and proliferation. Mechanistically, PD-L1 was found to promote EMT through the ERK signaling molecule pathway and interacted with the 1-86 aa fragment of KRAS to transduce signals. Collectively, our study demonstrated the role of PD-L1 after binding to PD-L1 in CRC, thereby providing a new theoretical basis for further improving immunotherapy with anti-PD-L1 antibodies. This article is protected by copyright. All rights reserved.
    Keywords:  Cell adhesion; Colorectal cancer (CRC); ERK; KRAS; Migration; PD-L1
    DOI:  https://doi.org/10.1111/1440-1681.13717
  8. Front Immunol. 2022 ;13 959705
    Deciphering colorectal cancer immune microenvironment transcriptional landscape on single cell resolution - A role for immunotherapy.
    Francis Yew Fu Tieng, Learn-Han Lee, Nurul-Syakima Ab Mutalib.
      Single cell RNA sequencing (scRNA-seq) is a novel high-throughput technique that enables the investigation of a single cell's entire transcriptome. It elucidates intricate cellular networks and generates indices that will eventually enable the development of more targeted and personalized medications. The importance of scRNA-seq has been highlighted in complex biological systems such as cancer and the immune system, which exhibit significant cellular heterogeneity. Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of cancer-related death globally. Chemotherapy continues to be used to treat these patients. However, 5-FU has been utilized in chemotherapy regimens with oxaliplatin and irinotecan since the 1960s and is still used today. Additionally, chemotherapy-resistant metastatic CRCs with poor prognoses have been treated with immunotherapy employing monoclonal antibodies, immune checkpoint inhibitors, adoptive cell therapy and cancer vaccines. Personalized immunotherapy employing tumor-specific neoantigens allows for treating each patient as a distinct group. Sequencing and multi-omics approaches have helped us identify patients more precisely in the last decade. The introduction of modern methods and neoantigen-based immunotherapy may usher in a new era in treating CRC. The unmet goal is to better understand the cellular and molecular mechanisms that contribute to CRC pathogenesis and resistance to treatment, identify novel therapeutic targets, and make more stratified and informed treatment decisions using single cell approaches. This review summarizes current scRNA-seq utilization in CRC research, examining its potential utility in the development of precision immunotherapy for CRC.
    Keywords:  colorectal cancer; immune landscape; immunotherapy; metastasis; precision medicine; single cell; tumor immune microenvironment
    DOI:  https://doi.org/10.3389/fimmu.2022.959705
  9. Sci Transl Med. 2022 Aug 31. 14(660): eabi8633
    Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation.
    Conner Kummerlowe, Simutanyi Mwakamui, Travis K Hughes, Nolawit Mulugeta, Victor Mudenda, Ellen Besa, Kanekwa Zyambo, Jessica E S Shay, Ira Fleming, Marko Vukovic, Ben A Doran, Toby P Aicher, Marc H Wadsworth, Juliet Tongue Bramante, Amiko M Uchida, Rabiah Fardoos, Osaretin E Asowata, Nicholas Herbert, Ömer H Yilmaz, Henrik N Kløverpris, John J Garber, José Ordovas-Montañes, Zev J Gartner, Thomas Wallach, Alex K Shalek, Paul Kelly.
      Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle-income countries. It is thought to be a key contributing factor to childhood malnutrition, growth stunting, and diminished oral vaccine responses. Although EE has been shown to be the by-product of a recurrent enteric infection, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE by performing high-throughput, single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE in Lusaka, Zambia (eight HIV-negative and three HIV-positive), six adults without EE in Boston, United States, and two adults in Durban, South Africa, which we complemented with published data from three additional individuals from the same clinical site. We analyzed previously defined bulk-transcriptomic signatures of reduced villus height and decreased microbial translocation in EE and showed that these signatures may be driven by an increased abundance of surface mucosal cells-a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract. In addition, we determined cell subsets whose fractional abundances associate with EE severity, small intestinal region, and HIV infection. Furthermore, by comparing duodenal EE samples with those from three control cohorts, we identified dysregulated WNT and MAPK signaling in the EE epithelium and increased proinflammatory cytokine gene expression in a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells in the EE cohort. Together, our work elucidates epithelial and immune correlates of EE and nominates cellular and molecular targets for intervention.
    DOI:  https://doi.org/10.1126/scitranslmed.abi8633
  10. Cancer Sci. 2022 Sep 02.
    3D matrix promotes cell dedifferentiation into colorectal cancer stem cells via integrin/cytoskeleton/glycolysis signaling.
    Tong Han, Yuhong Jiang, Xiaobo Wang, Shuangya Deng, Yongjun Hu, Qianqian Jin, Dongju Long, Kuijie Liu.
      The potential for tumor occurrence triggered by cancer stem cells (CSCs) has emerged as a significant challenge for human colorectal cancer therapy. However, the underlying mechanism of CSC development remains controversial. Our study provided evidence that the bulk of tumor cells could dedifferentiate to CSCs and reacquire CSCs-like phenotypes if cultured in the presence of extracellular matrix reagents, such as Matrigel and fibrin gels. In these 3D gels, CD133- colorectal cancer cells can regain tumorigenic potential and stem-like phenotypes. Mechanistically, the 3D extracellular matrix could mediate cytoskeletal F-actin bundling through biomechanical force associated receptors integrin β1 (ITGB1), contributing to the release of E3 ligase Tripartite motif protein 11 (TRIM11) from cytoskeleton and degradation of the glycolytic rate-limiting enzyme phosphofructokinase (PFK). Consequently, PFK inhibition resulted in enhanced glycolysis and upregulation of hypoxia-inducible factor 1 (HIF1α), thereby promoting the reprogramming of stem cell transcription factors and facilitating tumor progression in patients. This study provided novel insights into the role of the extracellular matrix in the regulation of CSC dedifferentiation in a cytoskeleton/glycolysis-dependent manner.
    Keywords:  colorectal cancer; dedifferentiation; extracellular matrix; glycolysis; integrin β1
    DOI:  https://doi.org/10.1111/cas.15548
  11. J Cancer Res Clin Oncol. 2022 Aug 30.
    Preventive cancer stem cell-based vaccination modulates tumor development in syngeneic colon adenocarcinoma murine model.
    Leila Eini, Marzieh Naseri, Feridoun Karimi-Busheri, Mahmood Bozorgmehr, Roya Ghods, Zahra Madjd.
       BACKGROUND: Cancer stem cells (CSCs), a rare sub-fraction of tumor cells, with the capability of self-renewal and strong oncogenicity are tightly responsible for chemo and radio resistance and tumor metastasis in colorectal cancer. Hence, CSCs targeting would improve the efficacy of therapeutic strategies and clinical outcomes.
    METHODS: Here, using three-dimensional CSC spheroids and syngeneic mice model, we evaluated the cancer preventive impact of CSCs-based vaccination. CSCs enrichment was performed via colonosphere formation from CT-26 cell line and CT-26-derived tumor biopsy and characterized by confirming high expression of key stemness genes (OCT4, SOX2, and NANOG) and CSC-related surface biomarkers (CD166, DCLK1, and CD133) via real-time PCR and flow cytometry, respectively. Then, the stemness phenotype and self-renewal in CSC-enriched spheroids were further confirmed by showing serial sphere formation capacity, clonogenicity potential, and enhanced in vivo tumorigenic capacity compared to their parental counterparts. CSCs lysates were used as vaccines in prophylactic settings compared to the parental cell lysate and PBS groups.
    RESULT: Immunization of syngeneic mice with CSCs lysates was effective in the prevention of tumor establishment and significantly decreased tumor growth rate accompanied by an improvement in survival rate in tumor-bearing mice compared to groups subjected to parental cells lysate and PBS. These results, for the first time, showed that mice immunized with cell lysate from tumor biopsy-derived spheroids are resistant to tumor induction. Immunofluorescence staining indicated that only the serum antibodies from CSC-vaccinated mice reacted with colonospheres.
    CONCLUSIONS: These findings represent CSCs lysate-based vaccination as a potential approach to hampering immunotherapy failure of colorectal cancer which along with other traditional therapies may effectively apply to prevent the establishment of aggressive tumors harboring stemness features.
    Keywords:  Cancer stem cells (CSCs); Colorectal cancer (CRC); Immunotherapy; Preventive CSCs-based vaccination; Spheroid formation
    DOI:  https://doi.org/10.1007/s00432-022-04303-8
  12. Cell Mol Gastroenterol Hepatol. 2022 Aug 26. pii: S2352-345X(22)00186-2. [Epub ahead of print]
    Culture Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids.
    Rachel D Edgar, Francesca Perrone, April R Foster, Felicity Payne, Sophia Lewis, Komal M Nayak, Judith Kraiczy, Aurélie Cenier, Franco Torrente, Camilla Salvestrini, Robert Heuschkel, Kai O Hensel, Rebecca Harris, D Leanne Jones, Daniel R Zerbino, Matthias Zilbauer.
       BACKGROUND & AIMS: Human intestinal epithelial organoids (IEO) are a powerful tool to model major aspects of intestinal development, health and diseases, as patient derived cultures retain many features found in-vivo. A necessary aspect of the organoid model is the requirement to expand cultures in-vitro through several rounds of passaging. This is of concern, as the passaging of cells has been shown to affect cell morphology, ploidy, and function.
    METHODS: Here we have analysed 173 human IEO lines derived from the small and large bowel and examined the effect of culture duration on DNA methylation (DNAm). Furthermore, we tested the potential impact of DNAm changes on gene expression and cellular function.
    RESULTS: Our analyses revealed a reproducible effect of culture duration on DNAm in a large discovery cohort as well as two publicly available validation cohorts generated in different laboratories. Although methylation changes were only seen in approximately 8% of tested CpGs and global cellular function remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokines exposure and withdrawal of Wnt agonists. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication indicating similarities to malignant transformation.
    CONCLUSIONS: Our study reveals distinct culture-associated epigenetic changes in mucosa derived human IEO, some of which appear to impact gene transcriptomic and cellular function. These findings highlight the need for future studies in this area and the importance of considering passage number as a potentially confounding factor.
    Keywords:  Organoid; culture conditions; epigenetics; intestinal epithelium
    DOI:  https://doi.org/10.1016/j.jcmgh.2022.08.008
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