bims-instec Biomed News
on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration
Issue of 2023–06–18
twenty-six papers selected by
Maria-Virginia Giolito, Free University of Brussels
  1. Generation of Fetal Intestinal Organoids and Their Maturation into Adult Intestinal Cells.
  2. Visualization of Differentiated Cells in 3D and 2D Intestinal Organoid Cultures.
  3. Identifying Cell Differentiation in Colorectal Cancer.
  4. TCF7L1 Controls the Differentiation of Tuft Cells in Mouse Small Intestine.
  5. Directed Differentiation of Murine and Human Small Intestinal Organoids Toward All Mature Lineages.
  6. Modeling Notch Activity and Lineage Decisions Using Intestinal Organoids.
  7. The oncogenic function of PLAGL2 is mediated via ASCL2 and IGF2 and a Wnt-independent mechanism in colorectal cancer.
  8. Glutamine boosts intestinal stem cell-mediated small intestinal epithelial development during early weaning: Involvement of WNT signaling.
  9. p57Kip2 acts as a transcriptional corepressor to regulate intestinal stem cell fate and proliferation.
  10. β-catenin drives butyrophilin-like molecule loss and γδ T-cell exclusion in colon cancer.
  11. Treatment outcome comparisons of first-line targeted therapy in patients with KRAS wild-type metastatic colorectal cancer: A nationwide database study.
  12. Co-culturing Human Intestinal Enteroid Monolayers with Innate Immune Cells.
  13. The remodeling roles of lipid metabolism in colorectal cancer cells and immune microenvironment.
  14. Epigenetic and Transcriptional Dynamics of Notch Program in Intestinal Differentiation.
  15. LIN28B promotes cell invasion and colorectal cancer metastasis via CLDN1 and NOTCH3.
  16. In Vitro Morphogenesis and Differentiation of Human Intestinal Epithelium in a Gut-on-a-Chip.
  17. Differentiated Epithelial Cells of the Gut.
  18. Single-cell and spatial transcriptome analysis reveals the cellular heterogeneity of liver metastatic colorectal cancer.
  19. Identification of Differentiated Intestinal Epithelial Cells Using Immunostaining and Fluorescence Microscopy.
  20. Negative correlation between acetyl-CoA acyltransferase 2 and cetuximab resistance in colorectal cancer.
  21. Mutual impact of adipocytes and colorectal cancer cells growing in co-culture conditions.
  22. Wild-Type KRAS Is Required for Panitumumab Efficacy in Patients With Metastatic Colorectal Cancer.
  23. In Vitro and in Vivo Assays for Testing Retinoids Effect on Intestinal Progenitors' Lineage Commitments.
  24. Organoids are not organs: Sources of variation and misinformation in organoid biology.
  25. Short chain fatty acids prime colorectal cancer cells to activate antitumor immunity.
  26. Organoid cultures for cancer modeling.
  1. Methods Mol Biol. 2023 ;2650 133-140
    Generation of Fetal Intestinal Organoids and Their Maturation into Adult Intestinal Cells.
    Masamichi Imajo, Akira Hirota, Shinya Tanaka.
      During embryonic development, the gut tube undergoes massive morphological changes from the simple tube structure composed of the pseudostratified epithelium into the mature intestinal tract composed of the columnar epithelium and characterized by the unique crypt-villus structures. In mice, maturation of fetal gut precursor cells into adult intestinal cells starts around embryonic day (E) 16.5, during which adult intestinal stem cells and their differentiated progenies are generated. In contrast to adult intestinal cells that form budding organoids containing both the crypt-like and villus-like regions, fetal intestinal cells can be cultured as simple spheroid-shaped organoids that show a uniform proliferation pattern. The fetal intestinal spheroids can undergo spontaneous maturation into adult budding organoids that contain intestinal stem cells and differentiated cells, including enterocytes, goblet, enteroendocrine, and Paneth cells, recapitulating intestinal cell maturation in vitro. Here, we provide detailed methods for establishment of fetal intestinal organoids and their differentiation into adult intestinal cells. These methods enable in vitro recapitulation of intestinal development and would be useful to reveal mechanisms that regulate the transition from fetal to adult intestinal cells.
    Keywords:  Fetal intestine; Intestinal development; Intestinal epithelial cells; Intestinal stem cells; Organoid culture
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_11
  2. Methods Mol Biol. 2023 ;2650 141-153
    Visualization of Differentiated Cells in 3D and 2D Intestinal Organoid Cultures.
    Hikaru Hanyu, Shinya Sugimoto, Toshiro Sato.
      The intestinal epithelium maintains self-renewal and differentiation capacities via coordination of key signaling pathways, including the Wnt, bone morphogenetic protein (BMP), epidermal growth factor (EGF), and Notch signaling pathways. Based on this understanding, a combination of stem cell niche factors, EGF, Noggin, and the Wnt agonist R-spondin was shown to enable the growth of mouse intestinal stem cells and the formation of organoids with indefinite self-renewal and full differentiation capacity. Two small-molecule inhibitors, including a p38 inhibitor and a TGF-beta inhibitor, were added to propagate cultured human intestinal epithelium but at the cost of differentiation capacity. There have been improvements in culture conditions to overcome these issues. Substitution of the EGF and a p38 inhibitor with insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) enabled multilineage differentiation. Monolayer culture with mechanical flow to the apical epithelium promoted the formation of villus-like structures with mature enterocyte gene expression. Here, we summarize our recent technological improvements in human intestinal organoid culture that will deepen the understanding of intestinal homeostasis and diseases.
    Keywords:  Colon; Differentiation; Human; Immunostaining; Intestinal stem cells; LGR5; Organoids; Small intestine; Whole-mount staining
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_12
  3. Methods Mol Biol. 2023 ;2650 227-233
    Identifying Cell Differentiation in Colorectal Cancer.
    Isabel Puig, Irene Chicote, Héctor G Pálmer.
      The intestinal epithelium is a rapid self-renewing tissue. Stem cells at the bottom of the crypts first give rise to a proliferative progeny that finally differentiates to a variety of cell types. These terminally differentiated intestinal cells are mostly present in the villi of the intestinal wall and serve as functional units to sustain the main purpose of the organ: food absorption. But for a balance homeostasis, the intestine is composed not only by absorptive enterocytes but also by other cell types such as goblet cells that secrete mucus to lubricate the intestinal lumen, Paneth cells that secrete antimicrobial peptides to control microbiome, and others. Many relevant conditions affecting the intestine including chronic inflammation, Crohn's disease, or cancer can alter the composition of these different functional cell types. As a consequence, they can lose their specialized activity as functional units and further contribute to disease progression and malignancy. Measuring the amount of these different cell populations in the intestine is essential to understand the bases of these diseases and their specific contribution to their malignancy. Interestingly, patient-derived xenograft (PDX) models faithfully recapitulate patients' tumors including the proportion of the different cell lineages present in the original tumor. Here we expose some protocols for evaluating the differentiation of intestinal cells in colorectal tumors.
    Keywords:  Colorectal cancer; Differentiated intestinal cells; Imaging; Immunofluorescence; Patient-derived xenografts
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_17
  4. Cells. 2023 May 23. pii: 1452. [Epub ahead of print]12(11):
    TCF7L1 Controls the Differentiation of Tuft Cells in Mouse Small Intestine.
    Valeriya V Zinina, Melanie Sauer, Lira Nigmatullina, Nastasja Kreim, Natalia Soshnikova.
      Continuous and rapid renewal of the intestinal epithelium depends on intestinal stem cells (ISCs). A large repertoire of transcription factors mediates the correct maintenance and differentiation of ISCs along either absorptive or secretory lineages. In the present study, we addressed the role of TCF7L1, a negative regulator of WNT signalling, in embryonic and adult intestinal epithelium using conditional mouse mutants. We found that TCF7L1 prevents precocious differentiation of the embryonic intestinal epithelial progenitors towards enterocytes and ISCs. We show that Tcf7l1 deficiency leads to upregulation of the Notch effector Rbp-J, resulting in a subsequent loss of embryonic secretory progenitors. In the adult small intestine, TCF7L1 is required for the differentiation of secretory epithelial progenitors along the tuft cell lineage. Furthermore, we show that Tcf7l1 promotes the differentiation of enteroendocrine D- and L-cells in the anterior small intestine. We conclude that TCF7L1-mediated repression of both Notch and WNT pathways is essential for the correct differentiation of intestinal secretory progenitors.
    Keywords:  Notch; TCF7L1; WNT; enteroendocrine cells; small intestine; tuft cells
    DOI:  https://doi.org/10.3390/cells12111452
  5. Methods Mol Biol. 2023 ;2650 107-122
    Directed Differentiation of Murine and Human Small Intestinal Organoids Toward All Mature Lineages.
    A Martinez-Silgado, J Beumer, H Clevers.
      Intestinal organoids are three-dimensional structures derived from tissue-resident adult stem cells. These organoids recapitulate key aspects of epithelial biology and can be used to study homeostatic turnover of the corresponding tissue. Organoids can be enriched for the various mature lineages which allows studies of the respective differentiation processes and of the diverse cellular functions. Here we describe mechanisms of intestinal fate specification and how these can be exploited to drive mouse and human small intestinal organoids into each of the functionally mature lineages.
    Keywords:  Adult stem cell-derived organoids; Differentiation; Intestinal lineages; Organoids; Small intestine
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_9
  6. Methods Mol Biol. 2023 ;2650 123-132
    Modeling Notch Activity and Lineage Decisions Using Intestinal Organoids.
    Yifan Qiu, Sabrina K Phanor, Subin Pyo, Chia-Wei Cheng.
      Organoid cultures have been developed to model intestinal stem cell (ISC) function in self-renewal and differentiation. Upon differentiation, the first fate decision for ISC and early progenitors to make is between secretory (Paneth cell, goblet cell, enteroendocrine cell, or tuft cell) and absorptive (enterocyte and M cell) lineages. Using genetic and pharmacological approaches, in vivo studies in the past decade have revealed that Notch signaling functions as a binary switch for the secretory vs. absorptive lineage decision in adult intestine. Recent breakthroughs in organoid-based assays enable real-time observation of smaller-scale and higher-throughput experiments in vitro, which have begun contributing to new understandings of mechanistic principles underlying intestinal differentiation. In this chapter, we summarize the in vivo and in vitro tools for modulating Notch signaling and assess its impact on intestinal cell fate. We also provide example protocols of how to use intestinal organoids as functional assays to study Notch activity in intestinal lineage decisions.
    Keywords:  Lineage; Notch; Organoids
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_10
  7. Am J Physiol Gastrointest Liver Physiol. 2023 Jun 13.
    The oncogenic function of PLAGL2 is mediated via ASCL2 and IGF2 and a Wnt-independent mechanism in colorectal cancer.
    Anthony D Fischer, Daniel A Veronese-Paniagua, Shriya Swaminathan, Hajime Kashima, Deborah C Rubin, Blair B Madison.
      Colorectal cancer (CRC) tumorigenesis and progression are linked to common oncogenic mutations, especially in the tumor suppressor APC, whose loss triggers the deregulation of TCF4/β-Catenin activity. CRC tumorigenesis is also driven by multiple epi-mutational modifiers, such as transcriptional regulators. We describe the common (and near-universal) activation of the zinc finger transcription factor and Let-7 target PLAGL2 in CRC and find that it is a key driver of intestinal epithelial transformation. PLAGL2 drives proliferation, cell cycle progression, and anchorage-independent growth in CRC cell lines and non-transformed intestinal cells. Investigating effects of PLAGL2 on downstream pathways revealed very modest effects on canonical Wnt signaling. Alternatively, we find pronounced effects on the direct PLAGL2 target genes IGF2, a fetal growth factor, and ASCL2, an intestinal stem cell-specific bHLH transcription factor. Inactivation of PLAGL2 in CRC cell lines has pronounced effects on ASCL2 reporter activity. Furthermore, ASCL2 expression can partially rescue deficits of proliferation and cell cycle progression caused by depletion of PLAGL2 in CRC cell lines. Thus, the oncogenic effects of PLAGL2 appear to be mediated via core stem cell and onco-fetal pathways, with minimal effects on downstream Wnt signaling.
    Keywords:  Colorectal Cancer; IGF2; PLAGL2; Tumorigenesis; Wnt
    DOI:  https://doi.org/10.1152/ajpgi.00058.2022
  8. Stem Cell Reports. 2023 May 27. pii: S2213-6711(23)00191-1. [Epub ahead of print]
    Glutamine boosts intestinal stem cell-mediated small intestinal epithelial development during early weaning: Involvement of WNT signaling.
    Junquan Tian, Yuying Li, Xuetai Bao, Fan Yang, Xiongzhuo Tang, Qian Jiang, Chengbo Yang, Yulong Yin, Kang Yao.
      Early weaning usually causes small intestine epithelial development abnormality, increasing the risk of gastrointestinal diseases. Glutamine (Gln), enriching in plasma and milk, is widely reported to benefit intestinal health. However, whether Gln affects intestinal stem cell (ISC) activity in response to early weaning is unclear. Here, both the early weaning mice and intestinal organoids were used to study the role of Gln in regulating ISC activities. Results showed that Gln ameliorated early weaning-induced epithelial atrophy and augmented the ISC-mediated epithelial regeneration. Gln deprivation disabled ISC-mediated epithelial regeneration and crypt fission in vitro. Mechanistically, Gln augmented WNT signaling in a dose-dependent manner to regulate ISC activity, while WNT signaling blockage abolished the effects of Gln on ISCs. Together, Gln accelerates stem cell-mediated intestinal epithelial development associated with the augmentation of WNT signaling, which provides novel insights into the mechanism by which Gln promotes intestinal health.
    Keywords:  WNT signaling; early weaning; glutamine; intestinal epithelial development; intestinal stem cell
    DOI:  https://doi.org/10.1016/j.stemcr.2023.05.012
  9. Cell Rep. 2023 Jun 15. pii: S2211-1247(23)00670-8. [Epub ahead of print]42(6): 112659
    p57Kip2 acts as a transcriptional corepressor to regulate intestinal stem cell fate and proliferation.
    Justine Creff, Ada Nowosad, Anne Prel, Anne Pizzoccaro, Marion Aguirrebengoa, Nicolas Duquesnes, Caroline Callot, Thomas Jungas, Christine Dozier, Arnaud Besson.
      p57Kip2 is a cyclin/CDK inhibitor and a negative regulator of cell proliferation. Here, we report that p57 regulates intestinal stem cell (ISC) fate and proliferation in a CDK-independent manner during intestinal development. In the absence of p57, intestinal crypts exhibit an increased proliferation and an amplification of transit-amplifying cells and of Hopx+ ISCs, which are no longer quiescent, while Lgr5+ ISCs are unaffected. RNA sequencing (RNA-seq) analyses of Hopx+ ISCs show major gene expression changes in the absence of p57. We found that p57 binds to and inhibits the activity of Ascl2, a transcription factor critical for ISC specification and maintenance, by participating in the recruitment of a corepressor complex to Ascl2 target gene promoters. Thus, our data suggest that, during intestinal development, p57 plays a key role in maintaining Hopx+ ISC quiescence and repressing the ISC phenotype outside of the crypt bottom by inhibiting the transcription factor Ascl2 in a CDK-independent manner.
    Keywords:  Ascl2; CDKN1C; CP: Stem cell research; Hopx; Lgr5; intestinal stem cells; p57(Kip2); transcriptional regulation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112659
  10. Cancer Immunol Res. 2023 Jun 13. pii: CIR-22-0644. [Epub ahead of print]
    β-catenin drives butyrophilin-like molecule loss and γδ T-cell exclusion in colon cancer.
    Toshiyasu Suzuki, Anna Kilbey, Nuria Casa Rodriguez, Amy Lawlor, Anastasia Georgakopoulou, Hannah Hayman, Kyi Lai Yin Swe, Anna Nordin, Claudio Cantù, Pierre Vantourout, Rachel A Ridgway, Ryan M Byrne, Lei Chen, Michael P Verzi, David M Gay, Ester Gil Vazquez, Hayley L Belnoue-Davis, Kathryn Gilroy, Anne Helene Kostner, Christian Kersten, Chanitra Thuwajit, Ditte K Andersen, Robert Wiesheu, Anett Jandke, Karen Blyth, Antonia K Roseweir, Simon J Leedham, Philip D Dunne, Joanne Edwards, Adrian Hayday, Owen J Sansom, Seth B Coffelt.
      Intraepithelial lymphocytes (IELs) expressing γδ T-cell receptors (γδTCRs) play key roles in elimination of colon cancer. However, the precise mechanisms by which progressing cancer cells evade immunosurveillance by these innate T cells are unknown. Here, we investigated how loss of the Apc tumor suppressor in gut tissue could enable nascent cancer cells to escape immunosurveillance by cytotoxic γδIELs. In contrast with healthy intestinal or colonic tissue, we found that γδIELs were largely absent from the microenvironment of both mouse and human tumors, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR interactions, were also downregulated in tumors. We then demonstrated that β-catenin activation through loss of Apc rapidly suppressed expression of the mRNA encoding the HNF4A and HNF4G transcription factors, preventing their binding to promoter regions of Btnl genes. Re-expression of BTNL1 and BTNL6 in cancer cells increased γδIEL survival and activation in co-culture assays but failed to augment their cancer-killing ability in vitro or their recruitment to orthotopic tumors. However, inhibition of β-catenin signaling via genetic deletion of Bcl9/Bcl9l in either Apc-deficient or mutant β-catenin mouse models restored Hnf4a, Hnf4g, and Btnl gene expression and γδ T-cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts γδIEL immunosurveillance and furthers cancer progression.
    DOI:  https://doi.org/10.1158/2326-6066.CIR-22-0644
  11. Cancer Med. 2023 Jun 16.
    Treatment outcome comparisons of first-line targeted therapy in patients with KRAS wild-type metastatic colorectal cancer: A nationwide database study.
    Yi-Hsin Liang, Kuo-Hsing Chen, Yu-Yun Shao.
       BACKGROUND: The first-line systemic therapy for metastatic colorectal cancer (mCRC) is a combination of one targeted therapy agent and a chemotherapy doublet. Whether bevacizumab or anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (mAb) is the more effective addition to a chemotherapy doublet as the first-line treatment for inoperable KRAS wild-type mCRC remains controversial in prior clinical trials. Moreover, the association between the sidedness of primary tumors and the efficacy of anti-EGFR mAb needs to be addressed.
    METHODS: We established a cohort of patients with KRAS wild-type mCRC who were treated with first-line targeted therapy plus doublet chemotherapy between 2013 and 2018 using Taiwan's National Health Insurance Research Database. Secondary surgery was defined as either resection of primary tumors, liver metastases, lung metastases, or radiofrequency ablation.
    RESULTS: A total of 6482 patients were included; bevacizumab and anti-EGFR mAb were the first-line targeted therapies in 3334 (51.4%) and 3148 (48.6%) patients, respectively. Compared with those who received bevacizumab, patients who received anti-EGFR mAb exhibited significantly longer overall survival (OS; median, 23.1 vs. 20.2 months, p = 0.012) and time to treatment failure (TTF; median, 11.3 vs. 10 months, p < 0.001). Among left-sided primary tumors, the OS and TTF benefits of anti-EGFR mAb remained. Among right-sided primary tumors, the OS and TTF were similar regardless of the type of targeted therapy. In multivariate analyses, first-line anti-EGFR mAb therapy remained an independent predictor of longer OS and TTF for left-sided primary tumors. Patients who received anti-EGFR mAb were more likely to receive secondary surgery (29.6% vs. 22.6%, p < 0.0001) than patients who received bevacizumab.
    CONCLUSION: For patients who received first-line doublet chemotherapy for KRAS wild-type mCRC, adding anti-EGFR mAb was associated with significantly longer OS and TTF, especially for left-sided primary tumors.
    Keywords:  EGFR; bevacizumab; cetuximab; metastatic colorectal cancer; panitumumab; secondary surgery
    DOI:  https://doi.org/10.1002/cam4.6196
  12. Methods Mol Biol. 2023 ;2650 207-223
    Co-culturing Human Intestinal Enteroid Monolayers with Innate Immune Cells.
    Janet F Staab, Jose M Lemme-Dumit, Rachel Latanich, Marcela F Pasetti, Nicholas C Zachos.
      The coordinated interaction between the intestinal epithelium and immune cells is required to maintain proper barrier function and mucosal host defenses to the harsh external environment of the gut lumen. Complementary to in vivo models, there is a need for practical and reproducible in vitro models that employ primary human cells to confirm and advance our understanding of mucosal immune responses under physiologic and pathophysiologic conditions. Here we describe the methods to co-culture human intestinal stem cell-derived enteroids grown as confluent monolayers on permeable supports with primary human innate immune cells (e.g., monocyte-derived macrophages and polymorphonuclear neutrophils). This co-culture model reconstructs the cellular framework of the human intestinal epithelial-immune niche with distinct apical and basolateral compartments to recreate host responses to luminal and submucosal challenges, respectively. Enteroid-immune co-cultures enable multiple outcome measures to interrogate important biological processes such as epithelial barrier integrity, stem cell biology, cellular plasticity, epithelial-immune cells crosstalk, immune cell effector functions, changes in gene expression (i.e., transcriptomic, proteomic, epigenetic), and host-microbiome interactions.
    Keywords:  Co-culture; Enteroids; Intestinal organoids; Macrophages; Monolayer; Neutrophils
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_16
  13. Oncol Res. 2022 ;30(5): 231-242
    The remodeling roles of lipid metabolism in colorectal cancer cells and immune microenvironment.
    Jiateng Zhong, Jingyu Guo, Xinyu Zhang, Shuang Feng, Wenyu DI, Yanling Wang, Huifang Zhu.
      Lipid is a key component of plasma membrane, which plays an important role in the regulation of various cell biological behaviors, including cell proliferation, growth, differentiation and intracellular signal transduction. Studies have shown that abnormal lipid metabolism is involved in many malignant processes, including colorectal cancer (CRC). Lipid metabolism in CRC cells can be regulated not only by intracellular signals, but also by various components in the tumor microenvironment, including various cells, cytokines, DNA, RNA, and nutrients including lipids. In contrast, abnormal lipid metabolism provides energy and nutrition support for abnormal malignant growth and distal metastasis of CRC cells. In this review, we highlight the remodeling roles of lipid metabolism crosstalk between the CRC cells and the components of tumor microenvironment.
    Keywords:  Colorectal cancer; Lipid metabolism; Tumor microenvironment (TME)
    DOI:  https://doi.org/10.32604/or.2022.027900
  14. Methods Mol Biol. 2023 ;2650 77-88
    Epigenetic and Transcriptional Dynamics of Notch Program in Intestinal Differentiation.
    Shahadat Rahman, Xi Lan, Christopher Terranova, Rayan El-Kholdi, Omer H Yilmaz, Chia-Wei Cheng.
      The equilibrium between stem cell self-renewal and differentiation followed by proper lineage specification of progenitor cells is considered imperative for maintaining intestinal homeostasis. In the hierarchical model, intestinal differentiation is defined by the stepwise acquisition of lineage-specific mature cell features, where Notch signaling and lateral inhibition instructively regulate the cell-fate decisions. Recent studies reveal a broadly permissive intestinal chromatin underlies the lineage plasticity and adaptation to diet mediated by Notch transcriptional program. Here, we review the conventional understanding of Notch programming in intestinal differentiation and describe how new data from epigenetic and transcriptional analyses may refine or revise the current view. We provide instructions on sample preparation and data analysis and explain how to use ChIP-seq and scRNA-seq in combination of lineage tracing assay to determine the dynamics of Notch program and intestinal differentiation in the context of dietary and metabolic regulation of cell-fate decisions.
    Keywords:  Cell-fate decisions; ChIP-seq; Notch; scRNA-seq
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_7
  15. JCI Insight. 2023 Jun 15. pii: e167310. [Epub ahead of print]
    LIN28B promotes cell invasion and colorectal cancer metastasis via CLDN1 and NOTCH3.
    Kensuke Sugiura, Yasunori Masuike, Kensuke Suzuki, Alice E Shin, Nozomu Sakai, Hisahiro Matsubara, Masayuki Ohtsuka, Peter A Sims, Christopher J Lengner, Anil K Rustgi.
      The RNA-binding protein LIN28B is overexpressed in over 30% of patients with colorectal cancer (CRC) and is associated with poor prognosis. In the present study, we unravel a novel mechanism by which LIN28B regulates colonic epithelial cell-cell junctions and CRC metastasis. Using human CRC cells (DLD-1, Caco-2 and LoVo) with either knockdown or overexpression of LIN28B, we identified Claudin 1 (CLDN1) tight junction protein as a direct downstream target and effector of LIN28B. RNA immunoprecipitation revealed that LIN28B directly binds to and post-transcriptionally regulates CLDN1 mRNA. Furthermore, using in vitro assays and a novel murine model of metastatic CRC, we show that LIN28B-mediated CLDN1 expression enhances collective invasion, cell migration, and metastatic liver tumor formation. Bulk RNA-sequencing of the metastatic liver tumors identified NOTCH3 as a downstream effector of the LIN28B-CLDN1 axis. Additionally, genetic and pharmacologic manipulation of NOTCH3 signaling revealed that NOTCH3 was necessary for invasion and metastatic liver tumor formation. In summary, our results suggest that LIN28B promotes invasion and liver metastasis of CRC by post-transcriptionally regulating CLDN1 and activating NOTCH3 signaling. This discovery offers a promising new therapeutic option for metastatic CRC to the liver, an area where therapeutic advancements have been relatively scarce.
    Keywords:  Cancer; Colorectal cancer; Gastroenterology; Oncology
    DOI:  https://doi.org/10.1172/jci.insight.167310
  16. Methods Mol Biol. 2023 ;2650 197-206
    In Vitro Morphogenesis and Differentiation of Human Intestinal Epithelium in a Gut-on-a-Chip.
    Woojung Shin, Hyun Jung Kim.
      The establishment of a three-dimensional (3D) epithelial structure and cytodifferentiation in vitro is necessary to recapitulate in vivo-relevant structure and function of the human intestine. Here, we describe an experimental protocol to build an organomimetic gut-on-a-chip microdevice that allows inducing 3D morphogenesis of human intestinal epithelium using Caco-2 cells or intestinal organoid cells. Under physiological flow and physical motions, intestinal epithelium spontaneously recreates 3D epithelial morphology in a gut-on-a-chip that offers enhanced mucus production, epithelial barrier, and longitudinal host-microbe co-culture. This protocol may provide implementable strategies to advance traditional in vitro static cultures, human microbiome studies, and pharmacological testing.
    Keywords:  Gut-on-a-chip; Human intestinal epithelium; In vitro model; Morphogenesis; Organoid
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_15
  17. Methods Mol Biol. 2023 ;2650 3-16
    Differentiated Epithelial Cells of the Gut.
    Andrea Bonilla-Díaz, Paloma Ordóñez-Morán.
      The intestine is a prime example of self-renewal where stem cells give rise to progenitor cells called transit-amplifying cells which differentiate into more specialized cells. There are two intestinal lineages: the absorptive (enterocytes and microfold cells) and the secretory (Paneth cells, enteroendocrine, goblet cells, and tuft cells). Each of these differentiated cell types has a role in creating an "ecosystem" to maintain intestinal homeostasis. Here, we summarize the main roles of each cell type.
    Keywords:  Colon; Differentiation; Enterocytes; Enteroendocrine cells; Goblet cells; Intestine; Microfold cells; Paneth cells; Tuft cells
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_1
  18. Sci Adv. 2023 Jun 16. 9(24): eadf5464
    Single-cell and spatial transcriptome analysis reveals the cellular heterogeneity of liver metastatic colorectal cancer.
    Fei Wang, Jie Long, Liang Li, Zi-Xin Wu, Tian-Tian Da, Xiao-Qing Wang, Chuan Huang, Yi-Hua Jiang, Xue-Qing Yao, Hai-Qing Ma, Zhe-Xiong Lian, Zhi-Bin Zhao, Jie Cao.
      In this study, we comprehensively charted the cellular landscape of colorectal cancer (CRC) and well-matched liver metastatic CRC using single-cell and spatial transcriptome RNA sequencing. We generated 41,892 CD45- nonimmune cells and 196,473 CD45+ immune cells from 27 samples of six CRC patients, and found that CD8_CXCL13 and CD4_CXCL13 subsets increased significantly in liver metastatic samples that exhibited high proliferation ability and tumor-activating characterization, contributing to better prognosis of patients. Distinct fibroblast profiles were observed in primary and liver metastatic tumors. F3+ fibroblasts enriched in primary tumors contributed to worse overall survival by expressing protumor factors. However, MCAM+ fibroblasts enriched in liver metastatic tumors might promote generation of CD8_CXCL13 cells through Notch signaling. In summary, we extensively analyzed the transcriptional differences of cell atlas between primary and liver metastatic tumors of CRC by single-cell and spatial transcriptome RNA sequencing, providing different dimensions of the development of liver metastasis in CRC.
    DOI:  https://doi.org/10.1126/sciadv.adf5464
  19. Methods Mol Biol. 2023 ;2650 17-34
    Identification of Differentiated Intestinal Epithelial Cells Using Immunostaining and Fluorescence Microscopy.
    Jessica R Digrazia, Melinda A Engevik, Amy C Engevik.
      Immunofluorescence imaging enables visualization of a wide range of molecules in diverse cells and tissues. Determining the localization and endogenous protein levels in cells using immunostaining can be highly informative for researchers studying cell structure and function. The small intestinal epithelium is composed of numerous cell types including absorptive enterocytes, mucus-producing goblet cells, lysozyme positive Paneth cells, proliferative stem cells, chemosensing tuft cells, and hormone-producing enteroendocrine cells. Each cell type in the small intestine has unique functions and structures that are critical for maintaining intestinal homeostasis and identifiable by immunofluorescence labeling. In this chapter we provide a detailed protocol and representative images of immunostaining of paraffin-embedded mouse small intestinal tissue. The method highlights antibodies and micrographs that identify differentiated cell types. These details are important because quality immunofluorescence imaging can provide novel insights and a greater understanding of healthy and disease states.
    Keywords:  Antibody; Enterocytes; Enteroendocrine cells; Goblet cells; Immunofluorescence; Immunostaining; Intestine; Microscopy; Paneth cells; Tuft cells
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_2
  20. Acta Biochim Biophys Sin (Shanghai). 2023 Jun 13.
    Negative correlation between acetyl-CoA acyltransferase 2 and cetuximab resistance in colorectal cancer.
    Yitao Yuan, Xun Sun, Mengling Liu, Suyao Li, Yu Dong, Keshu Hu, Jiayu Zhang, Bei Xu, Sining Ma, Hesheng Jiang, Pengcong Hou, Yufu Lin, Lu Gan, Tianshu Liu.
      The emergence of anti-EGFR therapy has revolutionized the treatment of colorectal cancer (CRC). However, not all patients respond consistently well. Therefore, it is imperative to conduct further research to identify the molecular mechanisms underlying the development of cetuximab resistance in CRC. In this study, we find that the expressions of many metabolism-related genes are downregulated in cetuximab-resistant CRC cells compared to their sensitive counterparts. Specifically, acetyl-CoA acyltransferase 2 (ACAA2), a key enzyme in fatty acid metabolism, is downregulated during the development of cetuximab resistance. Silencing of ACAA2 promotes proliferation and increases cetuximab tolerance in CRC cells, while overexpression of ACAA2 exerts the opposite effect. RTK-Kras signaling might contribute to the downregulation of ACAA2 expression in CRC, and ACAA2 predicts CRC prognosis in patients with Kras mutations. Collectively, our data suggest that modulating ACAA2 expression contributes to secondary cetuximab resistance in Kras wild-type CRC patients. ACAA2 expression is related to Kras mutation and demonstrates a prognostic role in CRC patients with Kras mutation. Thus, ACAA2 is a potential target in CRC with Kras mutation.
    Keywords:  ACAA2; cetuximab resistance; colorectal cancer; mutation
    DOI:  https://doi.org/10.3724/abbs.2023111
  21. Cell Commun Signal. 2023 Jun 14. 21(1): 130
    Mutual impact of adipocytes and colorectal cancer cells growing in co-culture conditions.
    Joanna Olszańska, Katarzyna Pietraszek-Gremplewicz, Mikołaj Domagalski, Dorota Nowak.
       BACKGROUND: Colorectal cancer (CRC) is the third most common malignancy worldwide. CRC cells are situated in an adipocyte-rich microenvironment, which leads to interactions between adipocytes and CRC cells. Upon exposure to cancer cells, adipocytes transform into cancer-associated adipocytes (CAAs), and as a result, they gain features that promote tumor progression. The aim of this research was to shed more light on the detailed role of interactions between adipocytes and CRC cells associated with cancer progression in the context of these alterations.
    METHODS: To implement adipocyte-CRC cell interaction, a co-culture model was applied. The analyses mainly focused on the metabolic modifications within CAAs and CRC cells, as well as the proliferation and migration potential of CRC cells. The impact of CRC on adipocytes was investigated by qRT-PCR analysis and Oil Red O staining. Proliferation and migration of CRC cells upon co-culture were tested with videomicroscopy, XTT, and a wound healing assay. Metabolic changes within CAAs and CRC cells were investigated based on lipid droplet formation, cell cycle analysis, gene and protein expression by qRT-PCR, and western blotting techniques.
    RESULTS: CRC cells induced reprogramming of adipocytes into CAAs, which was connected with downregulation of lipid droplet formation in CAAs and alteration in adipocyte features. CAAs showed decreased metabolism-related gene expression, phosphorylation of Akt, ERK kinases, STAT3, and lactate secretion in comparison to the control. CAAs also promoted the migration, proliferation, and lipid droplet accumulation of CRC cells. After co-culturing with adipocytes, there was a shift to the G2/M phase of the cell cycle according to the differences in cyclin expression.
    CONCLUSION: There are complex bidirectional interactions between adipocytes and CRC cells that may be connected with the induction of CRC cell progression. Video Abstract.
    Keywords:  Adipokines; Cancer-associated adipocytes (CAAs); Colorectal cancer (CRC); Lipid droplets; Metabolic reprogramming; Tumor microenvironment (TME)
    DOI:  https://doi.org/10.1186/s12964-023-01155-8
  22. J Clin Oncol. 2023 Jun 20. 41(18): 3278-3286
    Wild-Type KRAS Is Required for Panitumumab Efficacy in Patients With Metastatic Colorectal Cancer.
    Rafael G Amado, Michael Wolf, Marc Peeters, Eric Van Cutsem, Salvatore Siena, Daniel J Freeman, Todd Juan, Robert Sikorski, Sid Suggs, Robert Radinsky, Scott D Patterson, David D Chang.
       PURPOSE: Panitumumab, a fully human antibody against the epidermal growth factor receptor (EGFR), has activity in a subset of patients with metastatic colorectal cancer (mCRC). Although activating mutations in KRAS, a small G-protein downstream of EGFR, correlate with poor response to anti-EGFR antibodies in mCRC, their role as a selection marker has not been established in randomized trials.
    PATIENTS AND METHODS: KRAS mutations were detected using polymerase chain reaction on DNA from tumor sections collected in a phase III mCRC trial comparing panitumumab monotherapy to best supportive care (BSC). We tested whether the effect of panitumumab on progression-free survival (PFS) differed by KRAS status.
    RESULTS: KRAS status was ascertained in 427 (92%) of 463 patients (208 panitumumab, 219 BSC). KRAS mutations were found in 43% of patients. The treatment effect on PFS in the wild-type (WT) KRAS group (hazard ratio [HR], 0.45; 95% CI: 0.34 to 0.59) was significantly greater (P < .0001) than in the mutant group (HR, 0.99; 95% CI, 0.73 to 1.36). Median PFS in the WT KRAS group was 12.3 weeks for panitumumab and 7.3 weeks for BSC. Response rates to panitumumab were 17% and 0%, for the WT and mutant groups, respectively. WT KRAS patients had longer overall survival (HR, 0.67; 95% CI, 0.55 to 0.82; treatment arms combined). Consistent with longer exposure, more grade III treatment-related toxicities occurred in the WT KRAS group. No significant differences in toxicity were observed between the WT KRAS group and the overall population.
    CONCLUSION: Panitumumab monotherapy efficacy in mCRC is confined to patients with WT KRAS tumors. KRAS status should be considered in selecting patients with mCRC as candidates for panitumumab monotherapy.
    DOI:  https://doi.org/10.1200/JCO.22.02758
  23. Methods Mol Biol. 2023 ;2650 53-61
    In Vitro and in Vivo Assays for Testing Retinoids Effect on Intestinal Progenitors' Lineage Commitments.
    Krishna R Gajera, Kathryn L Fair, Gordon W Moran, Nicholas R F Hannan, Joerg Huelsken, Paloma Ordóñez-Morán.
      The intestine consists of epithelial cells surrounded by a complex environment as mesenchymal cells and the gut microbiota. With its impressive stem cell regeneration capability, the intestine is able to constantly replenish cells lost through apoptosis or abrasion by food passing through. Over the past decade, researchers have identified signaling pathways involved in stem cell homeostasis such as retinoids pathway. Retinoids are also involved in cell differentiation of healthy and cancer cells. In this study, we describe several approaches in vitro and in vivo to further investigate the effect of retinoids on stem cells, progenitors, and differentiated intestinal cells.
    Keywords:  Absorptive; Differentiation; Intestine; Organoids; Retinoids; Secretory; Stem cells
    DOI:  https://doi.org/10.1007/978-1-0716-3076-1_5
  24. Stem Cell Reports. 2023 Jun 13. pii: S2213-6711(23)00188-1. [Epub ahead of print]18(6): 1255-1270
    Organoids are not organs: Sources of variation and misinformation in organoid biology.
    Kim Bak Jensen, Melissa Helen Little.
      In the past decade, the term organoid has moved from obscurity to common use to describe a 3D in vitro cellular model of a tissue that recapitulates structural and functional elements of the in vivo organ it models. The term organoid is now applied to structures formed as a result of two distinct processes: the capacity for adult epithelial stem cells to re-create a tissue niche in vitro and the ability to direct the differentiation of pluripotent stem cells to a 3D self-organizing multicellular model of organogenesis. While these two organoid fields rely upon different stem cell types and recapitulate different processes, both share common challenges around robustness, accuracy, and reproducibility. Critically, organoids are not organs. This commentary serves to discuss these challenges, how they impact genuine utility, and shine a light on the need to improve the standards applied to all organoid approaches.
    DOI:  https://doi.org/10.1016/j.stemcr.2023.05.009
  25. Front Immunol. 2023 ;14 1190810
    Short chain fatty acids prime colorectal cancer cells to activate antitumor immunity.
    Courtney Mowat, Jasmine Dhatt, Ilsa Bhatti, Angela Hamie, Kristi Baker.
       Introduction: Colorectal cancer (CRC) is a leading cause of death worldwide and its growth can either be promoted or inhibited by the metabolic activities of intestinal microbiota. Short chain fatty acids (SCFAs) are microbial metabolites with potent immunoregulatory properties yet there is a poor understanding of how they directly regulate immune modulating pathways within the CRC cells.
    Methods: We used engineered CRC cell lines, primary organoid cultures, orthotopic in vivo models, and patient CRC samples to investigate how SCFA treatment of CRC cells regulates their ability to activate CD8+ T cells.
    Results: CRC cells treated with SCFAs induced much greater activation of CD8+ T cells than untreated CRC cells. CRCs exhibiting microsatellite instability (MSI) due to inactivation of DNA mismatch repair were much more sensitive to SCFAs and induced much greater CD8+ T cell activation than chromosomally instable (CIN) CRCs with intact DNA repair, indicating a subtype-dependent response to SCFAs. This was due to SCFA-induced DNA damage that triggered upregulation of chemokine, MHCI, and antigen processing or presenting genes. This response was further potentiated by a positive feedback loop between the stimulated CRC cells and activated CD8+ T cells in the tumor microenvironment. The initiating mechanism in the CRCs was inhibition of histone deacetylation by the SCFAs that triggered genetic instability and led to an overall upregulation of genes associated with SCFA signaling and chromatin regulation. Similar gene expression patterns were found in human MSI CRC samples and in orthotopically grown MSI CRCs independent of the amount of SCFA producing bacteria in the intestine.
    Discussion: MSI CRCs are widely known to be more immunogenic than CIN CRCs and have a much better prognosis. Our findings indicate that a greater sensitivity to microbially produced SCFAs contributes to the successful activation of CD8+ T cells by MSI CRCs, thereby identifying a mechanism that could be therapeutically targeted to improve antitumor immunity in CIN CRCs.
    Keywords:  HDAC; antitumor immunity; colorectal cancer; microbiota; microsatellite instability; short chain fatty acid (SCFA)
    DOI:  https://doi.org/10.3389/fimmu.2023.1190810
  26. Cell Stem Cell. 2023 Jun 12. pii: S1934-5909(23)00182-0. [Epub ahead of print]
    Organoid cultures for cancer modeling.
    Helen H N Yan, April S Chan, Frank Pui-Ling Lai, Suet Yi Leung.
      Organoids derived from adult stem cells (ASCs) and pluripotent stem cells (PSCs) are important preclinical models for studying cancer and developing therapies. Here, we review primary tissue-derived and PSC-derived cancer organoid models and detail how they have the potential to inform personalized medical approaches in different organ contexts and contribute to the understanding of early carcinogenic steps, cancer genomes, and biology. We also compare the differences between ASC- and PSC-based cancer organoid systems, discuss their limitations, and highlight recent improvements to organoid culture approaches that have helped to make them an even better representation of human tumors.
    Keywords:  cancer; organoids
    DOI:  https://doi.org/10.1016/j.stem.2023.05.012
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