bims-instec 2023-06-04 papers

bims-instec

Biomed News

on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration

Issue of 2023–06–04
eight papers selected by
Maria-Virginia Giolito, Free University of Brussels

Intestinal organoids as an in vitro platform to characterize disposition, metabolism, and safety profile of small molecules.
Maternal high-fat diet increases the susceptibility of offspring to colorectal cancer via the activation of intestinal inflammation.
Single-cell sequencing technology in colorectal cancer: a new technology to disclose the tumor heterogeneity and target precise treatment.
Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer.
CDK4/6 inhibition confers protection of normal gut epithelia against gemcitabine and the active metabolite of irinotecan.
MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer.
Activating transcription factor 5 (ATF5) controls intestinal tuft and goblet cell expansion upon succinate-induced type 2 immune responses in mice.
Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures.

Eur J Pharm Sci. 2023 May 25. pii: S0928-0987(23)00111-2. [Epub ahead of print] 106481

Intestinal organoids as an in vitro platform to characterize disposition, metabolism, and safety profile of small molecules.

Stephanie Kourula, Merel Derksen, Ferran Jardi, Sophie Jonkers, Marjolein van Heerden, Peter Verboven, Veronique Theuns, Stijn Van Asten, Tinne Huybrechts, Annett Kunze, Ewa Frazer-Mendelewska, Ka Wai Lai, René Overmeer, Jamie Lee Roos, Robert G J Vries, Sylvia F Boj, Mario Monshouwer, Farzin Pourfarzad, Jan Snoeys.

  Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species. Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available. Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids. In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.

Keywords:  ADME; Drug absorption; Drug disposition; Intestinal in vitro tools; Intestinal metabolism; Intestinal organoids; Intestinal toxicity

DOI:  https://doi.org/10.1016/j.ejps.2023.106481
Front Nutr. 2023 ;10 1191206

Maternal high-fat diet increases the susceptibility of offspring to colorectal cancer via the activation of intestinal inflammation.

Shimin Zheng, Jianbin Yin, Hui Yue, Lifu Li.

  A high-fat diet plays a key role in the pathogenesis of colorectal cancer, and this effect on the gut can also occur in the offspring of mothers with a high-fat diet. In this review, we discuss the role of a high-fat diet in the pathogenesis of colorectal cancer and summarize the effects of a maternal high-fat diet on the activation of inflammation and development of colorectal cancer in offspring. Studies have found that a maternal high-fat diet primarily induces an inflammatory response in the colorectal tissue of both the mother herself and the offspring during pregnancy. This leads to the accumulation of inflammatory cells in the colorectal tissue and the release of inflammatory cytokines, which further activate the NF-κb and related inflammatory signaling pathways. Research suggests that high levels of lipids and inflammatory factors from mothers with a high-fat diet are passed to the offspring through the transplacental route, which induces colorectal inflammation, impairs the intestinal microecological structure and the intestinal barrier, and interferes with intestinal development in the offspring. This in turn activates the NF-κb and related signaling pathways, which further aggravates intestinal inflammation. This process of continuous inflammatory stimulation and repair may promote the uncontrolled proliferation of colorectal mucosal cells in the offspring, thus increasing their susceptibility to colorectal cancer.

Keywords:  colorectal cancer; high-fat diet; intestinal inflammation; maternal; offspring

DOI:  https://doi.org/10.3389/fnut.2023.1191206
Front Immunol. 2023 ;14 1175343

Single-cell sequencing technology in colorectal cancer: a new technology to disclose the tumor heterogeneity and target precise treatment.

Rongbo Wen, Leqi Zhou, Zhiying Peng, Hao Fan, Tianshuai Zhang, Hang Jia, Xianhua Gao, Liqiang Hao, Zheng Lou, Fuao Cao, Guanyu Yu, Wei Zhang.

  Colorectal Cancer (CRC) is one of the most common gastrointestinal tumors, and its high tumor heterogeneity makes traditional sequencing methods incapable of obtaining information about the heterogeneity of individual cancer cells in CRC. Therefore, single-cell sequencing technology can be applied to better analyze the differences in genetic and protein information between cells, to obtain genomic sequence information of single cells, and to more thoroughly analyze the cellular characteristics and interactions in the CRC microenvironment. This will provide a more comprehensive understanding of colorectal cancer development and metastasis and indicate the treatment plan and prognosis. In this study, we review the application of single-cell sequencing to analyze the tumor microenvironment of CRC, explore the mechanisms involved in CRC metastasis and progression, and provide a reference for potential treatment options.

Keywords:  colorectal cancer; precise treatment; single-cell sequencing; tumor heterogeneity; tumor microenvironment

DOI:  https://doi.org/10.3389/fimmu.2023.1175343
Front Immunol. 2023 ;14 1053920

Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer.

Esther Strating, Mathijs P Verhagen, Emerens Wensink, Ester Dünnebach, Liza Wijler, Itziar Aranguren, Alberto Sanchez De la Cruz, Niek A Peters, Joris H Hageman, Mirjam M C van der Net, Susanne van Schelven, Jamila Laoukili, Riccardo Fodde, Jeanine Roodhart, Stefan Nierkens, Hugo Snippert, Martijn Gloerich, Inne Borel Rinkes, Sjoerd G Elias, Onno Kranenburg.

   Background: Poor prognosis in colon cancer is associated with a high content of cancer-associated fibroblasts (CAFs) and an immunosuppressive tumor microenvironment. The relationship between these two features is incompletely understood. Here, we aimed to generate a model system for studying the interaction between cancer cells and CAFs and their effect on immune-related cytokines and T cell proliferation.
Methods: CAFs were isolated from colon cancer liver metastases and were immortalized to prolong lifespan and improve robustness and reproducibility. Established medium and matrix compositions that support the growth of patient-derived organoids were adapted to also support CAF growth. Changes in growth pattern and cellular re-organization were assessed by confocal microscopy, live cell imaging, and immunofluorescence. Single cell RNA sequencing was used to study CAF/organoid co-culture-induced phenotypic changes in both cell types. Conditioned media were used to quantify the production of immunosuppressive factors and to assess their effect on T cell proliferation.
Results: We developed a co-culture system in which colon cancer organoids and CAFs spontaneously organize into superstructures with a high capacity to contract and stiffen the extracellular matrix (ECM). CAF-produced collagen IV provided a basement membrane supporting cancer cell organization into glandular structures, reminiscent of human cancer histology. Single cell RNA sequencing analysis showed that CAFs induced a partial epithelial-to-mesenchymal-transition in a subpopulation of cancer cells, similar to what is observed in the mesenchymal-like consensus molecular subtype 4 (CMS4) colon cancer. CAFs in co-culture were characterized by high expression of ECM components, ECM-remodeling enzymes, glycolysis, hypoxia, and genes involved in immunosuppression. An expression signature derived from CAFs in co-culture identified a subpopulation of glycolytic myofibroblasts specifically residing in CMS1 and CMS4 colon cancer. Medium conditioned by co-cultures contained high levels of the immunosuppressive factors TGFβ1, VEGFA and lactate, and potently inhibited T cell proliferation.
Conclusion: Co-cultures of organoids and immortalized CAFs recapitulate the histological, biophysical, and immunosuppressive features of aggressive mesenchymal-like human CRC. The model can be used to study the mechanisms of immunosuppression and to test therapeutic strategies targeting the cross-talk between CAFs and cancer cells. It can be further modified to represent distinct colon cancer subtypes and (organ-specific) microenvironments.

Keywords:  CMS4; cancer-associated fibroblast (CAF); colorectal cancer; immunosuppressive; microenvironment

DOI:  https://doi.org/10.3389/fimmu.2023.1053920
Cell Cycle. 2023 Jun 02. 1-20

CDK4/6 inhibition confers protection of normal gut epithelia against gemcitabine and the active metabolite of irinotecan.

Joshua Blume, Luisa Claus, Tamara Isermann, Antje Dickmanns, Lena-Christin Conradi, Ramona Schulz-Heddergott, Matthias Dobbelstein.

  Cancer chemotherapy relies on a high ratio of toxicity toward cancer cells vs. nonmalignant cells, making it desirable to protect normal cells. Among the nonmalignant cells, epithelia of the gut belong to the most vulnerable ones toward chemotherapeutics. Here, we use a murine intestinal organoid model to assess a strategy for protecting such epithelia against chemotherapy. Cell cycle progression was first stalled by palbociclib, a clinically established cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Washout of the drug allowed subsequent outgrowth of gut organoids. This transient cell cycle arrest conferred near-complete protection of the cells toward the nucleoside analogue gemcitabine. Moreover, pre-treatment with palbociclib protected the organoids against SN-38, the topoisomerase I-inhibiting metabolite of irinotecan, which is otherwise known for its severe gastrointestinal toxicities. In contrast, RB1-mutated cancer cells were not protected against gemcitabine or SN-38 when pre-treated with palbociclib. Taken together, these results outline a strategy for protecting nonmalignant cells against the toxicities of chemotherapeutics commonly used to treat advanced colorectal and pancreatic cancer. We propose that this strategy is particularly promising to protect the gut when treating RB1-deficient tumors that fail to arrest the cell cycle in response to CDK4/6 inhibitors. [Figure: see text].

Keywords:  CDK4/6; cyclotherapy; gemcitabine; intestinal organoids; irinotecan; palbociclib

DOI:  https://doi.org/10.1080/15384101.2023.2217003
Elife. 2023 Jun 01. pii: e80854. [Epub ahead of print]12

MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer.

Changyu Zhu, Yadira M Soto-Feliciano, John P Morris, Chun-Hao Huang, Richard P Koche, Yu-Jui Ho, Ana Banito, Chun-Wei David Chen, Aditya Shroff, Sha Tian, Geulah Livshits, Chi-Chao Chen, Myles Fennell, Scott A Armstrong, C David Allis, Darjus F Tschaharganeh, Scott W Lowe.

  Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.

Keywords:  cancer biology; genetics; genomics; human; mouse

DOI:  https://doi.org/10.7554/eLife.80854
Cell Tissue Res. 2023 May 31.

Activating transcription factor 5 (ATF5) controls intestinal tuft and goblet cell expansion upon succinate-induced type 2 immune responses in mice.

Haruo Nakano, Ayano Hata, Usato Ishimura, Ryo Kosugi, Eina Miyamoto, Kota Nakamura, Takumi Muramatsu, Moe Ogasawara, Motohiro Yamada, Mariko Umemura, Shigeru Takahashi, Yuji Takahashi.

  Intestinal tuft cells, a chemosensory cell type in mucosal epithelia that secrete interleukin (IL)-25, play a pivotal role in type 2 immune responses triggered by parasitic infections. Tuft cell-derived IL-25 activates type 2 innate lymphoid cells (ILC2) to secrete IL-13, which, in turn, acts on intestinal stem or transient amplifying cells to expand tuft cells themselves and mucus-secreting goblet cells. However, the molecular mechanisms of tuft cell differentiation under type 2 immune responses remain unclear. The present study investigated the effects of the deletion of activating transcription factor 5 (ATF5) on the type 2 immune response triggered by succinate (a metabolite of parasites) in mice. ATF5 mRNAs were expressed in the small intestine, and the loss of the ATF5 gene did not affect the gross morphology of the tissue or the basal differentiation of epithelial cell subtypes. Succinate induced marked increases in tuft and goblet cell numbers in the ATF5-deficient ileum. Tuft cells in the ATF5-deficient ileum are assumed to be a subtype of intestinal tuft cells (Tuft-2 cells) marked by the transcription factor Spib. Exogenous IL-25 induced similar increases in tuft and goblet cell numbers in wild-type and ATF5-deficient ilea. IL-13 at a submaximal dose enhanced tuft cell differentiation more in ATF5-deficient than in wild-type intestinal organoids. These results indicate that the loss of ATF5 enhanced the tuft cell-ILC2 type 2 immune response circuit by promoting tuft cell differentiation in the small intestine, suggesting its novel regulatory role in immune responses against parasitic infections.

Keywords:  Activating transcription factor; Interleukin 25; Intestines; Tuft cell; Type 2 immune response

DOI:  https://doi.org/10.1007/s00441-023-03781-7
Nat Commun. 2023 May 30. 14(1): 3128

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures.

Anna Urciuolo, Giovanni Giuseppe Giobbe, Yixiao Dong, Federica Michielin, Luca Brandolino, Michael Magnussen, Onelia Gagliano, Giulia Selmin, Valentina Scattolini, Paolo Raffa, Paola Caccin, Soichi Shibuya, Dominic Scaglioni, Xuechun Wang, Ju Qu, Marko Nikolic, Marco Montagner, Gabriel L Galea, Hans Clevers, Monica Giomo, Paolo De Coppi, Nicola Elvassore.

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape.

DOI: https://doi.org/10.1038/s41467-023-37953-4