bims-instec 2023-03-19 papers

bims-instec

Biomed News

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

Issue of 2023–03–19
twelve papers selected by
Maria-Virginia Giolito, Free University of Brussels

Fibroblast-derived EGF ligand Neuregulin-1 induces fetal-like reprogramming of the intestinal epithelium without supporting tumorigenic growth.
Smooth muscle contributes to the development and function of a layered intestinal stem cell niche.
Establishment of Epithelial Inflammatory Injury Model Using Intestinal Organoid Cultures.
Plasmatic BRAF-V600E allele fraction as a prognostic factor in metastatic colorectal cancer treated with BRAF combinatorial treatments.
Loss of LGR5 through therapy-induced downregulation or gene ablation is associated with resistance and enhanced MET-STAT3 signaling in colorectal cancer cells.
E2F1 inhibits cellular senescence and promotes oxaliplatin resistance in colorectal cancer.
Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids.
Precision oncology for KRASG12C-mutant colorectal cancer.
Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.
European expert panel consensus on the clinical management of BRAFV600E-mutant metastatic colorectal cancer.
Regulation of adult stem cell quiescence and its functions in the maintenance of tissue integrity.
CDX-2 expression correlates with clinical outcomes in MSI-H metastatic colorectal cancer patients receiving immune checkpoint inhibitors.

Dis Model Mech. 2023 Mar 13. pii: dmm.049692. [Epub ahead of print]

Fibroblast-derived EGF ligand Neuregulin-1 induces fetal-like reprogramming of the intestinal epithelium without supporting tumorigenic growth.

Toni T Lemmetyinen, Emma W Viitala, Linnea Wartiovaara, Tuomas Kaprio, Jaana Hagström, Caj Haglund, Pekka Katajisto, Timothy C Wang, Eva Domènech-Moreno, Saara Ollila.

  Growth factors secreted by stromal fibroblasts regulate the intestinal epithelium. Stroma-derived Epidermal growth factor (EGF) family ligands are implicated in epithelial regeneration and tumorigenesis, but their specific contributions and associated mechanisms remain unclear. Here, we use primary intestinal organoids modeling homeostatic, injured, and tumorigenic epithelium to assess how fibroblast-derived EGF family ligands Neuregulin-1 (NRG1) and Epiregulin (EREG) regulate the intestinal epithelium. NRG1 was expressed exclusively in the stroma, robustly increased crypt budding and protected intestinal epithelial organoids from radiation-induced damage. NRG1 also induced regenerative features in the epithelium including a fetal-like transcriptome, suppression of the Lgr5+ stem cell pool, and remodeling of the epithelial actin cytoskeleton. Intriguingly, unlike EGF and EREG, NRG1 failed to support the growth of pre-tumorigenic intestinal organoids lacking the tumor suppressor Apc, commonly mutated in human colorectal cancer (CRC). Interestingly, high expression of stromal NRG1 was associated with improved survival in CRC cohorts, suggesting a tumor suppressive function. Our results highlight the power of stromal NRG1 in transcriptional reprogramming and protection of the intestinal epithelium from radiation injury without promoting tumorigenesis.

Keywords:  Colorectal cancer; EGF; EREG; Intestinal regeneration; NRG1

DOI:  https://doi.org/10.1242/dmm.049692
Dev Cell. 2023 Mar 08. pii: S1534-5807(23)00070-9. [Epub ahead of print]

Smooth muscle contributes to the development and function of a layered intestinal stem cell niche.

Neil McCarthy, Guodong Tie, Shariq Madha, Ruiyang He, Judith Kraiczy, Adrianna Maglieri, Ramesh A Shivdasani.

  Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The in vivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.

Keywords:  crypt fission; epithelial-mesenchymal co-cultures; intestinal crypt morphogenesis; intestinal stem cell niche; intestinal sub-epithelial myofibroblasts; single-cell RNA profiles; smooth muscle; trophocytes

DOI:  https://doi.org/10.1016/j.devcel.2023.02.012
Stem Cells Int. 2023 ;2023 3328655

Establishment of Epithelial Inflammatory Injury Model Using Intestinal Organoid Cultures.

Chengfeng Xing, Guili Liang, Xin Yu, Anxing Zhang, Xiang Luo, Yu Liu, Zengli Tang, Bian Wu, Zhengji Song, Danfeng Lan.

Intestinal epithelial dysfunction is critical in the development of inflammatory bowel disease (IBD). However, most cellular experiments related to epithelial barrier studies in IBD have been based on tumor cell line that lack a variety of intestinal epithelial cell types. Thus, intestinal organoids can present the three-dimensional structure and better simulate the physiological structure and function of the intestinal epithelium in vitro. Here, the crypts were isolated from the small intestine of mice; with the participation of major cytokines (EGF, Noggin, and R-Spondin 1 included), the intestinal organoids were established at a density of 100 crypts per well, containing intestinal stem cells (ISC), Paneth cells, goblet cells, and intestinal endocrine cells. We found that tumor necrosis factor-alpha (TNF-α) could induce the inflammatory response of intestinal organoids, and a dose of 10 ng/mL could maintain stable passaging of organoids for dynamic observation. After stimulation with TNF-α, the intestinal organoid cultures showed lower expression of the cell proliferation-related protein identified by monoclonal antibody Ki 67 (Ki67), the ISC marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), and the intestinal tight junction proteins occludin (Ocln) and claudin-1 (Cldn1) while higher expression of the inflammatory cytokine interleukin- (IL-) 15 and the chemokines C-X-C motif ligand 2 (Cxcl2) and Cxcl10 significantly. In this study, we successfully established an epithelial inflammatory injury model of intestinal organoids, which provides an effective in vitro model for studying the pathogenesis and treatment of IBD.

DOI: https://doi.org/10.1155/2023/3328655
Ann Oncol. 2023 Mar 13. pii: S0923-7534(23)00112-6. [Epub ahead of print]

Plasmatic BRAF-V600E allele fraction as a prognostic factor in metastatic colorectal cancer treated with BRAF combinatorial treatments.

J Ros, J Matito, G Villacampa, R Comas, A Garcia, G Martini, I Baraibar, N Saoudi, F Salvà, Á Martin, M Antista, R Toledo, E Martinelli, F Pietrantonio, A Boccaccino, C Cremolini, R Dientsmann, J Tabernero, A Vivancos, E Elez.

   BACKGROUND: Combination of a BRAF inhibitor (BRAFi) and an anti-EGFR, with or without a MEK inhibitor (MEKi) improves survival in BRAF-V600E-mutant metastatic colorectal cancer (mCRC) over standard chemotherapy. However, responses are heterogeneous and there are no available biomarkers to assess patient prognosis or guide doublet or triplet-based regimes. In order to better characterize the clinical heterogeneity observed, we assessed the prognostic and predictive role of the plasmatic BRAF allele fraction (AF) for these combinations.
PATIENTS AND METHODS: A prospective discovery cohort including 47 BRAF-V600E-mutant patients treated with BRAF inhibitor-anti-EGFR +/- MEK inhibitor in clinical trials and real-world practice was evaluated. Results were validated in an independent multicenter cohort (N=29). Plasmatic BRAF-V600E AF cut-off at baseline was defined in the discovery cohort with droplet digital PCR (ddPCR). All patients had tissue-confirmed BRAF-V600E mutations.
RESULTS: Patients with high AF have major frequency of liver metastases and more metastatic sites. In the discovery cohort, median PFS and OS were 5.2 and 11.5 months, respectively. Patients with high-BRAF AF (≥2%, n=23) showed worse PFS (HR=2.97, 95% CI 1.55-5.69; P=0.001) and worse OS (HR=3.28, 95% CI 1.58-6.81; P=0.001) than low-BRAF AF patients (<2%, n=24). In the multivariable analysis, BRAF AF levels maintained independent significance. In the validation cohort, high-BRAF AF was associated with worse PFS (HR=3.83, 95% CI 1.60-9.17; P=0.002) and a trend toward worse OS was observed (HR=1.86, 95% CI 0.80-4.34; P=0.15). An exploratory analysis of predictive value showed that high-BRAF AF patients (n=35) benefited more from triplet therapy than low-BRAF AF patients (n=41; PFS and OS interaction tests, P<0.01).
CONCLUSIONS: Plasmatic BRAF AF determined by ddPCR is a reliable surrogate of tumor burden and aggressiveness in BRAF-V600E-mutant mCRC treated with a BRAFi plus an anti-EGFR with or without a MEKi and identifies patients who may benefit from treatment intensification. Our results warrant further validation of plasmatic BRAF AF to refine clinical stratification and guide treatment strategies.

Keywords:  BRAF inhibitor; BRAF-V600E mutation; MEK inhibitor; anti-EGFR; colorectal cancer; mutant allele fraction

DOI:  https://doi.org/10.1016/j.annonc.2023.02.016
Mol Cancer Ther. 2023 Mar 15. pii: MCT-22-0415. [Epub ahead of print]

Loss of LGR5 through therapy-induced downregulation or gene ablation is associated with resistance and enhanced MET-STAT3 signaling in colorectal cancer cells.

Tressie A Posey, Joan Jacob, Ashlyn Parkhurst, Shraddha Subramanian, Liezl E Francisco, Zhengdong Liang, Kendra S Carmon.

LGR5 is highly expressed in colorectal cancer (CRC) and cancer stem cells that play important roles in tumor initiation, progression, and metastasis. Loss of LGR5 has been shown to enhance therapy resistance. However, the molecular mechanisms that mediate this resistance remain elusive. In this study, we demonstrate conversion of LGR5+ CRC cells to an LGR5- state in response to chemotherapy, LGR5-targeted antibody-drug conjugates (ADCs), or LGR5 gene ablation, led to activation of STAT3. Further investigation revealed increased STAT3 activation occurred a result of increased MET activity. LGR5 overexpression decreased MET-STAT3 activity and sensitized CRC cells to therapy. STAT3 inhibition suppressed MET phosphorylation, while constitutively active STAT3 reduced LGR5 levels and increased MET activity, suggesting a potential feedback mechanism. Combination treatment of MET-STAT3 inhibitors with irinotecan or ADCs substantiated synergistic effects in CRC cells and tumor organoids. In CRC xenografts, STAT3 inhibition combined with irinotecan enhanced tumor growth suppression and prolonged survival. These findings suggest a mechanism by which drug-resistant LGR5- CRC cells acquire a survival advantage through activation of MET-STAT3 and provide rationale for new treatment strategies to target CRC.

DOI: https://doi.org/10.1158/1535-7163.MCT-22-0415
Ann Transl Med. 2023 Feb 28. 11(4): 185

E2F1 inhibits cellular senescence and promotes oxaliplatin resistance in colorectal cancer.

Haiyang Gao, Fangyuan Zhou, Runze Li, Jiao Yuan, Lin Ye.

   Background: Doctors have always been overwhelmed by tumor drug resistance because it is a major challenge in the clinical treatment of tumors. Cellular senescence has a strong relationship with the development of tumor drug resistance. Herein, we aimed to explore new regulatory factors involved in the aging process of colorectal cancer (CRC) cells and assess the effect of cellular senescence on CRC drug resistance.
Methods: Genes associated with cellular senescence for anticipating regulatory factors were first used, and the regulatory molecules of survival significance were then identified based on the results of public database analysis. The effects of E2F translation factor 1 (E2F1) on CRC cell viability, invasion, migration, and cellular senescence processes were assessed through 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), 5-Ethynyl-2'-deoxyuridine (EdU), Transwell, scar repairining, β-galactosidase staining, and cell immunofluorescence assays, respectively. Overexpression or silencing plasmids were used for transfecting HCT116 or OXA-HCT116 to assess the effect of E2F1 on the senescence process and drug resistance in CRC cells.
Results: On combining the database analysis results with those of our studies, we found that E2F1 was a critical regulator of cellular senescence in CRC. In the in vitro experiments, the E2F1 overexpression significantly stimulated the proliferation, invasion, and migration of CRC cells and even reduced oxaliplatin-induced senescence, further enhancing their resistance to oxaliplatin. Conversely, the tumorigenesis of colorectal cancer was repressed after the suppression of E2F1. Furthermore, CRC cells, which were otherwise resistant to oxaliplatin, also showed senescent phenotypes.
Conclusions: Our results suggest that E2F1 suppresses the aging of CRC cells and tumor cells develop resistance to oxaliplatin through high E2F1 expression. Moreover, E2F1 may act as a possible target for oxaliplatin resistance studies.

Keywords:  E2F translation factor 1; cellular senescence; chemoresistance; colorectal cancer; oxaliplatin

DOI:  https://doi.org/10.21037/atm-22-4054
Front Immunol. 2023 ;14 1097383

Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids.

Shreya Gopalakrishnan, Ingunn Bakke, Marianne Doré Hansen, Helene Kolstad Skovdahl, Atle van Beelen Granlund, Arne K Sandvik, Torunn Bruland.

  There are many unanswered questions regarding responses to proinflammatory signals in intestinal epithelial cells (IECs). For example, chemokines secreted by IECs upon external stimuli play multifunctional roles in both homeostasis and during inflammation. Several chemokines are upregulated during active inflammatory bowel disease (IBD), which is associated with an increased influx of immune cells into the gut mucosa. Therefore, studies on how chemokines are regulated in the intestinal epithelium may identify putative treatment targets in IBD. More recently, patient-derived ex vivo models such as intestinal organoids have facilitated molecular analysis of epithelial alterations in IBD patients own cells. Here, we describe refined experimental protocols and methods for the generation and maintenance of IBD patient-derived colonic organoids (colonoids) culture. We also give detailed description of medium, and supplements needed for colonoid establishment, growth, and differentiation, including production of Wnt-3A and Rspondin1 enriched media. Further, we present protocols for RNA and protein isolation from human colonoids, and subsequent gene expression analysis and Western blotting for e.g., signal transduction studies. We also describe how to process colonoids for chemokine protein expression analysis such as immunostaining, confocal imaging, and detection of secreted chemokines by e.g., enzyme-linked immunosorbent assay (ELISA). As proof of principle, we give examples of how the chemoattractant CCL20 can be regulated and expressed in colonoids derived from IBD-patients and healthy controls upon ligands-driven inflammation.

Keywords:  chemokines; colonoids; gene expression; inflammatory bowel diseases; intestinal epithelial organoids; intestinal epithelium; protein expression analysis; staining of paraffin sections

DOI:  https://doi.org/10.3389/fimmu.2023.1097383
Nat Rev Clin Oncol. 2023 Mar 13.

Precision oncology for KRASG12C-mutant colorectal cancer.

Federica Di Nicolantonio, Alberto Bardelli.

DOI: https://doi.org/10.1038/s41571-023-00748-z
Immunity. 2023 Mar 07. pii: S1074-7613(23)00092-4. [Epub ahead of print]

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Jinzhi Duan, Juan D Matute, Lukas W Unger, Thomas Hanley, Alexandra Schnell, Xi Lin, Niklas Krupka, Paul Griebel, Conner Lambden, Brandon Sit, Joep Grootjans, Michal Pyzik, Felix Sommer, Sina Kaiser, Maren Falk-Paulsen, Helmut Grasberger, John Y Kao, Tobias Fuhrer, Hai Li, Donggi Paik, Yunjin Lee, Samuel Refetoff, Jonathan N Glickman, Adrienne W Paton, Lynn Bry, James C Paton, Uwe Sauer, Andrew J Macpherson, Philip Rosenstiel, Vijay K Kuchroo, Matthew K Waldor, Jun R Huh, Arthur Kaser, Richard S Blumberg.

  Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Keywords:  Citrobacter rodentium; ROS signals; TH17 cells; commensal bacterial; epithelial endoplasmic reticulum stress; inflammatory bowel disease; purine metabolism

DOI:  https://doi.org/10.1016/j.immuni.2023.02.018
Cancer Treat Rev. 2023 Mar 11. pii: S0305-7372(23)00032-4. [Epub ahead of print]115 102541

European expert panel consensus on the clinical management of BRAFV600E-mutant metastatic colorectal cancer.

Erika Martinelli, Dirk Arnold, Andres Cervantes, Sebastian Stintzing, Eric Van Cutsem, Josep Tabernero, Julien Taieb, Harpreet Wasan, Fortunato Ciardiello.

  Metastatic colorectal cancer (mCRC) is a heterogenous disease caused by various genetic alterations. The BRAFV600E mutation occurs in approximately 8-12% of patients and is characterised by an aggressive clinical course and poor prognosis. Here we review the current knowledge on BRAFV600E-mutant mCRC and provide a series of consensus statements on its clinical management. The treatment landscape for BRAFV600E-mutant mCRC has changed greatly due to the emergence of molecular targeted therapies (including BRAF inhibitors) and immune checkpoint inhibitors. A scientific literature search identified available data on molecular testing, treatments, and clinical monitoring of patients with BRAFV600E-mutant mCRC. Consensus statements were discussed and developed by a European expert panel. This manuscript provides consensus management guidance for different clinical presentations of BRAFV600E-mutant mCRC and makes recommendations regarding treatment sequencing choices. To guide appropriate clinical management and treatment decisions for mCRC patients, tumour tissue analysis for DNA mismatch repair/microsatellite status and, at a minimum, KRAS, NRAS, and BRAF mutational status is mandatory at the time of diagnosis. Finally, we discuss the rapidly evolving treatment landscape for BRAFV600E-mutant mCRC and define priorities for the development of novel therapeutic strategies that are needed to improve patient outcomes.

Keywords:  BRAF; Consensus; Metastatic colorectal cancer; Treatment

DOI:  https://doi.org/10.1016/j.ctrv.2023.102541
Nat Rev Mol Cell Biol. 2023 Mar 15.

Regulation of adult stem cell quiescence and its functions in the maintenance of tissue integrity.

Antoine de Morree, Thomas A Rando.

Adult stem cells are important for mammalian tissues, where they act as a cell reserve that supports normal tissue turnover and can mount a regenerative response following acute injuries. Quiescent stem cells are well established in certain tissues, such as skeletal muscle, brain, and bone marrow. The quiescent state is actively controlled and is essential for long-term maintenance of stem cell pools. In this Review, we discuss the importance of maintaining a functional pool of quiescent adult stem cells, including haematopoietic stem cells, skeletal muscle stem cells, neural stem cells, hair follicle stem cells, and mesenchymal stem cells such as fibro-adipogenic progenitors, to ensure tissue maintenance and repair. We discuss the molecular mechanisms that regulate the entry into, maintenance of, and exit from the quiescent state in mice. Recent studies revealed that quiescent stem cells have a discordance between RNA and protein levels, indicating the importance of post-transcriptional mechanisms, such as alternative polyadenylation, alternative splicing, and translation repression, in the control of stem cell quiescence. Understanding how these mechanisms guide stem cell function during homeostasis and regeneration has important implications for regenerative medicine.

DOI: https://doi.org/10.1038/s41580-022-00568-6
Sci Rep. 2023 Mar 16. 13(1): 4397

CDX-2 expression correlates with clinical outcomes in MSI-H metastatic colorectal cancer patients receiving immune checkpoint inhibitors.

Pina Ziranu, Andrea Pretta, Marta Pozzari, Antonio Maccioni, Manuela Badiali, Daniela Fanni, Eleonora Lai, Clelia Donisi, Mara Persano, Clara Gerosa, Marco Puzzoni, Fabio Bardanzellu, Rossano Ambu, Valeria Pusceddu, Marco Dubois, Giulia Cerrone, Marco Migliari, Sara Murgia, Dario Spanu, Gianluca Pretta, Valentina Aimola, Francesca Balconi, Stefania Murru, Gavino Faa, Mario Scartozzi.

Immune checkpoint inhibitors (ICIs) showed efficacy in metastatic colorectal cancer (mCRC) with mismatch-repair deficiency or high microsatellite instability (dMMR-MSI-H). Unfortunately, a patient's subgroup did not benefit from immunotherapy. Caudal-related homeobox transcription factor 2 (CDX-2) would seem to influence immunotherapy's sensitivity, promoting the chemokine (C-X-C motif) ligand 14 (CXCL14) expression. Therefore, we investigated CDX-2 role as a prognostic-predictive marker in patients with mCRC MSI-H. We retrospectively collected data from 14 MSI-H mCRC patients treated with ICIs between 2019 and 2021. The primary endpoint was the 12-month progression-free-survival (PFS) rate. The secondary endpoints were overall survival (OS), PFS, objective response rate (ORR), and disease control rate (DCR). The PFS rate at 12 months was 81% in CDX-2 positive patients vs 0% in CDX-2 negative patients (p = 0.0011). The median PFS was not reached (NR) in the CDX-2 positive group versus 2.07 months (95%CI 2.07-10.8) in CDX-2 negative patients (p = 0.0011). Median OS was NR in CDX-2-positive patients versus 2.17 months (95% Confidence Interval [CI] 2.17-18.7) in CDX2-negative patients (p = 0.026). All CDX-2-positive patients achieved a disease response, one of them a complete response. Among CDX-2-negative patients, one achieved stable disease, while the other progressed rapidly (ORR: 100% vs 0%, p = 0.0005; DCR: 100% vs 50%, p = 0.02). Twelve patients received 1st-line pembrolizumab (11 CDX-2 positive and 1 CDX-2 negative) not reaching median PFS, while two patients (1 CDX-2 positive and 1 CDX-2 negative) received 3rd-line pembrolizumab reaching a median PFS of 10.8 months (95% CI, 10.8-12.1; p = 0.036). Although our study reports results on a small population, the prognostic role of CDX-2 in CRC seems confirmed and could drive a promising predictive role in defining the population more sensitive to immunotherapy treatment. Modulating the CDX-2/CXCL14 axis in CDX-2-negative patients could help overcome primary resistance to immunotherapy.

DOI: https://doi.org/10.1038/s41598-023-31538-3