Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers

Scientific reports

2021 Sep 07

Lee, HJ;Myung, JK;Kim, HS;Lee, DH;Go, HS;Choi, JH;Koh, HM;Lee, SJ;Jang, B;
PMID: 34493772 | DOI: 10.1038/s41598-021-97351-y

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.

IκBζ controls IL-17-triggered gene expression program in intestinal epithelial cells that restricts colonization of SFB and prevents Th17-associated pathologies

Mucosal immunology

2022 Aug 24

Yamazaki, S;Inohara, N;Ohmuraya, M;Tsuneoka, Y;Yagita, H;Katagiri, T;Nishina, T;Mikami, T;Funato, H;Araki, K;Nakano, H;
PMID: 35999460 | DOI: 10.1038/s41385-022-00554-3

Control of gut microbes is crucial for not only local defense in the intestine but also proper systemic immune responses. Although intestinal epithelial cells (IECs) play important roles in cytokine-mediated control of enterobacteria, the underlying mechanisms are not fully understood. Here we show that deletion of IκBζ in IECs in mice leads to dysbiosis with marked expansion of segmented filamentous bacteria (SFB), thereby enhancing Th17 cell development and exacerbating inflammatory diseases. Mechanistically, the IκBζ deficiency results in decrease in the number of Paneth cells and impairment in expression of IL-17-inducible genes involved in IgA production. The decrease in Paneth cells is caused by aberrant activation of IFN-γ signaling and a failure of IL-17-dependent recovery from IFN-γ-induced damage. Thus, the IL-17R-IκBζ axis in IECs contributes to the maintenance of intestinal homeostasis by serving as a key component in a regulatory loop between the gut microbiota and immune cells.

USP7 inactivation suppresses APC-mutant intestinal hyperproliferation and tumor development

Stem cell reports

2022 Dec 29

Novellasdemunt, L;Kucharska, A;Baulies, A;Hutton, C;Vlachogiannis, G;Repana, D;Rowan, A;Suárez-Bonnet, A;Ciccarelli, F;Valeri, N;Li, VSW;
PMID: 36669491 | DOI: 10.1016/j.stemcr.2022.12.013

Adenomatous polyposis coli (APC) mutation is the hallmark of colorectal cancer (CRC), resulting in constitutive WNT activation. Despite decades of research, targeting WNT signaling in cancer remains challenging due to its on-target toxicity. We have previously shown that the deubiquitinating enzyme USP7 is a tumor-specific WNT activator in APC-truncated cells by deubiquitinating and stabilizing β-catenin, but its role in gut tumorigenesis is unknown. Here, we show in vivo that deletion of Usp7 in Apc-truncated mice inhibits crypt hyperproliferation and intestinal tumor development. Loss of Usp7 prolongs the survival of the sporadic intestinal tumor model. Genetic deletion, but not pharmacological inhibition, of Usp7 in Apc+/- intestine induces colitis and enteritis. USP7 inhibitor treatment suppresses growth of patient-derived cancer organoids carrying APC truncations in vitro and in xenografts. Our findings provide direct evidence that USP7 inhibition may offer a safe and efficacious tumor-specific therapy for both sporadic and germline APC-mutated CRC.

CXCL16 inhibits epithelial regeneration and promotes fibrosis during the progression of radiation enteritis

The Journal of pathology

2022 Nov 14

Cui, Y;Wu, H;Liu, Z;Ma, T;Liang, W;Zeng, Q;Chen, D;Qin, Q;Huang, B;Wang, MH;Huang, X;He, Y;Kuang, Y;Sugimoto, S;Sato, T;Wang, L;
PMID: 36373877 | DOI: 10.1002/path.6031

Radiation enteritis (RE) is a prevalent complication of radiotherapy for pelvic malignant tumors, characterized by severe intestinal epithelial destruction and progressive submucosal fibrosis. However, little is known about the pathogenesis of this disease and so far, there is no specific targeted therapy. Here, we report that CXCL16 is up-regulated in the injured intestinal tissues of RE patients and in a mouse model. Genetic deletion of Cxcl16 mitigates fibrosis and promotes intestinal stem cell-mediated epithelial regeneration after radiation injury in mice. Mechanistically, CXCL16 functions on myofibroblasts through its receptor CXCR6 and activates JAK3/STAT3 signaling to promote fibrosis, and meanwhile to transcriptionally modulate the levels of BMP4 and HGF in myofibroblasts. Moreover, we find that CXCL16 and CXCR6 auto- and cross-regulate themselves in positive feedback loops. Treatment with CXCL16 neutralizing monoclonal antibody attenuates fibrosis and improves the epithelial repair in RE mouse model. Our findings emphasize the important role of CXCL16 in the progression of RE, and suggest that CXCL16 signaling could be a potential therapeutic target for RE. This article is protected by

Modification of Diet to Reduce the Stemness and Tumorigenicity of Murine and Human Intestinal Cells

Molecular nutrition & food research

2022 Oct 01

May, S;Greenow, KR;Higgins, AT;Derrick, AV;Taylor, E;Pan, P;Konstantinou, M;Nixon, C;Wooley, TE;Sansom, OJ;Wang, LS;Parry, L;
PMID: 36045438 | DOI: 10.1002/mnfr.202200234

Black raspberries (BRBs) have colorectal cancer (CRC) chemo-preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs.Mice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB-supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the "crypt-progenitor" phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self-renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel.BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.

The circadian clock gene, Bmal1, regulates intestinal stem cell signaling and represses tumor initiation

Cellular and molecular gastroenterology and hepatology

2021 Sep 14

Stokes, K;Nunes, M;Trombley, C;Flôres, DEFL;Wu, G;Taleb, Z;Alkhateeb, A;Banskota, S;Harris, C;Love, OP;Khan, WI;Rueda, L;Hogenesch, JB;Karpowicz, P;
PMID: 34534703 | DOI: 10.1016/j.jcmgh.2021.08.001

Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known.We tested the non-redundant clock gene, Bmal1, in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer.Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod-disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal non-transformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA-sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal.Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors exhibit high Yap (Hippo signaling) activity but exhibit low Wnt activity. Intestinal organoid assays reveal that loss of Bmal1 increases self-renewal in a Yap-dependent manner.Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation.

A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

2023 Jun 01

Schaaf, CR;Polkoff, KM;Carter, A;Stewart, AS;Sheahan, B;Freund, J;Ginzel, J;Snyder, JC;Roper, J;Piedrahita, JA;Gonzalez, LM;
PMID: 37159340 | DOI: 10.1096/fj.202300223R

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/β-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

PDGFRα-induced stromal maturation is required to restrain postnatal intestinal epithelial stemness and promote defense mechanisms

Cell stem cell

2022 May 05

Jacob, JM;Di Carlo, SE;Stzepourginski, I;Lepelletier, A;Ndiaye, PD;Varet, H;Legendre, R;Kornobis, E;Benabid, A;Nigro, G;Peduto, L;
PMID: 35523143 | DOI: 10.1016/j.stem.2022.04.005

After birth, the intestine undergoes major changes to shift from an immature proliferative state to a functional intestinal barrier. By combining inducible lineage tracing and transcriptomics in mouse models, we identify a prodifferentiation PDGFRαHigh intestinal stromal lineage originating from postnatal LTβR+ perivascular stromal progenitors. The genetic blockage of this lineage increased the intestinal stem cell pool while decreasing epithelial and immune maturation at weaning age, leading to reduced postnatal growth and dysregulated repair responses. Ablating PDGFRα in the LTBR stromal lineage demonstrates that PDGFRα has a major impact on the lineage fate and function, inducing a transcriptomic switch from prostemness genes, such as Rspo3 and Grem1, to prodifferentiation factors, including BMPs, retinoic acid, and laminins, and on spatial organization within the crypt-villus and repair responses. Our results show that the PDGFRα-induced transcriptomic switch in intestinal stromal cells is required in the first weeks after birth to coordinate postnatal intestinal maturation and function.

LGR5 is a conserved marker of hair follicle stem cells in multiple species and is present early and throughout follicle morphogenesis

Scientific reports

2022 Jun 01

Polkoff, KM;Gupta, NK;Green, AJ;Murphy, Y;Chung, J;Gleason, KL;Simpson, SG;Walker, DM;Collins, B;Piedrahita, JA;
PMID: 35650234 | DOI: 10.1038/s41598-022-13056-w

Hair follicle stem cells are key for driving growth and homeostasis of the hair follicle niche, have remarkable regenerative capacity throughout hair cycling, and display fate plasticity during cutaneous wound healing. Due to the need for a transgenic reporter, essentially all observations related to LGR5-expressing hair follicle stem cells have been generated using transgenic mice, which have significant differences in anatomy and physiology from the human. Using a transgenic pig model, a widely accepted model for human skin and human skin repair, we demonstrate that LGR5 is a marker of hair follicle stem cells across species in homeostasis and development. We also report the strong similarities and important differences in expression patterns, gene expression profiles, and developmental processes between species. This information is important for understanding the fundamental differences and similarities across species, and ultimately improving human hair follicle regeneration, cutaneous wound healing, and skin cancer treatment.

Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR × LGR5 bispecific antibody with efficacy in epithelial tumors

Nature cancer

2022 Apr 01

Herpers, B;Eppink, B;James, MI;Cortina, C;Cañellas-Socias, A;Boj, SF;Hernando-Momblona, X;Glodzik, D;Roovers, RC;van de Wetering, M;Bartelink-Clements, C;Zondag-van der Zande, V;Mateos, JG;Yan, K;Salinaro, L;Basmeleh, A;Fatrai, S;Maussang, D;Lammerts van Bueren, JJ;Chicote, I;Serna, G;Cabellos, L;Ramírez, L;Nuciforo, P;Salazar, R;Santos, C;Villanueva, A;Stephan-Otto Attolini, C;Sancho, E;Palmer, HG;Tabernero, J;Stratton, MR;de Kruif, J;Logtenberg, T;Clevers, H;Price, LS;Vries, RGJ;Batlle, E;Throsby, M;
PMID: 35469014 | DOI: 10.1038/s43018-022-00359-0

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.

ADAMTS18+ villus tip telocytes maintain a polarized VEGFA signaling domain and fenestrations in nutrient-absorbing intestinal blood vessels

Nature communications

2022 Jul 09

Bernier-Latmani, J;Mauri, C;Marcone, R;Renevey, F;Durot, S;He, L;Vanlandewijck, M;Maclachlan, C;Davanture, S;Zamboni, N;Knott, GW;Luther, SA;Betsholtz, C;Delorenzi, M;Brisken, C;Petrova, TV;
PMID: 35810168 | DOI: 10.1038/s41467-022-31571-2

The small intestinal villus tip is the first point of contact for lumen-derived substances including nutrients and microbial products. Electron microscopy studies from the early 1970s uncovered unusual spatial organization of small intestinal villus tip blood vessels: their exterior, epithelial-facing side is fenestrated, while the side facing the villus stroma is non-fenestrated, covered by pericytes and harbors endothelial nuclei. Such organization optimizes the absorption process, however the molecular mechanisms maintaining this highly specialized structure remain unclear. Here we report that perivascular LGR5+ villus tip telocytes (VTTs) are necessary for maintenance of villus tip endothelial cell polarization and fenestration by sequestering VEGFA signaling. Mechanistically, unique VTT expression of the protease ADAMTS18 is necessary for VEGFA signaling sequestration through limiting fibronectin accumulation. Therefore, we propose a model in which LGR5+ ADAMTS18+ telocytes are necessary to maintain a "just-right" level and location of VEGFA signaling in intestinal villus blood vasculature to ensure on one hand the presence of sufficient endothelial fenestrae, while avoiding excessive leakiness of the vessels and destabilization of villus tip epithelial structures.

Lymphatics act as a signaling hub to regulate intestinal stem cell activity

Cell stem cell

2022 Jun 15

Niec, RE;Chu, T;Schernthanner, M;Gur-Cohen, S;Hidalgo, L;Pasolli, HA;Luckett, KA;Wang, Z;Bhalla, SR;Cambuli, F;Kataru, RP;Ganesh, K;Mehrara, BJ;Pe'er, D;Fuchs, E;
PMID: 35728595 | DOI: 10.1016/j.stem.2022.05.007

Barrier epithelia depend upon resident stem cells for homeostasis, defense, and repair. Epithelial stem cells of small and large intestines (ISCs) respond to their local microenvironments (niches) to fulfill a continuous demand for tissue turnover. The complexity of these niches and underlying communication pathways are not fully known. Here, we report a lymphatic network at the intestinal crypt base that intimately associates with ISCs. Employing in vivo loss of function and lymphatic:organoid cocultures, we show that crypt lymphatics maintain ISCs and inhibit their precocious differentiation. Pairing single-cell and spatial transcriptomics, we apply BayesPrism to deconvolve expression within spatial features and develop SpaceFold to robustly map the niche at high resolution, exposing lymphatics as a central signaling hub for the crypt in general and ISCs in particular. We identify WNT-signaling factors (WNT2, R-SPONDIN-3) and a hitherto unappreciated extracellular matrix protein, REELIN, as crypt lymphatic signals that directly govern the regenerative potential of ISCs.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

Search form

Probes for INS

Content for comparison

Gene

Product

Research area

Category

Advanced Cell Diagnostics

Bio-Techne

Advanced Cell Diagnostics China