Cell-matrix interface regulates dormancy in human colon cancer stem cells

Nature

2022 Jul 07

Ohta, Y;Fujii, M;Takahashi, S;Takano, A;Nanki, K;Matano, M;Hanyu, H;Saito, M;Shimokawa, M;Nishikori, S;Hatano, Y;Ishii, R;Sawada, K;Machinaga, A;Ikeda, W;Imamura, T;Sato, T;
PMID: 35798028 | DOI: 10.1038/s41586-022-05043-y

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells (CSCs)1, a lack of experimental platforms that enable prospective analysis of CSC dynamics with sufficient spatiotemporal resolution has hindered testing of this hypothesis. Here, we develop a live genetic lineage-tracing system that allows longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids and identify LGR5+ CSCs that display a dormant behavior in a chemo-naive state. Dormant LGR5+ cells are marked by p27 expression, and intravital imaging directly demonstrates the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals an upregulation of COL17A1, a cell adhesion molecule that strengthens hemidesmosome, in dormant LGR5+p27+ cells. COL17A1-knockout organoids lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, suggesting a role of cell-matrix interface in dormancy maintenance. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signaling restrains chemo-resistant cells from exiting dormancy and delays tumor regrowth, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome refractoriness of human colorectal cancer to conventional chemotherapy.

Multiplexed imaging mass cytometry of the chemokine milieus in melanoma characterizes features of the response to immunotherapy

Science immunology

2022 Apr 01

Hoch, T;Schulz, D;Eling, N;Gómez, JM;Levesque, MP;Bodenmiller, B;
PMID: 35363540 | DOI: 10.1126/sciimmunol.abk1692

Intratumoral immune cells are crucial for tumor control and antitumor responses during immunotherapy. Immune cell trafficking into tumors is mediated by binding of specific immune cell receptors to chemokines, a class of secreted chemotactic cytokines. To broadly characterize chemokine expression and function in melanoma, we used multiplexed mass cytometry-based imaging of protein markers and RNA transcripts to analyze the chemokine landscape and immune infiltration in metastatic melanoma samples. Tumors that lacked immune infiltration were devoid of most of the profiled chemokines and exhibited low levels of antigen presentation and markers of inflammation. Infiltrated tumors were characterized by expression of multiple chemokines. CXCL9 and CXCL10 were often localized in patches associated with dysfunctional T cells expressing the B lymphocyte chemoattractant CXCL13. In tumors with B cells but no B cell follicles, T cells were the sole source of CXCL13, suggesting that T cells play a role in B cell recruitment and potentially in B cell follicle formation. B cell patches and follicles were also enriched with TCF7+ naïve-like T cells, a cell type that is predictive of response to immune checkpoint blockade. Our data highlight the strength of targeted RNA and protein codetection to analyze tumor immune microenvironments based on chemokine expression and suggest that the formation of tertiary lymphoid structures may be accompanied by naïve and naïve-like T cell recruitment, which may contribute to antitumor activity.

Opposing effects of Wnt/β-catenin signaling on epithelial and mesenchymal cell fate in the developing cochlea

Development (Cambridge, England)

2021 Jun 01

Billings, SE;Myers, NM;Quiruz, L;Cheng, AG;
PMID: 34061174 | DOI: 10.1242/dev.199091

During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via β-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.

Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries

Proceedings of the National Academy of Sciences of the United States of America

2021 May 18

Meinsohn, MC;Saatcioglu, HD;Wei, L;Li, Y;Horn, H;Chauvin, M;Kano, M;Nguyen, NMP;Nagykery, N;Kashiwagi, A;Samore, WR;Wang, D;Oliva, E;Gao, G;Morris, ME;Donahoe, PK;Pépin, D;
PMID: 33980714 | DOI: 10.1073/pnas.2100920118

Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.

Prox1 promotes expansion of the colorectal cancer stem cell population to fuel tumor growth and ischemia resistance.

Cell Rep. 2014 Sep 25;8(6):1943-56.

Wiener Z, Högström J, Hyvönen V, Band AM, Kallio P, Holopainen T, Dufva O, Haglund C, Kruuna O, Oliver G, Ben-Neriah Y, Alitalo K.
PMID: 25242330 | DOI: 10.1016/j.celrep.2014.08.034.

Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of these cells. We show here that a subpopulation of Prox1-transcription-factor-expressing cells have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Prox1 deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth via induction of annexin A1 and reduction of the actin-binding protein filamin A, which has been implicated as a prognostic marker in CRC. Loss of Prox1 also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Prox1 is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut.

TGFβ pathway limits dedifferentiation following WNT and MAPK pathway activation to suppress intestinal tumourigenesis

Cell Death Differ.

2017 Jun 16

Cammareri P, Vincent DF, Hodder MC, Ridgway RA, Murgia C, Nobis M, Campbell AD, Varga J, Huels DJ, Subramani C, Prescott KLH, Nixon C, Hedley A, Barry ST, Greten FR, Inman GJ, Sansom OJ.
PMID: 28622298 | DOI: 10.1038/cdd.2017.92

Recent studies have suggested increased plasticity of differentiated cells within the intestine to act both as intestinal stem cells (ISCs) and tumour-initiating cells. However, little is known of the processes that regulate this plasticity. Our previous work has shown that activating mutations of Kras or the NF-κB pathway can drive dedifferentiation of intestinal cells lacking Apc. To investigate this process further, we profiled both cells undergoing dedifferentiation in vitro and tumours generated from these cells in vivo by gene expression analysis. Remarkably, no clear differences were observed in the tumours; however, during dedifferentiation in vitro we found a marked upregulation of TGFβ signalling, a pathway commonly mutated in colorectal cancer (CRC). Genetic inactivation of TGFβ type 1 receptor (Tgfbr1/Alk5) enhanced the ability of KrasG12D/+ mutation to drive dedifferentiation and markedly accelerated tumourigenesis. Mechanistically this is associated with a marked activation of MAPK signalling. Tumourigenesis from differentiated compartments is potently inhibited by MEK inhibition. Taken together, we show that tumours arising in differentiated compartments will be exposed to different suppressive signals, for example, TGFβ and blockade of these makes tumourigenesis more efficient from this compartment.

NoxO1 Controls Proliferation of Colon Epithelial Cells

Front. Immunol.

2018 May 08

Moll F, Walter M ,Rezende F, Helfinger V, Vasconez E, De Oliveira T, Greten FR, Olesch C, Weigert A, Radeke HH, Schröder K.
PMID: - | DOI: 10.3389/fimmu.2018.00973

Aim: Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut.

Results: NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells.

Conclusion: NoxO1 affects colon epithelium homeostasis and prevents inflammation.

T Helper Cell Cytokines Modulate Intestinal Stem Cell Renewal and Differentiation

Cell 175, 1–14, November 15, 2018

2018 Nov 01

Biton M, Haber AL, Rogel N, Burgin G, Beyaz S, Schnell A, Ashenberg O, Su CW, Smillie C, Shekhar K, Chen Z, Wu C, Ordovas-Montanes J, Alvarez D, Herbst RH, Zhang M, Tirosh I, Dionne D, Nguyen LT, Xifaras ME, Shalek AK, von Andrian UH, Graham DB, Rozenblatt-Rosen O, Shi HN, Kuchroo V, Yilmaz OH, Regev A, Xavier RJ.
PMID: - | DOI: 10.1016/j.cell.2018.10.008

Summary In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.

A novel tankyrase inhibitor, MSC2504877, enhances the effects of clinical CDK4/6 inhibitors.

The Institute of Cancer Research (2018)

2018 Dec 18

Menon M, Elliott RJ, Bowers L, Balan N, Rafiq R, Costa-Cabral S, Munkonge F, Trinidade I, Ashworth A, Lord C.

Inhibition of the PARP superfamily tankyrase enzymes suppresses Wnt/b-catenin signalling in tumour cells. Here, we describe here a novel, drug-like small molecule inhibitor of tankyrase MSC2504877 that inhibits the growth of APC mutant colorectal tumour cells. Parallel siRNA and drug sensitivity screens showed that the clinical CDK4/6 inhibitor palbociclib, causes enhanced sensitivity to MSC2504877. This tankyrase inhibitor-CDK4/6 inhibitor combinatorial effect is not limited to palbociclib and MSC2504877 and is elicited with other CDK4/6 inhibitors and toolbox tankyrase inhibitors. The addition of MSC2504877 to palbociclib enhances G1 cell cycle arrest and cellular senescence in tumour cells. MSC2504877 exposure suppresses the upregulation of Cyclin D2 and Cyclin E2 caused by palbociclib and enhances the suppression of phospho-Rb, providing a mechanistic explanation for these effects. The combination of MSC2504877 and palbociclib was also effective in suppressing the cellular hyperproliferative phenotype seen in Apc defective intestinal stem cells in vivo. However, the presence of an oncogenic Kras p.G12D mutation in mice reversed the effects of the MSC2504877/palbociclib combination, suggesting one molecular route that could lead to drug resistance.

Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning.

EMBO Rep. 2018 Dec 10.

2018 Dec 10

Navis M, Martins Garcia T, Renes IB, Vermeulen JLM, Meisner S, Wildenberg ME, van den Brink GR, van Elburg RM, Muncan V.
PMID: 30530633 | DOI: 10.15252/embr.201846221

During the suckling‐to‐weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium‐derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivo. In vitro postnatal development of the fetal‐derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo. Together, our data show that organoids derived from fetal epithelium undergo suckling‐to‐weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.

Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation.

Nat Commun.

2019 Mar 06

Zhao B, Chen Y, Jiang N, Yang L, Sun S, Zhang Y, Wen Z, Ray L, Liu H, Hou G, Lin X.
PMID: 30842416 | DOI: 10.1038/s41467-019-09060-w

Lgr5+ stem cells are crucial to gut epithelium homeostasis; however, how these cells are maintained is not fully understood. Zinc finger HIT-type containing 1 (Znhit1) is an evolutionarily conserved subunit of the SRCAP chromosome remodeling complex. Currently, the function of Znhit1 in vivo and its working mechanism in the SRCAP complex are unknown. Here we show that deletion of Znhit1 in intestinal epithelium depletes Lgr5+ stem cells thus disrupts intestinal homeostasis postnatal establishment and maintenance. Mechanistically, Znhit1 incorporates histone variant H2A.Z into TSS region of genes involved in Lgr5+ stem cell fate determination, including Lgr5, Tgfb1 and Tgfbr2, for subsequent transcriptional regulation. Importantly, Znhit1 promotes the interaction between H2A.Z and YL1 (H2A.Z chaperone) by controlling YL1 phosphorylation. These results demonstrate that Znhit1/H2A.Z is essential for Lgr5+ stem cell maintenance and intestinal homeostasis. Our findings identified a dominant role of Znhit1/H2A.Z in controlling mammalian organ development and tissue homeostasis in vivo.

Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches

Nat Commun

2020 Jan 17

Kim JE Fei L, Yin WC, Coquenlorge S, Rao-Bhatia A, Zhang X, Shi SSW, Lee JH, Hahn NA, Rizvi W, Kim KH, Sung HK, Hui CC, Guo G, Kim TH
PMID: 31953387 | DOI: 10.1038/s41467-019-14058-5

Stomach and intestinal stem cells are located in discrete niches called the isthmus and crypt, respectively. Recent studies have demonstrated a surprisingly conserved role for Wnt signaling in gastrointestinal development. Although intestinal stromal cells secrete Wnt ligands to promote stem cell renewal, the source of stomach Wnt ligands is still unclear. Here, by performing single cell analysis, we identify gastrointestinal stromal cell populations with transcriptome signatures that are conserved between the stomach and intestine. In close proximity to epithelial cells, these perictye-like cells highly express telocyte and pericyte markers as well as Wnt ligands, and they are enriched for Hh signaling. By analyzing mice activated for Hh signaling, we show a conserved mechanism of GLI2 activation of Wnt ligands. Moreover, genetic inhibition of Wnt secretion in perictye-like stromal cells or stromal cells more broadly demonstrates their essential roles in gastrointestinal regeneration and development, respectively, highlighting a redundancy in gastrointestinal stem cell niches.

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	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

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