ABSTRACT
The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).
Subject(s)
Bone and Bones/cytology , Intercellular Signaling Peptides and Proteins/metabolism , Intestine, Small/cytology , Mesenchymal Stem Cells/cytology , Animals , Cartilage/metabolism , Intestine, Small/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BLABSTRACT
Mammalian cells repress expression of repetitive genomic sequences by forming heterochromatin. However, the consequences of ectopic repeat expression remain unclear. Here we demonstrate that inhibitors of EZH2, the catalytic subunit of the Polycomb repressive complex 2 (PRC2), stimulate repeat misexpression and cell death in resting splenic B cells. B cells are uniquely sensitive to these agents because they exhibit high levels of histone H3 lysine 27 trimethylation (H3K27me3) and correspondingly low DNA methylation at repeat elements. We generated a pattern recognition receptor loss-of-function mouse model, called RIC, with mutations in Rigi (encoding for RIG-I), Ifih1 (MDA5), and Cgas. In both wildtype and RIC mutant B cells, EZH2 inhibition caused loss of H3K27me3 at repetitive elements and upregulated their expression. However, NF-κB-dependent expression of inflammatory chemokines and subsequent cell death was suppressed by the RIC mutations. We further show that inhibition of EZH2 in cancer cells requires the same pattern recognition receptors to activate an interferon response. Together, the results reveal chemokine expression induced by EZH2 inhibitors in B cells as a novel inflammatory response to genomic repeat expression. Given the overlap of genes induced by EZH2 inhibitors and Epstein-Barr virus infection, this response can be described as a form of viral mimicry.
Subject(s)
B-Lymphocytes , Enhancer of Zeste Homolog 2 Protein , Epstein-Barr Virus Infections , Animals , Mice , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , DNA Methylation , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Epstein-Barr Virus Infections/genetics , Herpesvirus 4, Human/genetics , Histones/metabolism , Repetitive Sequences, Nucleic AcidABSTRACT
PD-L1 plays a central role in immune recognition of cancer cells. In this issue of Molecular Cell, Jin et al. (2019) report that a phosphorylated retinoblastoma protein contacts the DNA-binding domain of p65 NF-κB, thereby blocking transcription of PD-L1.
Subject(s)
B7-H1 Antigen , Transcription Factor RelA/genetics , Gene Expression Regulation , NF-kappa B/genetics , Retinoblastoma Protein , Signal TransductionABSTRACT
BACKGROUND & AIMS: Colorectal cancer is a leading cause of cancer death, and a major risk factor is chronic inflammation. Despite the link between colitis and cancer, the mechanism by which inflammation leads to colorectal cancer is not well understood. METHODS: To investigate whether different forms of inflammation pose the same risk of cancer, we compared several murine models of colitis (dextran sodium sulfate [DSS], 2,4,6-trinitrobenzene sulfonic acid, 4-ethoxylmethylene-2-phenyloxazol-5-one, Citrobacter rodentium, Fusobacterium nucleatum, and doxorubicin) with respect to their ability to lead to colonic tumorigenesis. We attempted to correlate the severity of colitis and inflammatory profile with the risk of tumorigenesis in both azoxymethane-dependent and Dclk1/APCfl/fl murine models of colitis-associated cancer. RESULTS: DSS colitis reproducibly led to colonic tumors in both mouse models of colitis-associated cancer. In contrast, all other forms of colitis did not lead to cancer. When compared with the colitis not associated with tumorigenesis, DSS colitis was characterized by significantly increased CD11b+F4/80+Ly6Chigh macrophages and CD11b+Ly6G+ neutrophils. Interestingly, depletion of the CD11b+F4/80+Ly6Chigh macrophages inhibited tumorigenesis, whereas depletion of CD11b+Ly6G+ neutrophils had no effect on tumorigenesis. Furthermore, the macrophage-derived cytokines interleukin-1ß, tumor necrosis factor-α, and interleukin-6 were significantly increased in DSS colitis and promoted stemness of Dclk1+ tuft cells that serve as the cellular origin of cancer. CONCLUSIONS: We have identified CD11b+F4/80+Ly6Chigh macrophages as key mediators of cancer initiation in colitis-associated cancer. Development of new therapies that target these cells may provide an effective preventative strategy for colitis-associated cancer.
Subject(s)
Colitis-Associated Neoplasms , Colitis , Animals , Mice , Azoxymethane , Carcinogenesis/metabolism , Cell Plasticity , Colitis/chemically induced , Colitis/complications , Colitis/metabolism , Colitis-Associated Neoplasms/metabolism , Dextran Sulfate/toxicity , Disease Models, Animal , Inflammation/metabolism , Macrophages/metabolism , Mice, Inbred C57BLABSTRACT
During homeostasis, the colonic epithelium is replenished every 3-5 days by rapidly cycling Lgr5+ stem cells. However, various insults can lead to depletion of Lgr5+ stem cells, and colonic epithelium can be regenerated from Lgr5-negative cells. While studies in the small intestine have addressed the lineage identity of the Lgr5-negative regenerative cell population, in the colon this question has remained unanswered. Here, we set out to identify which cell(s) contribute to colonic regeneration by performing genetic fate-mapping studies of progenitor populations in mice. First, using keratin-19 (Krt19) to mark a heterogeneous population of cells, we found that Lgr5-negative cells can regenerate colonic crypts and give rise to Lgr5+ stem cells. Notch1+ absorptive progenitor cells did not contribute to epithelial repair after injury, whereas Atoh1+ secretory progenitors did contribute to this process. Additionally, while colonic Atoh1+ cells contributed minimally to other lineages during homeostasis, they displayed plasticity and contributed to epithelial repair during injury, independent of Lgr5+ cells. Our findings suggest that promotion of secretory progenitor plasticity could enable gut healing in colitis.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Colitis/prevention & control , Colon/cytology , Intestine, Small/cytology , Receptors, G-Protein-Coupled/metabolism , Regeneration , Stem Cells/cytology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cells, Cultured , Colitis/chemically induced , Colitis/pathology , Colon/physiology , Homeostasis , Intestine, Small/physiology , Keratin-19/genetics , Keratin-19/metabolism , Mice , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Receptors, G-Protein-Coupled/genetics , Stem Cells/physiologyABSTRACT
BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor ß was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.
Subject(s)
Cancer-Associated Fibroblasts/pathology , Cancer-Associated Fibroblasts/physiology , Carcinogenesis/pathology , Cell Lineage , Colorectal Neoplasms/pathology , Mesenchymal Stem Cells/physiology , Actins/genetics , Actins/metabolism , Adult , Aged , Aged, 80 and over , Animals , CD146 Antigen/genetics , CD146 Antigen/metabolism , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Differentiation , Cell Proliferation , Colorectal Neoplasms/metabolism , Disease Models, Animal , Female , Humans , Intestinal Mucosa/pathology , Ki-67 Antigen/metabolism , Male , Mice , Mice, Transgenic , Middle Aged , Organoids/pathology , Organoids/physiology , Prognosis , Receptors, Leptin/genetics , Receptors, Leptin/metabolism , Sequence Analysis, RNA , Survival Rate , Tumor MicroenvironmentABSTRACT
BACKGROUND & AIMS: The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated. METHODS: We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apcflox/flox, LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5-positive cells, were irradiated, or were given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues, we analyzed the fate of progeny that expressed Bhlha15. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using The Cancer Genome Atlas data set. RESULTS: Bhlha15 identified Paneth cells and short-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15+ cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15+ secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15+ secretory precursors to long-lived enterocyte progenitors. Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with enterocyte progenitors, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15+ precursors toward those of ISCs. Bhlha15+ enterocyte progenitors with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells after dextran sodium sulfate-induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon. CONCLUSIONS: In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. These cells could mediate tumor formation in addition to traditional ISCs.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Colonic Neoplasms/genetics , Enterocytes/pathology , Intestinal Mucosa/metabolism , Receptors, Notch/metabolism , Stem Cells/metabolism , Transcriptome , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents, Hormonal/pharmacology , Basic Helix-Loop-Helix Transcription Factors/genetics , CD24 Antigen/metabolism , Calcium-Binding Proteins , Cell Cycle Proteins , Cell Plasticity , Chromogranin A/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Doxorubicin/pharmacology , Enterocytes/metabolism , Gene Expression , Gene Expression Profiling , Intercellular Signaling Peptides and Proteins/genetics , Intestinal Mucosa/drug effects , Intestinal Mucosa/pathology , Intestine, Small/cytology , Intestine, Small/metabolism , Mice , Neoplasm Proteins/genetics , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Pancreatitis-Associated Proteins , Paneth Cells , Phosphoproteins/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Stem Cells/drug effects , Stem Cells/physiology , Stem Cells/radiation effects , Tamoxifen/pharmacology , YAP-Signaling Proteins , src-Family Kinases/metabolismABSTRACT
OBJECTIVE: Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. DESIGN: We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. RESULTS: Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5(neg or low) cell population but was distinct from typical antral Lgr5(high) stem cells. Treatment with progastrin interconverts Lgr5(neg or low) CCK2R+ cells into Lgr5(high) cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. CONCLUSIONS: CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.
Subject(s)
Carcinogenesis , Pyloric Antrum/cytology , Receptor, Cholecystokinin B/physiology , Stem Cells/physiology , Animals , Cells, Cultured , Gastrins/physiology , Mice , Protein Precursors/physiologyABSTRACT
There are two major stem cell populations in the intestinal crypt region that express either Bmi1 or Lgr5; however, it has been shown that other populations in the crypt can regain stemness. In this study, we demonstrate that the transcription factor NK2 homeobox 2 (Nkx2.2) is expressed in enteroendocrine cells located in the villus and crypt of the intestinal epithelium and is coexpressed with the stem cell markers Bmi1 and Lgr5 in a subset of crypt cells. To determine whether Nkx2.2-expressing enteroendocrine cells display cellular plasticity and stem cell potential, we performed genetic lineage tracing of the Nkx2.2-expressing population using Nkx2.2(Cre/+);R26RTomato mice. These studies demonstrated that Nkx2.2+ cells are able to give rise to all intestinal epithelial cell types in basal conditions. The proliferative capacity of Nkx2.2-expressing cells was also demonstrated in vitro using crypt organoid cultures. Injuring the intestine with irradiation, systemic inflammation, and colitis did not enhance the lineage potential of Nkx2.2-expressing cells. These findings demonstrate that a rare mature enteroendocrine cell subpopulation that is demarcated by Nkx2.2 expression display stem cell properties during normal intestinal epithelial homeostasis, but is not easily activated upon injury.
Subject(s)
Cell Differentiation , Cell Lineage , Cell Proliferation , Enteroendocrine Cells/metabolism , Homeodomain Proteins/metabolism , Intestinal Mucosa/metabolism , Pluripotent Stem Cells/metabolism , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Biomarkers/metabolism , Cells, Cultured , Enteroendocrine Cells/pathology , Enteroendocrine Cells/radiation effects , Genotype , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/genetics , Intestinal Mucosa/pathology , Intestinal Mucosa/radiation effects , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Phenotype , Polycomb Repressive Complex 1/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Transcription Factors/genetics , Whole-Body Irradiation , Zebrafish Proteins , Red Fluorescent ProteinABSTRACT
BACKGROUND & AIMS: Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R)-partly by increasing the number of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R. METHODS: We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene, gastrin knockout mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2; levels of ß-arrestin 1 and 2 were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division. RESULTS: The BMP pathway was down-regulated in the colons of human progastrin mice compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by ß-arrestin 1 and 2. In mouse colonic epithelial cells, down-regulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of inhibitor of DNA binding 4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44(+), bromodeoxyuridine+, and NUMB(+) cells, indicating an increase in symmetric divisions of putative cancer stem cells. CONCLUSIONS: Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and ß-arrestin 1 and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division.
Subject(s)
Arrestins/physiology , Bone Morphogenetic Protein 2/antagonists & inhibitors , Cell Proliferation/drug effects , Colon/drug effects , Gastrins/pharmacology , Protein Precursors/pharmacology , Receptor, Cholecystokinin B/physiology , Animals , Bone Morphogenetic Protein 2/physiology , Colon/cytology , Mice , Mice, Inbred C57BL , Signal Transduction , Stem Cells/drug effects , beta-Arrestin 1 , beta-ArrestinsABSTRACT
BACKGROUND & AIMS: Interleukin (IL)-8 has an important role in initiating inflammation in humans, attracting immune cells such as neutrophils through their receptors CXCR1 and CXCR2. IL-8 has been proposed to contribute to chronic inflammation and cancer. However, mice do not have the IL-8 gene, so human cancer cell lines and xenograft studies have been used to study the role of IL-8 in colon and gastric carcinogenesis. We generated mice that carry a bacterial artificial chromosome that encompasses the entire human IL-8 gene, including its regulatory elements (IL-8Tg mice). METHODS: We studied the effects of IL-8 expression in APCmin(+/-) mice and IL-8Tg mice given azoxymethane and dextran sodium sulfate (DSS). We also examined the effects of IL-8 expression in gastric cancer in INS-GAS mice that overexpress gastrin and IL-8Tg mice infected with Helicobacter felis. RESULTS: In IL-8Tg mice, expression of human IL-8 was controlled by its own regulatory elements, with virtually no messenger RNA or protein detectable under basal conditions. IL-8 was strongly up-regulated on systemic or local inflammatory stimulation, increasing mobilization of immature CD11b(+)Gr-1(+) myeloid cells (IMCs) with thioglycolate-induced peritonitis, DSS-induced colitis, and H. felis-induced gastritis. IL-8 was increased in colorectal tumors from patients and IL-8Tg mice compared with nontumor tissues. IL-8Tg mice developed more tumors than wild-type mice following administration of azoxymethane and DSS. Expression of IL-8 increased tumorigenesis in APCmin(+/-) mice compared with APCmin(+/-) mice that lack IL-8; this was associated with increased numbers of IMCs and angiogenesis in the tumors. CONCLUSIONS: IL-8 contributes to gastrointestinal carcinogenesis by mobilizing IMCs and might be a therapeutic target for gastrointestinal cancers.
Subject(s)
Cell Movement/drug effects , Cell Transformation, Neoplastic/metabolism , Colitis/metabolism , Colonic Neoplasms/metabolism , Gastritis/metabolism , Interleukin-8/metabolism , Myeloid Cells/drug effects , RNA, Messenger/metabolism , Animals , Azoxymethane , Cell Line, Tumor , Colitis/chemically induced , Colonic Neoplasms/chemically induced , Colonic Neoplasms/pathology , Dendritic Cells/metabolism , Dextran Sulfate , Gastritis/microbiology , Helicobacter Infections/complications , Helicobacter felis , Humans , Interleukin-8/genetics , Interleukin-8/pharmacology , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Mice , Mice, Transgenic , Myeloid Cells/metabolism , Primary Cell Culture , Tumor Burden , Up-Regulation/drug effectsABSTRACT
OBJECTIVE: Stromal cell-derived factor-1 (SDF-1/CXCL12), the main ligand for CXCR4, is overexpressed in human cancer. This study addressed the precise contribution of SDF-1 to gastric carcinogenesis. DESIGN: SDF-1 transgenic mice were created and a Helicobacter-induced gastric cancer model was used in combination with H/K-ATPase-IL-1ß mice. Gastric tissue was analysed by histopathology and cells isolated from the stomach were analysed by molecular biological methods. RESULTS: Analysis of the H/K-ATPase/SDF-1 transgenic (SDF-Tg) mice showed that SDF-1 overexpression results in significant gastric epithelial hyperproliferation, mucous neck cell hyperplasia and spontaneous gastric dysplasia (wild-type mice 0/15 (0%) vs SDF-Tg mice 4/14 (28.6%), p=0.042, Fisher exact test) but has minimal effects on inflammation. SDF-Tg mice also showed a dramatic expansion of α-smooth muscle actin-positive myofibroblasts and CXCR4-expressing gastric epithelial cells in the progenitor zone, both of which preceded the development of significant gastritis or dysplasia. Gremlin 1-expressing mesenchymal stem cells, the putative precursors of myofibroblasts, were also increased within the dysplastic stomachs of SDF-Tg mice and showed chemotaxis in response to SDF-1 stimulation. SDF-1 overexpression alone resulted in minimal recruitment of haematopoietic cells to the gastric mucosa, although macrophages were increased late in the disease. When SDF-Tg mice were crossed with H/K-ATPase-IL-1ß mice or infected with Helicobacter felis, however, there were dramatic synergistic effects on recruitment of bone marrow-derived cells and progression to preneoplasia. CONCLUSION: Activation of the SDF-1/CXCR4 axis can contribute to early stages of carcinogenesis primarily through recruitment of stromal cells and modulation of the progenitor niche.
Subject(s)
Chemokine CXCL12/genetics , Gene Expression Regulation/physiology , Mesenchymal Stem Cells/pathology , Myofibroblasts/pathology , Precancerous Conditions/genetics , Stomach Neoplasms/genetics , Animals , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Gastric Mucosa/pathology , Immunohistochemistry , Intercellular Signaling Peptides and Proteins/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myofibroblasts/metabolism , Precancerous Conditions/pathology , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Receptors, CXCR4/metabolism , Stomach Neoplasms/pathologyABSTRACT
Background: Cannabis is used by patients with Crohn's disease (CD) and ulcerative colitis (UC) as an alternative to, or in combination with, conventional therapies to treat symptoms such as abdominal pain, poor sleep, and reduced appetite. The clinical efficacy of cannabis for these disorders is controversial, with some studies showing harmful outcomes associated with its use. Previous studies suggest that cannabis is used by ~12% of patients with UC and ~16% of patients with CD in the USA despite legal prohibition. Methods: We conducted a prospective cohort study of adult patients with inflammatory bowel diseases (IBD) followed in a Canadian tertiary care center. Patients completed an online 40-question survey that included demographics, IBD disease history, cannabis use, and the Short Inflammatory Bowel Disease Questionnaire (SIBDQ). Results: Completed surveys were obtained from 254 participants (148 with CD, 90 with UC, and 16 with indeterminate colitis). Recent cannabis use was reported by 41% of CD and 31% of UC participants. Interestingly, only 46% of participants who used cannabis discussed their use with their physician. Participants who recently used cannabis reported more abdominal pain, poor appetite, and flatulence, and importantly this was associated with lower SIBDQ scores (recent use 37 vs non-recent use 40). Conclusions: Cannabis use among patients with IBD has more than doubled since its legalization. Cannabis use is associated with worse abdominal symptoms and quality of life. Physicians should inquire about cannabis use and optimize symptom control with evidence-based therapies.
ABSTRACT
While adult pancreatic stem cells are thought not to exist, it is now appreciated that the acinar compartment harbors progenitors, including tissue-repairing facultative progenitors (FPs). Here, we study a pancreatic acinar population marked by trefoil factor 2 (Tff2) expression. Long-term lineage tracing and single-cell RNA sequencing (scRNA-seq) analysis of Tff2-DTR-CreERT2-targeted cells defines a transit-amplifying progenitor (TAP) population that contributes to normal homeostasis. Following acute and chronic injury, Tff2+ cells, distinct from FPs, undergo depopulation but are eventually replenished. At baseline, oncogenic KrasG12D-targeted Tff2+ cells are resistant to PDAC initiation. However, KrasG12D activation in Tff2+ cells leads to survival and clonal expansion following pancreatitis and a cancer stem/progenitor cell-like state. Selective ablation of Tff2+ cells prior to KrasG12D activation in Mist1+ acinar or Dclk1+ FP cells results in enhanced tumorigenesis, which can be partially rescued by adenoviral Tff2 treatment. Together, Tff2 defines a pancreatic TAP population that protects against Kras-driven carcinogenesis.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/genetics , Trefoil Factor-2/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Pancreas/metabolism , Acinar Cells/metabolism , Carcinogenesis/genetics , Carcinogenesis/metabolismABSTRACT
Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.
Subject(s)
Chromosomes, Artificial, Bacterial/genetics , Gastrin-Secreting Cells/metabolism , Gastrins/genetics , Green Fluorescent Proteins/genetics , Helicobacter felis/genetics , Aging/metabolism , Animals , Azoxymethane , Carcinogens , Colonic Neoplasms/chemically induced , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Dextran Sulfate , Down-Regulation , Fasting , Fetus/metabolism , Gastric Acid/metabolism , Gastrins/deficiency , Gene Expression Regulation, Developmental , Genes, Reporter , Green Fluorescent Proteins/metabolism , Helicobacter Infections/genetics , Helicobacter Infections/metabolism , Mice , Mice, Transgenic , Pyloric Antrum/metabolism , Pyloric Antrum/pathology , Somatostatin/administration & dosage , Tissue Distribution , Transcription, Genetic , Transgenes , Up-RegulationABSTRACT
Bone marrow mesenchymal stem cells (MSCs) have been shown to have immune modulatory effects. Despite efforts to identify these cells in vivo, to date, MSCs have been defined mainly by their in vitro cell characteristics. Here, we show that Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells make up approximately 0.5%-1% of murine whole bone marrow cells and yield nearly an equal amount of fibroblastic colony-forming units (CFU-F) as whole bone marrow. After transplantation into lethally irradiated recipients, Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells engrafted in the bone marrow long-term and demonstrated characteristics of MSCs, including capacity to differentiate into osteoblasts and adipocytes. To examine whether Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells have immune modulatory effects, in vitro coculture with activated CD4+ T-cells resulted in decreased Th17 cell differentiation by Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells. Furthermore, serial infusions with Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells reduced the progression to low-grade gastric dysplasia in mice infected with chronic Helicobacter felis (p = .038). This correlated with reduced gastric interleukin (IL)-17F, IL-22, and ROR-gammat gene expression in responding mice (p < .05). These data suggest that bone marrow derived Lin(-)CD44(hi)Sca1(-)cKit+CD34(-) cells have characteristics of MSCs and reduce progression of early gastric tumorigenesis induced by chronic H. felis infection. The prevention of dysplastic changes may occur through inhibition of Th17-dependent pathways.
Subject(s)
Bone Marrow Cells/physiology , Helicobacter felis/physiology , Mesenchymal Stem Cells/physiology , Stomach Neoplasms/prevention & control , Stomach Neoplasms/therapy , Animals , Bone Marrow Cells/cytology , Bone Marrow Transplantation , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Female , Flow Cytometry , Helicobacter Infections/pathology , Male , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred C57BL , Stem Cells , Stomach Neoplasms/microbiologyABSTRACT
BACKGROUND: Ensuring competency of trainees is a challenge for residency programs. The American Society for Gastrointestinal Endoscopy (ASGE) recommends that a minimum of 130 EGDs and 140 colonoscopies be performed to assess competency. OBJECTIVE: We assessed the number of endoscopies performed by surgery and gastroenterology residents during their training. Endoscopy patterns were also assessed for staff gastroenterology specialists and general surgeons in Alberta, Canada. DESIGN: Physician billing data were used to determine endoscopic practice patterns, and the number of endoscopies performed by gastroenterology fellows and surgery residents were obtained. SETTING: Major teaching hospital. MAIN OUTCOME MEASUREMENT: Procedure numbers. RESULTS: In large cities, the number of colonoscopies performed by gastroenterologists increased ( approximately 2-fold) over the study period (there was minimal change in endoscopy numbers by surgeons). In contrast, in smaller communities, EGDs and colonoscopies by surgeons increased about 2-fold (from approximately 4065 to 7288) and about 4-fold (from approximately 1909 to approximately 7629), respectively (with only a minimal increase in colonoscopies ( approximately 3000), by gastroenterologists. During training, gastroenterology fellows performed significantly more procedures (EGDs, 29 +/- 5.6 by surgery residents vs 363.9 +/- 12.7 by gastroenterology fellows; colonoscopies, 91 +/- 14.2 by surgery residents vs 247.8 +/- 21.6 by gastroenterology fellows). LIMITATION: All training data are from a single teaching center. CONCLUSIONS: All gastroenterology fellows, but none of the surgery residents, achieved the minimum number of endoscopic procedures recommended by the ASGE to assess competency. These data suggest that we must reexamine our training programs and/or our methods for evaluating endoscopic competence.
Subject(s)
Clinical Competence , Endoscopy, Gastrointestinal/statistics & numerical data , General Surgery/education , Internship and Residency , Alberta , Endoscopy, Gastrointestinal/standardsABSTRACT
We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers.
Subject(s)
Bone Marrow Cells/pathology , Cell Transformation, Neoplastic/chemically induced , Cell Transformation, Neoplastic/pathology , Epithelial Cells/pathology , Skin Neoplasms/pathology , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Bone Marrow Cells/cytology , Bone Marrow Transplantation , Bone Morphogenetic Protein 5/metabolism , Cell Movement , Cell Plasticity/physiology , Coculture Techniques , Epithelial Cells/cytology , Female , HMGB1 Protein/metabolism , Hair Follicle/cytology , Keratinocytes/pathology , Keratins/metabolism , Male , Mice , Mice, Inbred C57BL , Neoplasm Invasiveness/pathology , Papilloma/pathology , Stem Cells/cytology , Stem Cells/pathology , Tetradecanoylphorbol Acetate/toxicity , Tumor Cells, CulturedABSTRACT
Syk kinase is best known as a critical component of immunoreceptor signaling in leukocytes. Activation of Syk following cross-linking of Fcgamma and Fcepsilon receptors on macrophages, mast cells, and other cells induces various inflammatory events. We hypothesized that Syk is involved in inflammatory responses induced by the lipopolysaccharide (LPS). We studied the role of Syk using its inhibition by antisense oligonucleotides, or small interfering RNA. Our data demonstrated that in vivo inhibition of Syk caused down-regulation of LPS-induced responses in rat alveolar macrophages. In in vitro experiments, inhibition of Syk in rat peritoneal macrophages, as well as in human myelomonocyte cell line THP-1 also caused a decrease in LPS-induced cytokine release. Our data support the hypothesis that, in macrophages, Syk is involved in LPS-induced intracellular signaling pathways leading to the release of pro-inflammatory mediators. Understanding the role of Syk in LPS-induced signaling may help in developing new therapeutic tools for inflammatory disorders.
Subject(s)
Cytokines/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Lipopolysaccharides/metabolism , Macrophages, Alveolar/metabolism , Macrophages, Peritoneal/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Blotting, Western , Cell Line , Cytokines/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Inflammation Mediators/metabolism , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/immunology , Lipopolysaccharides/immunology , Liposomes , Macrophages, Alveolar/enzymology , Macrophages, Alveolar/immunology , Macrophages, Peritoneal/enzymology , Macrophages, Peritoneal/immunology , Male , Monocytes/immunology , Monocytes/metabolism , Nitric Oxide/metabolism , Oligonucleotides, Antisense/metabolism , Oligonucleotides, Antisense/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/immunology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Syk Kinase , Transfection , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND AND STUDY AIMS: Adenoma detection rate (ADR) is an important measure of colonoscopy quality, as are polyp, advanced ADR, and adenocarcinoma detection rates. We investigated whether performance report cards improved these outcome measures. PATIENTS AND METHODS: Endoscopists were given report cards comparing their detection rates to the institutional mean on an annual basis. Detection rates were evaluated at baseline, 1 year after report cards (Year 1), and 2 years after report cards (Year 2). Endoscopists were unaware of the study and received no other interventions. The primary outcome was ADR and secondary outcomes were polyp detection rate (PDR), advanced ADR, and adenocarcinoma detection rate. Multivariate regression was performed to adjust for temporal trends in patient, endoscopists, and procedural factors. RESULTS: Seventeen physicians performed 3,118 screening colonoscopies in patients with positive FOBT or family history of colon cancer. The ADR increased from 34.5â% (baseline) to 39.4â% (Year 1) and 41.2â% (Year 2) ( P â=â0.0037). The PDR increased from 45â% (baseline) to 48.8â% (Year 1) and 51.8â% (Year 2) ( P â=â0.011). There was no significant improvement in advanced ADR or adenocarcinoma detection rates. On multivariate analysis, the ADR increased by 22â% in Year 1 ( P â=â0.03) and 30â% in Year 2 ( P â=â0.008). Among physicians with a baseline ADRâ<â25â%, improvement in ADR was even greater, increasing 2.2 times by the end of the study ( P â=â0.004). Improvements in ADR were not correlated with specialty although gastroenterologists were 52â% more likely to find an adenoma than general surgeons. CONCLUSIONS: Annual performance report cards increased adenoma detection rates, especially among physicians with low ADRâ<â25â%.