Secretion Of Stem Cell Factor Research Articles

Oxidative stress-induced hypermethylation and low expression of ANXA2R: Novel insights into the dysfunction of melanocytes in vitiligo

BackgroundVitiligo is a skin disorder with melanocyte destruction caused by complex interplay between multiple genetic and environmental factors. Recent studies have suggested DNA methylation is involved in the melanocyte damage, but the underlying mechanism remains unknown. ObjectiveTo explore the abnormal DNA methylation patterns in vitiligo lesional and nonlesional skin, and the mechanism of DNA methylation involved in vitiligo pathogenesis. MethodsInitially, the genome-wide aberrant DNA methylation profiles in lesional and nonlesional skin of vitiligo were detect via Illumina methylation EPIC 850k Beadchip. Subsequently, a comprehensive analysis was conduct to investigate the genomic characteristics of differentially methylated regions (DMRs). Furthermore, the effects of key aberrant methylated genes on cell apoptosis and function of both melanocytes and keratinocytes were further identified and validated by western bloting, ELISA, and immunofluorescence. ResultsCompared with nonlesional skins, we discovered 79 significantly differentially methylated CpG sites in vitiligo lesions. These DMRs were mainly located in the gene body and the TS1500 region. Annexin A2 receptor (ANXA2R), a crucial gene in cell apoptosis, was hypermethylated in vitiligo lesions. Furthermore, we showed that ANXA2R displayed hypermethylation and low expression levels in both keratinocytes and melanocytes of vitiligo patients, and the hypermethylated-triggered downregulation of ANXA2R under oxidative stress induced melanocyte apoptosis, and inhibited the secretion of stem cell factor (SCF) from keratinocytes thus impaired the survival of melanocytes. ConclusionsOur study illustrates the DNA methylation modification in vitiligo, and further demonstrates the molecular mechanism of hypermethylated ANXA2R in the dysfunction of melanocytes under oxidative stress.

Targeting senescent dermal fibroblasts responsible for hyperactive melanocytes in melasma

Targeting senescent dermal fibroblasts responsible for hyperactive melanocytes in melasma

High-Risk Cytokine Profiles Predict Lower Survivals in AML after Allotransplantation

Introduction: In a context of allogeneic hematopoietic stem cell transplantation (Allo-HSCT) for acute myeloid leukemia (AML), the impact of peripheral cytokine levels has been poorly studied so far. Methods: The aim of this prospective study (NCT02693899) was to analyze peripheral levels of 8 cytokines in 35 AML patients (pts) before conditioning (BC) then at Day (D) 0, 30, 60 and 100 post-Allo-HSCT by comparison to median levels from 20 healthy controls (16 males, median age 48.5 yo). ELISA (Bio-Techne, Minneapolis, MN) was used to assay the levels of FLt3-ligand (FL), TNF-alpha, stem-cell factor (SCF), c-kit, IL-1 beta, IL-6, IL-10 and granulocyte-monocyte colony-stimulating factor (GM-CSF). A total of 1313 and 157 samples were analyzed in pts and controls, respectively. The impact of BC, D0 and D30 levels and secretion (secretors = pts with detectable cytokine levels) on overall (OS), disease free (DFS) and GVHD and relapse free survival (GRFS) was also assessed. Results: Pts characteristics and cytokine levels are given in Tables 1 and 2. At D0, IL-6 (p=0.0006), FL (p<0.001) and SCF (p=0.003) levels were significantly higher in pts than controls. This remained true for SCF levels on D30 (p=0.0009), D60 (p=0.001) and D100 (p=0.01). Median GM-CSF and c-kit levels were significantly lower in pts at any time (p<0.001). Two-by-two paired cytokine comparisons showed no correlation, especially considering c-kit and its ligand SCF. ELN2017 classification and disease-risk index (DRI) were significantly associated with OS and LFS by univariate analysis (UA), only the former remaining associated with both OS (HR 8.51; 95%CI 1.18-61.54, p=0.034) and LFS (HR 8.10; 95%CI 1.20-54.82, p=0.032) in multivariate analysis (MA). Only SCF, IL-6 and c-kit levels impacted survivals. Higher pre-transplant SCF levels were associated with lower OS (p=0.02), DFS (p=0.01) and GRFS (P<0.001) by UA and remained associated with lower GRFS (HR 3.19; 95%CI 1.48-6.88, p=0.003) in MA, with a trend for lower LFS (HR 1.96; 95%CI 0.94-4.08, p=0.07). There was also an association between the absence of SCF secretion and better OS, LFS and GRFS (p=0.02, p=0.02, p=0.03) while c-kit secretion was associated with better OS (p=0.03) in UA. This remained true in MA for OS (HR 0.30; 95%CI 0.10-0.92, p=0.03) and there was a trend between no SCF secretion and GRFS (HR 0.45; 95%CI 0.18-1.14, p=0.09) by MA. Higher IL-6 levels at D0 were associated with lower OS (p=0.01) by UA and lower LFS both by UA (p=0.01) and MA (HR: 2.83; 95%CI: 1.04-7.70, p=0.04), with a trend for lower OS (HR: 2.55; 95%CI: 0.90-7.29, p=0.07) in MA. ROC curve analysis identified 7pg/mL as the best cut-off for D0 IL-6 levels which resulted, for pts under this level, in significantly better 4-year OS (80% (62-1) vs 35.7% (18-69), p=0.02) and LFS (73.3% (54-99) vs 28.6% (13-63) p=0.01; Figure). For alive patients at D30, higher c-kit and lower SCF levels were associated with better OS (p=0.003 and p=0.01) and LFS (p=0.009 and p=0.04) by UA while c-kit levels remained associated with LFS (HR: 0.37; 95%CI: 0.17-0.80, p=0.01) by MA. The absence of SCF secretion was associated with better OS (p=0.03) and a trend for better LFS (p=0.07). c-kit secretion was associated with better OS (p=0.008), LFS (p=0.01) and a trend for better GRFS (p=0.07). Only c-kit secretion remained associated with better OS (HR: 0.15; 95%CI: 0.04-0.57, p=0.005), LFS (HR: 0.19; 95%CI: 0.06-0.62, p=0.006) and GRFS (HR: 0.40.95%CI: 0.16-1.00, p=0.05) by MA. Conclusion: In this cohort, early higher IL-6 and SCF and lower c-kit levels appeared to be significantly associated with lower survivals in AML pts after Allo-HSCT. These high-risk cytokine profiles might allow to propose therapeutic intervention to decrease relapses in selected pts, either by modulating cytokine levels or by starting early maintenance treatment post-Allo-HSCT. These results have to be confirmed on larger studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Microbiota-derived lactate promotes hematopoiesis and erythropoiesis by inducing stem cell factor production from leptin receptor+ niche cells

Although functional interplay between intestinal microbiota and distant sites beyond the gut has been identified, the influence of microbiota-derived metabolites on hematopoietic stem cells (HSCs) remains unclear. This study investigated the role of microbiota-derived lactate in hematopoiesis using mice deficient in G-protein-coupled receptor (Gpr) 81 (Gpr81−/−), an established lactate receptor. We detected significant depletion of total HSCs in the bone marrow (BM) of Gpr81−/− mice compared with heterogenic (Gpr81+/−) mice in a steady state. Notably, the expression levels of stem cell factor (SCF), which is required for the proliferation of HSCs, decreased significantly in leptin receptor-expressing (LepR+) mesenchymal stromal cells (MSCs) around the sinusoidal vessels of the BM from Gpr81−/− mice compared with Gpr81+/− mice. Hematopoietic recovery and activation of BM niche cells after irradiation or busulfan treatment also required Gpr81 signals. Oral administration of lactic acid-producing bacteria (LAB) activated SCF secretion from LepR+ BM MSCs and subsequently accelerated hematopoiesis and erythropoiesis. Most importantly, LAB feeding accelerated the self-renewal of HSCs in germ-free mice. These results suggest that microbiota-derived lactate stimulates SCF secretion by LepR+ BM MSCs and subsequently activates hematopoiesis and erythropoiesis in a Gpr81-dependent manner.

β-estradiol promotes the growth of primary human fetal spermatogonial stem cells via the induction of stem cell factor in Sertoli cells.

Mammalian spermatogenesis is responsible for male fertility and is supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). Sertoli cells provide a supportive microenvironment for SSCs, in part by the production of stem cell factor (SCF), which is a potent regulator of spermatogonia proliferation and survival. We investigated the novel role of β-estradiol in modulating the proliferation and apoptosis of fetal SSCs via the regulation of SCF secretion in Sertoli cells isolated from human fetal testes. The proliferation of SSCs in the co-culture system was determined by colony formation and BrdU incorporation assays. TUNEL assay was used to measure SSC apoptosis in co-culture in response to treatment with control, β-estradiol, or the combination of β-estradiol and the estrogen receptor inhibitor ICI 182780. In the system with purified human fetal Sertoli cells (MIS+/c-Kit-/AP-), β-estradiol upregulated the production of SCF in a dose- and time-dependent manner. In the co-culture system of primary human fetal SSCs (c-Kit+/SSEA-4+/Oct-4+/AP+) and Sertoli cells (MIS+), β-estradiol markedly increased the proliferation of SSCs. Moreover, SSC apoptosis was significantly inhibited by β-estradiol and was completely reversed by the combination of β-estradiol and ICI 182780. Here we report, for the first time, that β-estradiol can induce the increase of SCF expression in human fetal Sertoli cells and regulates the growth and survival of human fetal SSCs. These novel findings provide new perspectives on the current understanding of the role of estrogen in human spermatogenesis.

Cell Sheet Comprised of Mesenchymal Stromal Cells Overexpressing Stem Cell Factor Promotes Epicardium Activation and Heart Function Improvement in a Rat Model of Myocardium Infarction.

Cell therapy of the post-infarcted myocardium is still far from clinical use. Poor survival of transplanted cells, insufficient regeneration, and replacement of the damaged tissue limit the potential of currently available cell-based techniques. In this study, we generated a multilayered construct from adipose-derived mesenchymal stromal cells (MSCs) modified to secrete stem cell factor, SCF. In a rat model of myocardium infarction, we show that transplantation of SCF producing cell sheet induced activation of the epicardium and promoted the accumulation of c-kit positive cells in ischemic muscle. Morphometry showed the reduction of infarct size (16%) and a left ventricle expansion index (0.12) in the treatment group compared to controls (24–28%; 0.17–0.32). The ratio of viable myocardium was more than 1.5-fold higher, reaching 49% compared to the control (28%) or unmodified cell sheet group (30%). Finally, by day 30 after myocardium infarction, SCF-producing cell sheet transplantation increased left ventricle ejection fraction from 37% in the control sham-operated group to 53%. Our results suggest that, combining the genetic modification of MSCs and their assembly into a multilayered construct, we can provide prolonged pleiotropic effects to the damaged heart, induce endogenous regenerative processes, and improve cardiac function.

Microbiota-Derived Lactate Controls Hematopoiesis and Erythropoiesis Through Regulating Stem Cell Factor from Leptin Receptor+ Niche Cells in the Bone Marrow

Although functional interplay between intestinal microbiota and the distant sites beyond the gut has been identified, it is unclear how microbiota-derived metabolites influence the fate of hematopoietic stem cells (HSCs). In this study, we investigated the role of microbiota-derived lactate in hematopoiesis using G-protein–coupled receptor (Gpr) 81, a known lactate receptor, deficient (Gpr81-/-) mice. We found that the total number of HSCs was significantly reduced in the bone marrow (BM) of Gpr81-/- mice as compared with heterogenic (Gpr81+/-) mice in a steady state. Notably, the expression levels of stem cell factor (SCF), which is required for proliferation of HSCs, were significantly decreased in leptin-receptor expressing (LepR+) mesenchymal stromal cells (MSCs) around the sinusoidal vessels of BM obtained from Gpr81-/- mice as compared with Gpr81+/- mice. Hematopoietic recovery and activation of BM niche cells after irradiation or busulfan treatment required Gpr81 signals. Oral administration of lactic acid-producing bacteria (LAB) activated the secretion of SCF from LepR+ BM MSCs and subsequently accelerated hematopoiesis and erythropoiesis. Most importantly, LAB feeding accelerated the self-renewal of HSCs in germ-free mice. These results suggested that microbiota-derived lactate stimulates SCF secretion by LepR+ BM MSCs and subsequently activates hematopoiesis and erythropoiesis in a Gpr81-dependent manner.

Microbiota-derived lactate control hematopoiesis and erythropoiesis through regulating stem cell factor from leptin receptor+ niche cells in the bone marrow

Abstract Although the functional interplay between the intestinal microbiota and the distant sites beyond the gut have been identified, it is still unveiled how microbiota-derived metabolites are involved in determining the fate of hematopoietic stem cells (HSC). In this study, we are aiming to investigate a role of microbiota-derived lactate in hematopoiesis using G-protein-coupled receptor (Gpr) 81, a known lactate receptor, deficient (Gpr81−/−) mice. We found that total numbers of hematopoietic cells and numbers of Lin−c-Kit+Sca-1+ HSC were significantly reduced in the bone marrow (BM) from Gpr81−/−mice when compared to heterogenic (Gpr81+/−) mice in steady-state condition. Of note, expression levels of stem cell factor (SCF), which is required for maintenance of HSC and erythropoiesis, were significantly decreased in Leptin-receptor expressing (LepR+) stromal cells around sinusoidal vascular of BM tissue obtained from Gpr81−/−mice when compared to those from Gpr81+/− mice. In addition, Gpr81deletion considerably reduced hematopoietic recovery and SCF expression in the BM damaged by irradiation. Oral administration with probiotics or lactate stimulated SCF secretion from LepR+ BM stromal cells and increased hematopoiesis and erythropoiesis. Furthermore, colonization of lactate-producing bacteria in GF mice also accelerated HSC number and SCF expression. Taken together, these results demonstrate that microbiota-derived lactate stimulates SCF secretion by LepR+ BM stromal cells and subsequently activate hematopoiesis and erythropoiesis in a Gpr81-dependent manner.

Downregulation of hepatic stem cell factor by Vivo-Morpholino treatment inhibits mast cell migration and decreases biliary damage/senescence and liver fibrosis in Mdr2−/− mice

Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis. MethodsFVB/NJ and Mdr2−/− mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber. ResultsBiliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2−/− mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2−/− mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation. ConclusionCholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression.

Tumor infiltrating mast cells determine oncogenic HIF-2α-conferred immune evasion in clear cell renal cell carcinoma.

Hypoxia-inducible factor 2α (HIF-2α) overexpression leads to activation of angiogenic pathways. However, little is known about the association between HIF-2α expression and anti-tumor immunity in clear cell renal cell carcinoma (ccRCC). We aimed to explore how HIF-2α influenced the microenvironment and the underlying mechanisms. We immunohistochemically evaluated immune cells infiltrations and prognostic value of HIF-2α expression in a retrospective Zhongshan Hospital cohort of 280 ccRCC patients. Fresh tumor samples, non-tumor tissues and autologous peripheral blood for RT-PCR, ELISA and flow cytometry analyses were collected from patients who underwent nephrectomy in Zhongshan Hospital from September 2017 to April 2018. The TCGA KIRC cohort and SATO cohort were assessed to support our findings. We demonstrated that ccRCC patients with HIF-2αhigh tumors exhibited reduced overall survival (p = 0.025) and recurrence-free survival (p < 0.001). Functions of CD8+ T cells were impaired in HIF-2αhigh patients. In ccRCC patients, HIF-2α induced the expression of stem cell factor (SCF), which served as chemoattractant for mast cells. Tumor infiltrating mast cells impaired anti-tumor immunity partly by secreting IL-10 and TGF-β. HIF-2α mRNA level adversely associated with immunostimulatory genes expression in KIRC and SATO cohorts. HIF-2α contributed to evasion of anti-tumor immunity via SCF secretion and subsequent recruitment of mast cells in ccRCC patients.

Paneth Cells Respond to Inflammation and Contribute to Tissue Regeneration by Acquiring Stem-like Features through SCF/c-Kit Signaling

IBD syndromes such as Crohn's disease and ulcerative colitis result from the inflammation of specific intestinal segments. Although many studies have reported on the regenerative response of intestinal progenitor and stem cells to tissue injury, very little is known about the response of differentiated lineages to inflammatory cues. Here, we show that acute inflammation of the mouse small intestine is followed by a dramatic loss of Lgr5+ stem cells. Instead, Paneth cells re-enter the cell cycle, lose their secretory expression signature, and acquire stem-like properties, thus contributing to the tissue regenerative response to inflammation. Stem cell factor secretion upon inflammation triggers signaling through the c-Kit receptor and a cascade of downstream events culminating in GSK3β inhibition and Wnt activation in Paneth cells. Hence, the plasticity of the intestinal epithelium in response to inflammation goes well beyond stem and progenitor cells and extends to the fully differentiated and post-mitotic Paneth cells.

Anti-Müllerian hormone inhibits proliferation and induces apoptosis in epithelial ovarian cancer cells by regulating the cell cycle and decreasing the secretion of stem cell factor.

Anti-Müllerian hormone (AMH) has been demonstrated to exhibit an inhibitory effect on the proliferation, invasion, metastasis and drug resistance of ovarian cancer. However, the mechanisms underlying these effects remain unclear. In the present study, 10 µg/ml recombinant human AMH (rhAMH) was administered to human OVCAR3 and OVCAR8 epithelial ovarian cancer (EOC) cell lines. Cell proliferation, apoptosis and cell cycle were analyzed. The level of stem cell factor (SCF) was detected using a reverse transcription-quantitative polymerase chain reaction and an ELISA, respectively. The exogenous addition of rhAMH significantly reduced the proliferation of OVCAR3 and OVCAR8 cell lines compared with the control group (P<0.01). The apoptosis rate in the rhAMH treated group (48 h) significantly increased compared with in the control group (OVCAR3, P=0.035; OVCAR8, P=0.020). The apoptosis rate increased at 72 h but did not exhibit a significant difference when compared with the 48 h group (OVCAR3, P=0.145; OVCAR8, P=0.296). The percentage of cells in the G1 phase in the rhAMH treated group (48 h) increased but was not significantly different compared with the control group (OVCAR3, P=0.070; OVCAR8, P=0.051). However, there was a significant difference at 72 h compared with the control group (OVCAR3, P=0.016; OVCAR8, P=0.019). At 48 h, the rhAMH-treated group exhibited a statistically significant inhibition of SCF mRNA expression levels (P=0.008), but no significant difference in the protein expression levels (P=0.101) compared with the control, though a significant inhibition was exhibited at 72 h (mRNA expression levels, P=0.005; protein expression levels, P=0.036). The present study revealed that rhAMH may be able to inhibit the proliferation and induce the apoptosis of EOC cells via G1/S-phase cell cycle arrest and the decreased secretion of SCF.

Ultraviolet-irradiated endothelial cells secrete stem cell factor and induce epidermal pigmentation

Ultraviolet (UV)-associated hyperpigmented skins are characterized with increased vasculature underlying pigmentation, suggestive of the possible biological role of endothelial cells in the regulation of skin pigmentation during UV irradiation. In this study, we showed that UV-irradiated endothelial cells significantly increased the pigmentation of melanocytes through epithelial-mesenchymal crosstalk. The stimulatory effect of endothelial cells was further demonstrated using ex vivo human skin. RNA sequence analysis and enzyme-linked immunosorbent assay showed that endothelial cells secrete more stem cell factor (SCF) upon UV irradiation than non-irradiated cells. The increased pigmentation elicited by endothelial cells was abrogated following inhibition of SCF/c-KIT signaling. Together these results suggest that endothelial cells are activated upon UV exposure to release melanogenic factors such as SCF, which contributes to the development of skin hyperpigmentation during chronic sun exposure.

High mobility group box 1 (HMGB1) acts as an “alarmin” to promote acute myeloid leukaemia progression

ABSTRACTHigh mobility group box 1 (HMGB1) is a non-histone protein localised in the cell nucleus, where it interacts with DNA and promotes nuclear transcription events. HMGB1 levels are elevated during acute myeloid leukaemia (AML) progression followed by participation of this protein in triggering signalling events in target cells as a pro-inflammatory stimulus. This mechanism was hypothesised to be employed as a survival pathway by malignant blood cells and our aims were therefore to test this hypothesis experimentally. Here we report that HMGB1 triggers the release of tumour necrosis factor alpha (TNF-α) by primary human AML cells. TNF-α induces interleukin 1 beta (IL-1β) production by healthy leukocytes, leading to IL-1β-induced secretion of stem cell factor (SCF) by competent cells (for example endothelial cells). These results were verified in mouse bone marrow and primary human AML blood plasma samples. In addition, HMGB1 was found to induce secretion of angiogenic vascular endothelial growth factor (VEGF) and this process was dependent on the immune receptor Tim-3. We therefore conclude that HMGB1 is critical for AML progression as a ligand of Tim-3 and other immune receptors thus supporting survival/proliferation of AML cells and possibly the process of angiogenesis.

IPSC-derived mesenchymal stem cells exert SCF-dependent recovery of cigarette smoke-induced apoptosis/proliferation imbalance in airway cells.

Mesenchymal stem cells (MSCs) have emerged as a potential cell‐based therapy for pulmonary emphysema in animal models. Our previous study demonstrated that human induced pluripotent stem cell–derived MSCs (iPSC‐MSCs) were superior over bone marrow–derived MSCs (BM‐MSCs) in attenuating cigarette smoke (CS)‐induced airspace enlargement possibly through mitochondrial transfer. This study further investigated the effects of iPSC‐MSCs on inflammation, apoptosis, and proliferation in a CS‐exposed rat model and examined the effects of the secreted paracrine factor from MSCs as another possible mechanism in an in vitro model of bronchial epithelial cells. Rats were exposed to 4% CS for 1 hr daily for 56 days. At days 29 and 43, human iPSC‐MSCs or BM‐MSCs were administered intravenously. We observed significant attenuation of CS‐induced elevation of circulating 8‐isoprostane and cytokine‐induced neutrophil chemoattractant‐1 after iPSC‐MSC treatment. In line, a superior capacity of iPSC‐MSCs was also observed in ameliorating CS‐induced infiltration of macrophages and neutrophils and apoptosis/proliferation imbalance in lung sections over BM‐MSCs. In support, the conditioned medium (CdM) from iPSC‐MSCs ameliorated CS medium‐induced apoptosis/proliferation imbalance of bronchial epithelial cells in vitro. Conditioned medium from iPSC‐MSCs contained higher level of stem cell factor (SCF) than that from BM‐MSCs. Deprivation of SCF from iPSC‐MSC‐derived CdM led to a reduction in anti‐apoptotic and pro‐proliferative capacity. Taken together, our data suggest that iPSC‐MSCs may possess anti‐apoptotic/pro‐proliferative capacity in the in vivo and in vitro models of CS‐induced airway cell injury partly through paracrine secretion of SCF.

Inhibition of Mast Cell-Derived Histamine Decreases Human Cholangiocarcinoma Growth and Differentiation via c-Kit/Stem Cell Factor–Dependent Signaling

The tumor microenvironment of cholangiocarcinoma (CCA) is composed of numerous cells, including mast cells (MCs). MCs release histamine, which increases CCA progression and angiogenesis. Cholangiocytes secrete stem cell factor, which functions via the MC growth factor receptor c-Kit. Here, we show that cholangiocytes express histidine decarboxylase and its inhibition reduces CCA growth. MC recruitment in the tumor microenvironment increased CCA growth. MC infiltration and MC markers were detected by toluidine blue staining and real-time PCR in human biopsies and in tumors from athymic mice treated with saline, histamine, histidine decarboxylase inhibitor, or cromolyn sodium. Tumor growth, angiogenesis, and epithelial-mesenchymal transition (EMT)/extracellular matrix (ECM) markers were measured in mice treated with cromolyn sodium. Invitro, human CCA cells were treated with MC supernatant fluids before evaluating angiogenesis and EMT/ECM expression. Migration assays were performed with CCA cells treated with the stem cell factor inhibitor. MC supernatant fluids increased CCA histidine decarboxylase, vascular endothelial growth factor, and MC/EMT/ECM expression that decreased with pretreatment of cromolyn sodium. MCs were found in human biopsies. In mice treated with cromolyn sodium, MC infiltration and tumor growth decreased. Inhibition of CCA stem cell factor blocked MC migration and MC/EMT/ECM in CCA. MCs migrate into CCA tumor microenvironment via c-Kit/stem cell factor and increase tumor progression, angiogenesis, EMT switch, and ECM degradation.

Covering the Cover

Covering the Cover

Maintenance of Clonogenic KIT+ Human Colon Tumor Cells Requires Secretion of Stem Cell Factor by Differentiated Tumor Cells

Colon tumors contain a fraction of undifferentiated stem cell-like cancer cells with high tumorigenic potential. Little is known about the signals that maintain these stem-like cells. We investigated whether differentiated tumor cells provide support. We established undifferentiated colonosphere cultures from human colon tumors and used them to generate stably differentiated cell lines. Antibody arrays were used to identify secreted factors. Expression of genes involved in stemness, differentiation, and the epithelial to mesenchymal transition was measured using reverse transcription quantitative polymerase chain reaction. Expression of KIT in human tumors was analyzed with gene expression arrays and by immunohistochemistry. Colonospheres were injected into the livers of CBy.Cg-Foxn1nu/J mice. After liver tumors had formed, hypoxia was induced by vascular clamping. Differentiated cells from various tumors, or medium conditioned by them, increased the clonogenic capacity of colonospheres. Stem cell factor (SCF) was secreted by differentiated tumor cells and supported the clonogenic capacity of KIT(+) colonosphere cells. Differentiated tumor cells induced the epithelial to mesenchymal transition in colonosperes; this was prevented by inhibition of KIT or SCF. SCF prevented loss of clonogenic potential under differentiation-inducing conditions. Suppression of SCF or KIT signaling greatly reduced the expression of genes that regulate stemness and the epithelial to mesenchymal transition and inhibited clonogenicity and tumor initiation. Bioinformatic and immunohistochemical analyses revealed a correlation between expression of KIT- and hypoxia-related genes in colon tumors, which was highest in relapse-prone mesenchymal-type tumors. Hypoxia induced expression of KIT in cultured cells and in human colon tumor xenografts and this contributed to the clonogenic capacity of the tumor cells. Paracrine signaling from SCF to KIT, between differentiated tumor cells and undifferentiated stem-like tumor cells, helps maintain the stem-like features of tumor cells, predominantly under conditions of hypoxia.

Abstract 1045: Differentiated tumor cells secrete stem cell factor (SCF) to promote maintenance of cancer stem cells and induce EMT in colorectal tumors

Abstract Colorectal tumors are hierarchically organized in which cancer stem cells (CSCs) are at the top of the hierarchy and give rise to non-tumorigenic differentiated tumor cells (DTC). The maintenance of normal intestinal stem cells requires ‘niche’ signals that are provided, at least in the small intestine, by neighboring differentiated Paneth cells. CSC maintenance may also depend on niche signals although little is known about the nature of such signals and their potential source. For the present study we tested the hypothesis that DTCs could provide CSC-maintaining signals. We used CSC-enriched colonosphere cultures that were established from human colorectal tumors and CSC-derived stably differentiated offspring that was derived from them. We found that DTCs established from multiple colorectal tumors greatly increased the clone-forming capacity of CSCs. This effect was reproduced by DTC-conditioned medium, suggesting the involvement of a secreted factor. By using a cytokine array we identified stem cell factor (SCF) as a major DTC-secreted factor. While SCF was mainly produced by DTCs, its receptor, c-Kit, was mainly expressed by CSCs. Indeed, recombinant SCF enhanced clone-forming capacity to a similar extent as DTC-conditioned medium. Furthermore, basal and DTC-stimulated clone-forming capacity was strongly reduced in the presence of an SCF-neutralizing antibody or in the presence of the cKit inhibitors Imatinib or ISCK03. qPCR and western blot analysis showed that inhibition of cKit caused a strong reduction in the expression of stem cell genes, including LGR5, CD133, OLMF4 and SOX2. Furthermore, DTCs induce the E-to N-cadherin switch in CSCs and to a strong increase in the expression of EMT markers such as vimentin, ZEB1, ZEB2, SNAIL1 and fibronectin. cKIT inhibitors prevented EMT. Our results identify a critical role for non-tumorigenic DTCs in the support of colorectal CSCs. DTCs induce EMT and EMT-associated stem cell characteristics at least in part via paracrine SCF-cKit signaling. We conclude that cKit inhibitors that are widely used in the clinic to treat gastrointestinal stromal tumors (GIST) and acute myeloid leukemia (AML), may have therapeutic value in the treatment of (a subgroup of) colorectal tumors. Citation Format: Szabolcs Fatrai, Susanne J. van Schelven, Inne H.M. Borel Rinkes, Onno Kranenburg. Differentiated tumor cells secrete stem cell factor (SCF) to promote maintenance of cancer stem cells and induce EMT in colorectal tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2014-1045

Effects of salidroside on proliferation of bone marrow mesenchymal stem cells

This study was aimed to investigate the effect of salidroside on proliferation of bone marrow mesenchymal stem cells (MSC) and their secretion of stem cell factor (SCF). MSC were isolated and amplified in vitro via density gradient centrifugation and adherence screening method. MCS were identified by flow cytometry and osteogenic/adipogenic induction. The effects of salidroside on cell proliferation, cell cycle and the SCF secretion of MSC were detected by flow cytometry. The results showed that the salidroside could induce the proliferation of MSC, peaked at the concentration of 1.5 mg/ml and in a time-dependent manner (in 24 h, 48 h and 72 h). Salidroside at 1.5 mg/ml could more effectively increase the percentage of cells in S and G1/M phase. Co-cultured with salidroside at the concentration of 1.5 mg/ml for 48 h, the SCF and the expression levels of SCF mRNA in co-culture supernatant were both significantly increased (P < 0.01). It is concluded that salidroside in a range of certain concentration can obviously promote the proliferation of MSC and increase the expression and secretion of SCF.