Expression Of Stem Cell-associated Genes Research Articles

Advantages of photo-curable collagen-based cell-laden bioinks compared to methacrylated gelatin (GelMA) in digital light processing (DLP) and extrusion bioprinting

The development of cell-laden bioinks that possess high biocompatibility and printability is crucial in the field of bioprinting for the creation of cell-embedded tissue engineering scaffolds. As widely known, methacrylated gelatin (GelMA) is one of the most commonly used photo-crosslinkable bioink for cell-laden bioprinting with different printing methods, but GelMA is the derivative of gelatin, so it loses the unique triple-helix molecular structure of collagen and may not be able to successfully activate the cellular pathways or facilitate cell-matrix interaction as effectively as collagen. Recently, methacrylated collagen (CMA) was developed to be an alternative photocrosslinkable bioink with a good bioactivity, but its low printability and biocompatibility limited that application in tissue engineering. In this study, the synthetic process for CMA was improved by synthesizing under 4 °C and using acidic aqueous solution as solvent. Our CMA bioinks were demonstrated a similar printability as GelMA in extrusion bioprinting, while a better formability in digital light processing (DLP). To further analyze the bioactive properties, CMA bioinks were encapsulated with Schwann cells (SCs) and bone mesenchymal stem cells (BMSCs) for printing. SCs-laden CMA bioinks had a significantly higher proliferation rate and expression of neural stem cell-associated genes than GelMA in DLP bioprinting. While, BMSCs-laden CMA bioinks demonstrated >95% cellular viability, better cell spreading and higher expression of osteogenesis-related genes than that of GelMA. Overall, we speculate that the CMA-based bioink developed in this study could be potential bioinks for 3D cell-laden bioprinting in the future.

HK2 Is a Crucial Downstream Regulator of miR-148a for the Maintenance of Sphere-Forming Property and Cisplatin Resistance in Cervical Cancer Cells.

The acquisition of cancer stem-like properties is believed to be responsible for cancer metastasis and therapeutic resistance in cervical cancer (CC). CC tissues display a high expression level of hexokinase 2 (HK2), which is critical for the proliferation and migration of CC cells. However, little is known about the functional role of HK2 in the maintenance of cancer stem cell-like ability and cisplatin resistance of CC cells. Here, we showed that the expression of HK2 is significantly elevated in CC tissues, and high HK2 expression correlates with poor prognosis. HK2 overexpression (or knockdown) can promote (or inhibit) the sphere-forming ability and cisplatin resistance in CC cells. In addition, HK2-overexpressing CC cells show enhanced expression of cancer stem cell-associated genes (including SOX2 and OCT4) and drug resistance-related gene MDR1. The expression of HK2 is mediated by miR-145, miR-148a, and miR-497 in CC cells. Overexpression of miR-148a is sufficient to reduce sphere formation and cisplatin resistance in CC cells. Our results elucidate a novel mechanism through which miR-148a regulates CC stem cell-like properties and chemoresistance by interfering with the oncogene HK2, providing the first evidence that dysregulation of the miR-148a/HK2 signaling plays a critical role in the maintenance of sphere formation and cisplatin resistance of CC cells. Our findings may guide future studies on therapeutic strategies that reverse cisplatin resistance by targeting this pathway.

MSX2 represses tumor stem cell phenotypes within oral squamous cell carcinomas via SOX2 degradation.

Oral squamous cell carcinoma (OSCC) is the sixth malignancy in the world with high incidence. The MSX2 (muscle segment homeobox 2)-Sry-related high-mobility box 2 (SOX2) signaling pathway plays a significant role in maintaining cancer stem cells, which are the origin of malignancy, leading to unfavorable outcomes in several carcinomas. This study aims to elucidate the mechanisms through which the MSX2-SOX2 pathway controls the cancer stem cell-like characterization in OSCC. The results showed that MSX2 was remarkably downregulated in OSCC and that the MSX2 expression level was related to unfavorable outcomes in patients with OSCC. Meanwhile, the MSX2 expression level was lower in the CD44+/CD24- population than in the other populations of OSCC cells. The OSCC2 cells exhibited decreased percentage of CD44+/CD24- cells, owing to MSX2 overexpression but increased owing to MSX2 knockdown. Moreover, a negative correlation was observed between MSX2 expression and is SOX2 transcriptional levels in different populations within the OSCC cell lines. Regarding the loss and gain of function, cancer stem cell phenotypes such as tumor globular formation, CD44+ subpopulation cells, and stem cell-associated gene expression were enhanced by MSX2 knockdown in OSCC CD44+/CD24- cells but decreased by MSX2 overexpression in other OSCC populations. However, these events were counteracted by the co-knockdown or SOX2 overexpression. Cells with MSX2 overexpression or knockdown formed smaller or bigger cancers in vivo, thereby showing a lower or a higher tumor incidence, respectively. Thus, our results confirm that MSX2 has a tumor suppression effect on the cancer stem cell phenotypes of OSCC and indicate that the MSX2-SOX2 signaling pathway could be a useful target for OSCC treatment.

Abstract PS16-04: Estrogen receptor (ER) and NFkB activity determines cancer stem cell properties in ER positive breast cancer

Abstract Approximately 50% of estrogen receptor (ER) positive breast cancer patients will experience relapse. One explanation for this is the presence of breast cancer stem cells (BCSCs), which have self-renewal capability and can contribute to therapy resistance, metastasis, and relapse. Previously, we have shown that cells with active ER and NFκB gain stem cell properties. They are characterized by higher mammosphere (MS) forming efficiency and increased expression of stem-associated markers. NFκB has been previously described as a player in BCSC biology. However, the role of ER remains unclear since some studies have suggested that BCSCs are ER negative. In order to study the role of ER and NFκB in BCSCs in more detail, we utilized single cell RNA sequencing of MS. Dimensionality reduction of single cells identified 4 clusters with unique transcriptional signatures. In order to determine which cluster of these 4 represents BCSCs, we ran functional enrichment analysis (FEA) using MsigDB as a source of various stem cell signatures. Results of FEA showed 1 of 9 stem cell signatures enriched in Cluster 0. Whereas clusters 1, 2 and 3 demonstrated increasing enrichment of different stem cell signatures, respectively. Further analysis of stem cell signatures and genes revealed that cluster 1 included not only classical stem markers (ALDH1A3, KRT8, CD36), but also was enriched for PI3K and p53 signaling pathways. The cluster 2 stem cell signature was associated with DREAM complex activity and BRCA1 mutation. Cluster 3 was enriched for expression of NANOG and SOX2 targets. We also ran FEA to determine ER and NFκB activity in each population. Cluster 0 showed no activity of either ER or NFκB, whereas Cluster 1 was enriched for both, cluster 2 was enriched for ER only, and cluster 3 was enriched for NFκB only. Taken together this data suggests that ER and NFκB may play different roles in promoting BCSC properties. In order to determine how both pathways effect BCSC features, we made a dual reporter cell line expressing ERE-mCherry and NFκB-RE-GFP. It was found ERE activity was constant throughout MS formation, whereas NFκB-RE activity increased proportionally to MS growth over time, suggesting these pathways have different dynamics and further supporting unique roles for each in MS development. To determine which cell population has the capacity to seed MS, an indication of stemness, we sorted cells by reporter activity and tested MS forming efficiency. We discovered that ERE+ cells, either with or without NFκB activity, are more stem-like, whereas activation of NFκB alone is not sufficient to drive MS development. This finding was verified by stem cell-associated gene expression. These data suggest that ER activity is a key driver for BCSC properties and that NFκB may play a supporting role. Furthermore, a novel mechanism of ER regulating the DREAM complex, which is known to be involved in cancer progression and used to evaluate the risk of recurrence in breast cancer, is suggested as a potential ER-mediated mechanism for driving BCSCs. Taken together, our findings suggest novel roles for ER and NFκB in BCSC biology, which could be exploited to target BCSCs therapeutically in ER+ breast cancer patients in order to improve their outcome. Citation Format: Svetlana E. Semina, Irida Kastrati, Luis Alejo Cruz, Tara McCray, Mark Maienschein-Cline, Elaine T. Alarid, Jonna Frasor. Estrogen receptor (ER) and NFkB activity determines cancer stem cell properties in ER positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-04.

LOSS OF GLUTATHIONE PEROXIDASE 1 ATTENUATES COLITIS AND IS CRITICAL IN ACTIVATING EPITHELIAL REGENERATIVE RESPONSES

Many selenium-containing “selenoproteins” function as antioxidants, and work by our lab and others has demonstrated that selenoproteins often protect against intestinal inflammatory diseases, including colitis. Glutathione peroxidase 1 (GPx1) is a ubiquitous, mitochondrial and cytosolic selenoprotein which catalyzes the reduction of hydrogen peroxide by glutathione. Previously, we determined that despite its antioxidant role, loss of GPx1 greatly reduced disease severity in the dextran sodium sulfate (DSS) colitis model. Furthermore, GPx1 loss increased baseline intestinal cell proliferation, enhanced enteroid plating efficiency, and induced expression of stem cell-associated genes, such as Lgr5.

LOSS OF GLUTATHIONE PEROXIDASE 1 ATTENUATES COLITIS AND IS CRITICAL IN ACTIVATING EPITHELIAL REGENERATIVE RESPONSES

Abstract Many selenium-containing “selenoproteins” function as antioxidants, and work by our lab and others has demonstrated that selenoproteins often protect against intestinal inflammatory diseases, including colitis. Glutathione peroxidase 1 (GPx1) is a ubiquitous, mitochondrial and cytosolic selenoprotein which catalyzes the reduction of hydrogen peroxide by glutathione. Previously, we determined that despite its antioxidant role, loss of GPx1 greatly reduced disease severity in the dextran sodium sulfate (DSS) colitis model. Furthermore, GPx1 loss increased baseline intestinal cell proliferation, enhanced enteroid plating efficiency, and induced expression of stem cell-associated genes, such as Lgr5. Next, we aimed to determine the mechanism by which GPx1 modifies response to DSS. We observed that GPx1 is increased in colonic tissues from DSS-treated mice as compared to nontreated controls, suggesting that GPx1 may functionally contribute to intestinal injury responses. While GPx1 is expressed in both intestinal epithelial and immune cells, in situ hybridization to visualize Gpx1 identified epithelial cells as the most highly expressing cell type, with the greatest Gpx1 upregulation observed in wound-adjacent and regenerative crypts. Next, we investigated whether GPx1 loss affects stem cell function after injury. Here, we determined that both proliferation (p<0.01) and Lgr5 expression (p<0.05) were increased in the crypts of Gpx1-/- DSS-treated mice in comparison to WT controls. Similarly, organoids established from ulcerative colitis tissue displayed increased growth rates (p<0.01), expression of stem cell and Wnt target genes such as AXIN2 (p<0.0001) and LGR5 (p<0.01), and proliferation (p<0.05) following GPX1 knockdown. Together, these results indicate that GPx1 has an epithelial-cell autonomous role, and that its loss activates stem cell and proliferative responses which may both protect from intestinal injury and promote healing. Interestingly, recent research has highlighted the role of cellular metabolism in maintaining intestinal stem cell function, and GPx1 has previously been implicated in these processes. RNA-sequencing from DSS-treated mice and gene set enrichment analysis identified a positive association with oxidative phosphorylation-associated genes in Gpx1-/- mice (NES: 1.78; FDR q-val: 0.01), suggesting altered metabolism which may favor stem cell function. Further analysis of cellular metabolism using GPX1 knockdown colorectal cancer cells observed higher basal respiration (p<0.0001) and ATP generation (p<0.0001). Together, these results suggest that unlike other intestinal selenoproteins studied to date, loss of GPx1 augments stem cell injury responses to protect against intestinal inflammation, likely via augmenting epithelial regenerative responses.

Aryl hydrocarbon receptor mediates Jak2/STAT3 signaling for non-small cell lung cancer stem cell maintenance

Cancer stem cells (CSCs) play an important role in shaping the invasive cancer phenotype by contributing to tumor initiation, metastasis, relapse, and therapeutic resistance in non-small cell lung cancer (NSCLC). The Aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, which is well known for mediating the toxicity and tumorigenesis of a variety of environmental pollutants, has been extensively recognized as an important mediator in NSCLC development. Here, evidence showed that AhR was overexpressed in NSCLC tissues, and a high AhR protein level was associated with an aggressive tumor phenotype. Knockdown of AhR suppressed cell proliferation, invasion and migration, as well as CSC-like properties, while upregulation and activation of AhR enhanced CSC-like properties and increased stem cell-associated gene expression in NSCLC cells. Elevated and activated AhR leads to phosphorylation of janus kinase 2 (Jak2), as well as its downstream effector, activator of transcription 3 (STAT3), while inhibition of Jak2/STAT3 signaling by pharmacologic approach attenuates the effects of AhR-mediated NSCLC cell stemness, suggesting a role for the Jak2/STAT3 pathway in AhR-regulated NSCLC stemness. In summary, our study uncovers a transcriptional-independent mechanism of AhR through which AhR mediates NSCLC stemness via Jak2/STAT3 signaling pathway, indicating a promising target for the treatment of NSCLC.

Stem Cell Marker Expression in Early Stage Colorectal Cancer is Associated with Recurrent Intestinal Neoplasia.

Colorectal cancer (CRC) ranks second in cancer deaths worldwide and presents multiple management challenges, one of which is identifying high risk stage II disease that may benefit from adjuvant therapy. Molecular biomarkers, such as ones that identify stem cell activity, could better stratify high-risk cohorts for additional treatment. To identify possible biomarkers of high-risk disease in early-stage CRC, a discovery set (n = 66) of advanced-stage tumors were immunostained with antibodies to stemness proteins (CD166, CD44, CD26, and LGR5) and then digitally analyzed. Using a second validation cohort (n = 54) of primary CRC tumors, we analyzed protein and gene expression of CD166 across disease stages, and extended our analyses to CD166-associated genes (LGR5, ASCL2, BMI1, POSTN, and VIM) by qRT-PCR. Stage III and metastatic CRC tumors highly expressed stem cell-associated proteins, CD166, CD44, and LGR5. When evaluated across stages, CD166 protein expression was elevated in advanced-stage compared to early-stage tumors. Notably, a small subset of stage I and II cancers harbored elevated CD166 protein expression, which correlated with development of recurrent cancer or adenomatous polyps. Gene expression analyses of CD166-associated molecules revealed elevated ASCL2 in primary tumors from patients who recurred. We identified a protein signature prognostic of aggressive disease in early stage CRC. Stem cell-associated protein and gene expression identified a subset of early-stage tumors associated with cancer recurrence and/or subsequent adenoma formation. Signatures for stemness offer promising fingerprints for stratifying early-stage patients at high risk of recurrence.

Midkine-a Is Required for Cell Cycle Progression of Müller Glia during Neuronal Regeneration in the Vertebrate Retina

In the retina of zebrafish, Müller glia have the ability to reprogram into stem cells capable of regenerating all classes of retinal neurons and restoring visual function. Understanding the cellular and molecular mechanisms controlling the stem cell properties of Müller glia in zebrafish may provide cues to unlock the regenerative potential in the mammalian nervous system. Midkine is a cytokine/growth factor with multiple roles in neural development, tissue repair, and disease. In midkine-a loss-of-function mutants of both sexes, Müller glia initiate the appropriate reprogramming response to photoreceptor death by increasing expression of stem cell-associated genes, and entering the G1 phase of the cell cycle. However, transition from G1 to S phase is blocked in the absence of Midkine-a, resulting in significantly reduced proliferation and selective failure to regenerate cone photoreceptors. Failing to progress through the cell cycle, Müller glia undergo reactive gliosis, a pathological hallmark in the injured CNS of mammals. Finally, we determined that the Midkine-a receptor, anaplastic lymphoma kinase, is upstream of the HLH regulatory protein, Id2a, and of the retinoblastoma gene, p130, which regulates progression through the cell cycle. These results demonstrate that Midkine-a functions as a core component of the mechanisms that regulate proliferation of stem cells in the injured CNS.SIGNIFICANCE STATEMENT The death of retinal neurons and photoreceptors is a leading cause of vision loss. Regenerating retinal neurons is a therapeutic goal. Zebrafish can regenerate retinal neurons from intrinsic stem cells, Müller glia, and are a powerful model to understand how stem cells might be used therapeutically. Midkine-a, an injury-induced growth factor/cytokine that is expressed by Müller glia following neuronal death, is required for Müller glia to progress through the cell cycle. The absence of Midkine-a suspends proliferation and neuronal regeneration. With cell cycle progression stalled, Müller glia undergo reactive gliosis, a pathological hallmark of the mammalian retina. This work provides a unique insight into mechanisms that control the cell cycle during neuronal regeneration.

TMIC-22. IDENTIFICATION OF CANCER-ASSOCIATED FIBROBLASTS IN GLIOBLASTOMA and Defining Their Protumoral Effects

Abstract While cancer-associated fibroblasts (CAFs) and their pro-tumoral effects have been demonstrated in systemic cancers, CAFs had been presumed absent in glioblastoma given the lack of normal fibroblasts in the healthy brain. Here, we show that 5–26% (mean=12%) of cells in human glioblastomas express CAF markers α-SMA or PDGFR-β, morphologically resemble fibroblasts, and transcriptomically resemble by RNA-seq CAFs from other cancers. Glioblastoma CAFs were chemotactically attracted to glioblastoma-initiating stem cells (P=0.02). While glioblastoma CAFs did not affect differentiated glioblastoma cell proliferation (P=0.4), CAFs increased glioblastoma stem cell proliferation (P=0.002) and expression of glioblastoma stem cell-associated genes (P< 0.001). To identify mediators of CAF/glioblastoma stem cell interactions, we created a resource of inferred crosstalk by mapping the expression of receptors to that of their cognate ligands/agonists, using our RNA-seq results from glioblastoma CAFs and stem cells, revealing PDGF-β/PDGFR and osteopontin/CD44 to mediate stem cell recruitment of CAFs and CAF enrichment of stem cells, as confirmed by blocking antibodies (P=0.02–0.03). CAFs also render the glioblastoma microenvironment more pro-tumoral by promoting M2 polarization of tumor-associated macrophages (P=0.01), an effect we found to arise from unique CAF production of the EDA splice variant of fibronectin binding toll-like receptor 4 (TLR4), a known EDA receptor expressed by macrophages (P=0.02). In patient glioblastomas, CAFs were enriched 3-fold in the subventricular zone (SVZ) (P=0.04) which houses the neural stem cells that generate glioblastoma stem cells. SVZs from epilepsy cases or autopsies of glioblastoma-containing brains without ventricular involvement lacked CAFs. Depleting CAFs in xenografts derived from neurosphere-containing glioblastoma stem cells slowed their growth in vivo (P< 0.001). These findings are among the first to identify and profile glioblastoma CAFs. CAF recruitment by glioblastoma stem cells and creation of a pro-tumoral microenvironment in the perivascular niche housing glioblastoma stem cells, particularly in the SVZ, makes them an intriguing therapeutic target.

Interleukin 6 signaling maintains the stem-like properties of bladder cancer stem cells.

BackgroundThe relapse and metastasis of bladder cancer are due to its strong resistance to chemotherapeutic drugs after surgery as a result of the expansion and self-renewal of cancer stem cells (CSCs). However, the molecular mechanisms underlying the biology of bladder CSCs are unknown. This study aimed to investigate the role of interleukin 6 (IL6)/IL6 receptor (IL6R) in the stem-like characteristics of bladder CSCs.MethodsEnzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect IL6 expression in the supernatant and cells of bladder CSCs, respectively. Following that, self-renewal, stem cell-associated gene expression, invasion, metastasis, and tumorigenicity were assessed by sphere-forming assay, qRT-PCR, invasion and transwell assays, and tumor-forming experiment in NOD/SCID mice, respectively. Finally, Western blot and qRT-PCR were employed to examine the IL6 signaling pathway in regulating the stem-like properties of bladder CSCs.ResultsThe spheres, originating from the bladder cancer cell lines RT4 and J82, possessed a higher expression of stem-associated genes. The expression levels of IL6 were elevated in the supernatant and cells of the bladder CSCs. IL6R was also up-regulated in the bladder CSCs. Recombinant IL6 promoted the stem-like properties of the bladder CSCs, including self-renewal, expression of stem-associated genes, invasion, migration, and tumorigenicity. Mechanistically, IL6 exerted its biological effects by binding to IL6R, which enhanced the phosphorylation of STAT3 and triggered its activation. Furthermore, these effects were alleviated by the FDA-approved drug tocilizumab.ConclusionsOur findings demonstrate that IL6/IL6R/STAT3 maintains the stem-like properties of bladder CSCs. Furthermore, IL6R may serve as a potential therapeutic target for CSCs in bladder cancer.

Interleukin 6 signaling maintains the stem-like properties of bladder cancer stem cells

Background: The relapse and metastasis of bladder cancer are due to its strong resistance to chemotherapeutic drugs after surgery as a result of the expansion and self-renewal of cancer stem cells (CSCs). However, the molecular mechanisms underlying the biology of bladder CSCs are unknown. This study aimed to investigate the role of interleukin 6 (IL6)/IL6 receptor (IL6R) in the stem-like characteristics of bladder CSCs. Methods: Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect IL6 expression in the supernatant and cells of bladder CSCs, respectively. Following that, self-renewal, stem cell-associated gene expression, invasion, metastasis, and tumorigenicity were assessed by sphere-forming assay, qRT-PCR, invasion and transwell assays, and tumor-forming experiment in NOD/SCID mice, respectively. Finally, Western blot and qRT-PCR were employed to examine the IL6 signaling pathway in regulating the stem-like properties of bladder CSCs. Results: The spheres, originating from the bladder cancer cell lines RT4 and J82, possessed a higher expression of stem-associated genes. The expression levels of IL6 were elevated in the supernatant and cells of the bladder CSCs. IL6R was also up-regulated in the bladder CSCs. Recombinant IL6 promoted the stem-like properties of the bladder CSCs, including self-renewal, expression of stem-associated genes, invasion, migration, and tumorigenicity. Mechanistically, IL6 exerted its biological effects by binding to IL6R, which enhanced the phosphorylation of STAT3 and triggered its activation. Furthermore, these effects were alleviated by the FDA-approved drug tocilizumab. Conclusions: Our findings demonstrate that IL6/IL6R/STAT3 maintains the stem-like properties of bladder CSCs. Furthermore, IL6R may serve as a potential therapeutic target for CSCs in bladder cancer.

Blockade of TNFα to Improve Human CD34+ Cell Repopulating Activity in Allogeneic Stem Cell Transplantation.

Early release of TNFα after hematopoietic stem cell transplantation (HSCT) correlates with development of acute graft-vs.-host disease (GVHD). Here we tested the effect of TNFα and alloreactive T cells on early hematopoietic HSC genotype and function. Addition of TNFα (10 ng/ml) in liquid cultures with CD34+ cells for 6–72 h resulted in the downregulation of genes associated with stem cell activity, such as DNMT3A, DNMT3B, TET1, TET2, SOX2, NANOG, and OCT4, whereas no significant effect was observed on DNMT1 and GATA2 expression. These findings were reversed by using an anti-TNFα antibody. Similar gene downregulation was observed when CD34+ cells were co-cultured with alloreactive T cells CD34+ cells for 48–72 h, and this effect was partially prevented by rapamycin and an anti-TNFα antibody. CD34+ cells pre-incubated with TNFα for 48 h and transplanted in irradiated NOD-SCID ɤnull (NSG) mice showed a reduced myeloid engraftment compared to control mice. By using a xenograft model recently developed in our lab, we co-transplanted CD34+ cells and allogeneic T lymphocytes at 1:0.1 ratio in one group that also received etanercept (TNFα inhibitor) at 100 μg intra-peritoneum (i.p.) on days −1,+1,+3,+5 post-HSCT, and in the control group. At 6 weeks post-transplant, mice that received etanercept had a significantly higher number of marrow huCD45+CD34+CD38- early stem cells (p = 0.03) and a reduced number of huCD45+CD3+ splenic T cells (p = 0.04) compared to controls. The repopulating activity of marrow cells from mice treated with etanercept vs. controls was tested in secondary transplants. Although the overall engraftment was similar in the two groups, CD34+ cells isolated from recipients of marrow from the etanercept group showed a significantly greater expression of stem cell-associated genes and a higher number of CD45+CD34+CD38- cells than in controls (p = 0.03). Our findings suggest that early TNFα increase post-transplant can affect long-term stem cell engraftment, and that blockade of TNFα early after transplant may limit a cytokine-mediated suppressive effect on repopulating stem cell function.

The stem cell-associated gene expression signature allows risk stratification in pediatric acute myeloid leukemia.

Despite constant progress in prognostic risk stratification, children with acute myeloid leukemia (AML) still relapse. Treatment failure and subsequent relapse have been attributed to acute myeloid leukemia-initiating cells (LSC), which harbor stem cell properties and are inherently chemoresistant. Although pediatric and adult AML represent two genetically very distinct diseases, we reasoned that common LSC gene expression programs are shared and consequently, the highly prognostic LSC17 signature score recently developed in adults may also be of clinical interest in childhood AML. Here, we demonstrated prognostic relevance of the LSC17 score in pediatric non-core-binding factor AML using Nanostring technology (ELAM02) and RNA-seq data from the NCI (TARGET-AML). AML were stratified by LSC17 quartile groups (lowest 25%, intermediate 50% and highest 25%) and children with low LSC17 score had significantly better event-free survival (EFS: HR = 3.35 (95%CI = 1.64-6.82), P < 0.001) and overall survival (OS: HR = 3.51 (95%CI = 1.38-8.92), P = 0.008) compared with patients with high LSC17 scores. More importantly, the high LSC17 score was an independent negative EFS and OS prognosticator determined by multivariate Cox model analysis (EFS: HR = 3.42 (95% CI = 1.63-7.16), P = 0.001; OS HR = 3.02 (95%CI = 1.16-7.85), P = 0.026). In conclusion, we have demonstrated the broad applicability of the LSC17 score in the clinical management of AML by extending its prognostic relevance to pediatric AML.

Liver-enriched activator protein 1 as an isoform of CCAAT/enhancer-binding protein beta suppresses stem cell features of hepatocellular carcinoma

PurposeLiver cancer stem cells (CSCs) are known to be associated with the development, survival, proliferation, metastasis, and recurrence of liver tumors. The aim of this study was to investigate the association of liver-enriched activator protein 1 (LAP1) with hepatocellular carcinoma (HCC) and liver CSCs (LCSCs) and explore the impact of LAP1 on LCSCs.Materials and methodsDifferences in LAP1 expression in liver cancer tissues versus matched para-tumoral liver tissues and LCSCs versus non-CSCs were analyzed by Western blotting, real-time polymerase chain reaction, immunohistochemistry, and flow cytometry. The effect of LAP1 on liver cancer cells was evaluated by the expression of CSC markers, oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle distribution and the number of apoptotic cells were analyzed to assess cell cycle and cell apoptosis. Furthermore, a mouse subcutaneous tumor implant model was established to explore the role of LAP1 in the development of HCC in vivo. Finally, the expression of CSC markers in paraffin-embedded sections was evaluated by immunofluorescence.ResultsLAP1 was weakly expressed in HCC tumors and cell lines and even weaker in LCSCs. LAP1 inhibited the expression of stem cell–associated genes and reduced the abilities of oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle assay revealed that LAP1 induced G1/G0 arrest. Furthermore, LAP1 decreased subcutaneous tumor-formation ability and the expression of CSC markers and Ki67 in vivo.ConclusionLAP1 suppressed the stem cell features of HCC, indicating that it possessed an antitumor effect in liver cancer, both in vitro and in vivo; therefore, LAP1 may prove to be a potential target in liver CSC-targeted therapy.

Mesenchymal Stem Cell Protection of Neurons against Glutamate Excitotoxicity Involves Reduction of NMDA-Triggered Calcium Responses and Surface GluR1, and Is Partly Mediated by TNF.

Mesenchymal stem cells (MSC) provide therapeutic effects in experimental CNS disease models and show promise as cell-based therapies for humans, but their modes of action are not well understood. We previously show that MSC protect rodent neurons against glutamate excitotoxicity in vitro, and in vivo in an epilepsy model. Neuroprotection is associated with reduced NMDA glutamate receptor (NMDAR) subunit expression and neuronal glutamate-induced calcium (Ca2+) responses, and increased expression of stem cell-associated genes. Here, to investigate whether MSC-secreted factors modulate neuronal AMPA glutamate receptors (AMPAR) and gene expression, we performed longitudinal studies of enriched mouse cortical neurons treated with MSC conditioned medium (CM). MSC CM did not alter total levels of GluR1 AMPAR subunit in neurons, but its distribution, reducing cell surface levels compared to non-treated neurons. Proportions of NeuN-positive neurons, and of GFAP- and NG2-positive glia, were equal in untreated and MSC CM-treated cultures over time suggesting that neurons, rather than differentially-expanded glia, account for the immature gene profile previously reported in MSC CM-treated cultures. Lastly, MSC CM contained measurable amounts of tumor necrosis factor (TNF) bioactivity and pre-treatment of MSC CM with the TNF inhibitor etanercept reduced its ability to protect neurons. Together these results indicate that MSC-mediated neuroprotection against glutamate excitotoxicity involves reduced NMDAR and GluR1-containing AMPAR function, and TNF-mediated neuroprotection.

MiR-1301-3p promotes prostate cancer stem cell expansion by targeting SFRP1 and GSK3β

Cancer stem cells promote tumor progression, drug-resistance, and relapse, and many microRNAs (miRNAs) play critical roles in the expansion of cancer stem cells. In the present study, we investigated the role of miR-1301-3p in the expansion of prostate cancer stem cells; miR-1301-3p was significantly upregulated in prostate cancer cells and tissues compared with normal prostate cells and tissues. Sphere formation and side population assays suggested that miR-1301-3p promoted the expansion of prostate cancer stem cells, and increased the expression of prostate cancer stem cell-associated genes, such as OCT4, SOX2, NANOG, CD44, KLF4, c-MYC, and MMP2. MiR-1301-3p targeted Wnt pathway inhibitors, GSK3β and SFRP1, and inhibited their expression by directly binding to their 3′ untranslated regions. TOP/FOP luciferase assays suggested that miR-1301-3p activated the Wnt pathway, which was confirmed by increased β-catenin expression in the nucleus. Furthermore, the miR-1301-3p level correlated negatively with GSK3β and SFRP1 in prostate cancer tissues. In summary, we found that miR-1301-3p promoted the expansion of prostate cancer stem cells by inhibiting GSK3β and SFRP1, and activating the Wnt pathway.

Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133.

The present study aimed to isolate and identify the properties of the cluster of differentiation (CD)133+ subset in human gallbladder cancer cells. The CD133+ and CD133− subpopulations of the GBC-SD cell line were separated using immunomagnetic separation, and the biological features of the two subpopulations were analyzed in vitro and in vivo. In particular, the present study aimed to determine whether the two subpopulations were resistant to anti-tumor reagents and to identify the underlying molecular mechanisms involved. Following cell sorting of GBC-SD cells using immunomagnetic beads, 90.2±2% of cells were identified as CD133+. Immunofluorescence confirmed that CD133 was expressed at higher levels in the Cd133+ group compared with the CD133− group. The proliferation of the CD133+ group was significantly increased compared with the CD133− group in vitro and in vivo. Following treatment with fluorouracil or gemcitabine, cells in the CD133+ group exhibited a decreased sensitivity to these drugs. The number of transmembrane cells was significantly increased in the CD133+ group compared with the CD133− group. In addition, the expression levels of ATP binding cassette subfamily G member 2, CD44, C-X-C motif chemokine receptor 4 (CXCR4), phosphorylated-protein kinase B (Akt) and CD133 in the CD133+ group were significantly increased, compared with those in the CD133− group. In CD133+ GBC-SD cells, stromal cell-derived factor 1α (SDF-1α) or treatment with AMD3100, an inhibitor of CXCR4, promotes or suppresses the SDF-1α/CXCR4 axis, respectively, resulting in increased or decreased CD133 expression through the Akt signaling pathway. Inhibition of the Akt signaling pathway resulted in decreased CD133 expression in GBC-SD cells. Immunomagnetic beads were successfully used for isolation of the CD133+ subset from GBC-SD cells. Furthermore, the CD133+ subset revealed an increased potential for tumor formation, cell proliferation, invasion and resistance to chemotherapeutic agents with expression of stem cell-associated genes. Therefore, in GBC-SD cells, the CXCR4/Akt/CD133 signaling pathways may be activated.

WNT/β-Catenin Directs Self-Renewal Symmetric Cell Division of hTERThigh Prostate Cancer Stem Cells.

Cancer stem-like cells (CSC) drive cancer progression and recurrence. Self-renewal expansion of CSC is achieved through symmetric cell division, yet how external stimuli affect intracellular regulatory programs of CSC division modes and stemness remains obscure. Here, we report that the hTERThigh prostate cancer cells exhibit CSC properties, including a stem cell-associated gene expression signature, long-term tumor-propagating capacity and epithelial-to-mesenchymal transition. In promoting the self-renewal symmetric division of hTERThigh prostate cancer cells, WNT3a dramatically decreased the ratio of hTERThigh prostate cancer cells undergoing asymmetric division. Increased WNT/β-catenin signal activation was also detected in hTERThigh prostate cancer cells. hTERT-mediated CSC properties were at least partially dependent on β-catenin. These findings provide novel cellular and molecular mechanisms for the self-renewal of CSC orchestrated by tumor microenvironmental stimuli and intracellular signals. Cancer Res; 77(9); 2534-47. ©2017 AACR.

MP41-15 STEM CELL ACTIVATION IN THE MAJOR PELVIC GANGLION IS ENHANCED BY STROMAL DERIVED FACTOR-1 PENILE INJECTIONS FOLLOWING NERVE INJURY

You have accessJournal of UrologyStem Cell Research1 Apr 2017MP41-15 STEM CELL ACTIVATION IN THE MAJOR PELVIC GANGLION IS ENHANCED BY STROMAL DERIVED FACTOR-1 PENILE INJECTIONS FOLLOWING NERVE INJURY Nikolai Sopko, Hotaka Matsui, Takahiro Yoshida, Xiaopu Liu, Max Kates, and Trinity Bivalacqua Nikolai SopkoNikolai Sopko More articles by this author , Hotaka MatsuiHotaka Matsui More articles by this author , Takahiro YoshidaTakahiro Yoshida More articles by this author , Xiaopu LiuXiaopu Liu More articles by this author , Max KatesMax Kates More articles by this author , and Trinity BivalacquaTrinity Bivalacqua More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1283AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Stem cells are believed to play a role in nerve regeneration following nerve injury. Stromal Derived Factor-1 (SDF1) is potent stem cell chemokine. We investigated whether penile injections with SDF1 following bilateral nerve crush injury (BCNI) can enhance stem cell recruitment and activation in the major pelvic ganglion (MPG). METHODS Sprague Dawley (350g) male rats underwent sham procedure or BCNI (n=10/group). Starting on the day of surgery, BCNI animals received penile injections with 1ug of SDF1 or saline QOD for 5 injections. Sham animals received saline penile injections only. 2 weeks after BCNI, intracorporeal pressure (ICP) response to cavernous nerve stimulation was assessed (n=5/group) and MPGs were collected and RNA was isolated (n=5/group). Stem cell activity in the MPG was assessed using a stem cell expression array expressed as mean fold-change ± SE normalized to sham. RESULTS SDF1 penile injections resulted in improved ICP preservation following BCNI compared to animals receiving saline injections. Sham animals had significantly greater (P<0.05, 1-way ANOVA with Tukey’s post-hoc testing) than BCNI+saline at all voltages (2, 4, 6, 8v), whereas there was no significant difference between Sham and BCNI+SDF1 at any voltages. Additionally, BCNI+SDF1 had greater ICP at 2v and 4v compared to BCNI+saline (0.40±0.09 vs. 0.11±0.08, p<0.05 and 0.47±0.11 vs. 0.12±0.08, p<0.05, respectively). Stem cell-associated gene expression normalized to Shams was globally increased following BCNI and BCNI+SDF1 had a greater increase in expression compared to BCNI+saline (7.89±0.67 fold vs. 1.87±0.15 fold, p<0.0001). On subset analysis, stem cell pathways had significantly greater expression in BCNI+SDF1 compared to BCNI+saline including cytokines and growth factors (6.45±0.63 vs. 1.86±0.13, p<0.0001), Notch pathway (7.72±0.96 vs. 1.95±0.17, p<0.0001), Wnt pathway (7.01±0.68 vs. 1.87±0.12, p<0.0001), mesenchymal stem cell markers (6.33±1.16 vs. 1.82±0.40, p=0.0015), and neural cell lineage markers (6.63±0.51 vs. 1.85±0.14, p=0.0003). CONCLUSIONS SDF1 penile injections improve preservation of erectile function following BCNI and are associated with increased stem cell pathway activation in MPG. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e541 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Nikolai Sopko More articles by this author Hotaka Matsui More articles by this author Takahiro Yoshida More articles by this author Xiaopu Liu More articles by this author Max Kates More articles by this author Trinity Bivalacqua More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...