Marrow Stromal Cell Line Research Articles

Interference with PLA2G16 promotes cell cycle arrest and apoptosis and inhibits the reprogramming of glucose metabolism in multiple myeloma cells by modulating the Hippo/YAP signaling pathway.

Multiple myeloma, which is a clonal plasma cell tumor, derives from a postmitotic lymphoid B-cell lineage and remains untreatable. Group XVI phospholipase A2 (PLA2G16) can either be a tumor suppressor or an oncogene in different types of cancer. This study was intended to explore the role of PLA2G16 in multiple myeloma and to reveal the reaction mechanism. The mRNA and protein expressions of PLA2G16 in human bone marrow stromal cell line HS-5 and multiple myeloma cells were assessed using reverse transcription-quantitative PCR and western blot. The transfection efficacy of sh-PLA2G16 and oe-YAP was examined using reverse transcription-quantitative PCR and western blot. Through cell counting kit-8 assay and 5-ethynyl-2'- deoxyuridine staining, multiple myeloma cell viability and proliferation were detected. Flow cytometry was used to measure cell apoptosis and cell cycle distribution. Oxygen consumption rate, the activities of mitochondrial respiratory chain complexes I-V, and the activity of caspase-3 were estimated with Seahorse XF24 analyzer, oxidative phosphorylation activity assay kit, and caspase-3 assay kit, respectively. Lactate production and glucose consumption were evaluated usingcorresponding assay kits. Western blot was employed to meaure proteins associated with cell cycle, glycolysis, pentose phosphate pathway as well as Hippo/YAP signaling pathway. In this study, PLA2G16 expression was greatly increased in multiple myeloma cells and PLA2G16 silence inhibited cell proliferation, promoted cell apoptosis, facilitated cell cycle arrest, and suppressed the reprogramming of glucose metabolism in multiple myeloma. It was also identified that PLA2G16 depletion inhibited the Hippo/YAP signaling pathway. Further experiments revealed that the overexpression of YAP partially reversed the inhibitory effects of PLA2G16 silence on multiple myeloma cell malignant development and the reprogramming of glucose metabolism. Collectively, PLA2G16 silence impeded multiple myeloma progression and inhibited glucose metabolism reprogramming by blocking the Hippo/YAP signaling pathway.

IL-6 knockdown in a model of the human bone marrow, abrogates DNA damage induction in bystander cells post-chemotherapy induced cytokine release syndrome

Following infection or exposure to therapeutic agents, an aggressive immune response may result, termed cytokine storm (CS) or cytokine release syndrome. Here the innate immune system becomes uncontrolled, leading to serious consequences including possible death. Patients surviving CS are at greater risk for de novo tumorigenesis, but it is unclear if any specific cytokines are directly responsible for this outcome. De novo tumorigenesis has been observed in donated cells exposed to CS following haematopoietic stem cell transplant (HSCT).Modelling HSCT, we firstly demonstrated the release of CS levels from the HS-5 human bone marrow stromal cell line, post-exposure to chemotherapy. We then exposed the TK6 lymphoblast cell line to healthy and storm doses of IL-6 and measured increased genotoxicity via the micronucleus assay. During HSCT, haematopoietic cells are exposed to a complex mix of cytokines, so to determine if IL-6 was integral in a chemotherapy-induced bystander effect, we attempted to inhibit IL-6 from HS-5 cells using resatorvid or siRNA, treated with chlorambucil or mitoxantrone, and then co-cultured with bystander TK6 cells. Whilst resatorvid did not reduce IL-6 and did not reduce micronuclei in the bystander TK6 cells, siRNA inhibition reduced IL-6 to healthy in vivo levels, and micronuclei aligned with untreated controls.Our data suggests that exposure to high IL-6 (in the absence of inflammatory cells) has potential to induce genetic damage and may contribute to de novo tumorigenesis post-CS. We suggest that for individuals with a pro-inflammatory profile, anti-IL-6 therapy may be an appropriate intervention to prevent complications post-CS.

Effects of circ-SFMBT2 on Proliferation, Migration and Invasion of Acute Myeloid Leukemia Cells by Regulating miR-491-5p/HOXA9 Axis

To explore the effects and molecular mechanism of circ-SFMBT2 on the proliferation, migration and invasion of acute myeloid leukemia (AML) cells. Bone marrow samples from 35 pediatric AML patients and 35 healthy controls in Henan Provincial Children's Hospital from April 2015 to April 2017 and human bone marrow stromal cell lines (HS-5) and AML cell lines (HL-60, THP-1, U-937 and Kasumi-1) were collected. The expressions of circ-SFMBT2, miR-491-5p and homeobox A9 (HOXA9) in bone marrow samples and cells were detected by RT-qPCR and Western blot. The Pearson method was used to analyze the correlation of circ-SFMBT2, miR-491-5p and HOXA9 mRNA expression levels in bone marrow samples of AML patients. HL-60 cells were cultured in vitro and divided into 5 groups: Control, si-NC, si-circ-SFMBT2, si-circ-SFMBT2+anti-NC and si-circ-SFMBT2+anti-miR-491-5p, HL-60 cells were transfected with si-NC, si-circ-SFMBT2, anti-NC, and miR-491-5p inhibitor with Lipofectamine™ 3000. RT-qPCR and Western blot were performed to detect the expression levels of circ-SFMBT2, miR-491-5p and HOXA9 in cells of each group. The proliferation activity of HL-60 cells in each group was detected by CCK-8 assay at 24, 48 and 72 h after transfection, respectively. The apoptosis rate was detected by flow cytometry. The migration and invasion abilities of cells were detected by Transwell assay. The regulatory roles of circ-SFMBT2, miR-491-5p and HOXA9 in AML cells were verified by dual-luciferase reporter gene assay, RNA pull-down and RNA-binding protein immunoprecipitation (RIP) experiments. The expression levels of circ-SFMBT2 and HOXA9 mRNA were increased in bone marrow samples and cell lines (HL-60, THP-1, U-937 and Kasumi-1) of children with AML (P <0.001), while the expression level of miR-491-5p was significantly decreased (P <0.001). Pearson correlation analysis showed that the expression levels of circ-SFMBT2 and miR-491-5p in bone marrow samples of AML children were negatively correlated (r =-0.905), miR-491-5p was also negatively correlated with HOXA9 mRNA (r =-0.930), while the expression levels of HOXA9 mRNA and circ-SFMBT2 was positively correlated (r =0.911). The overall survival rate of AML children with high expression of circ-SFMBT2 was significantly decreased than those with low expression of circ-SFMBT2 (P <0.05). Silencing of circ-SFMBT2 could greatly up-regulate the expression of miR-491-5p, decrease the expression of HOXA9, inhibit the proliferation, migration and invasion of AML cells, and promote cell apoptosis (P <0.05). Down-regulation of miR-491-5p expression greatly attenuated the inhibitory effects of circ-SFMBT2 silencing on cell proliferation, migration and invasion (P <0.05). Dual-luciferase reporter gene assay, RNA pull-down and RIP experiments confirmed that circ-SFMBT2 could target miR-491-5p and negatively regulate the expression of miR-491-5p in AML, and HOXA9 was the target of miR-491-5p. Silencing of circ-SFMBT2 may inhibit the proliferation, migration and invasion of AML cells by regulating the miR-491-5p/HOXA9 axis.

Chaperon Protein GRP78 Released from MPN Cells Increase Expression of Lysyl Oxidase in Human Stroma Cell Line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found an increased expression of the chaperone protein glucose-regulated protein (GRP)78, which is a central regulator of ER stress in megakaryocytes from primary myelofibrosis (PMF) (n=7) or post-essential thrombocythemia myelofibrosis(POST-ET-MF) patients (n=2) regardless of the type of driver mutation. In accord with these notions, we found that GRP78 was overexpressed in JAK2V617F-harboring cell lines (HEL and SET-2) and in UT-7/TPO cells overexpressing either type1 or type2 mutant form of calreticulin. To address the mechanism responsible for the induction of GRP78, we treated the HEL and SET-2 cells with 4-phenyl butyric acid and tauroursodeoxycholic acid both of which are well known inhibitors of ER stress, however, these inhibitors did not affect the expression levels of GRP78. These results indicated that overexpression of GRP78 shown in these cells were not a result of activation of ER stress. In contrast, ruxolitinib, an inhibitor of JAK2V617F, blocked GRP78 expression in these cells. We also found ruxolitinib blocked nuclear accumulation of the transcription factor 4 (ATF4), one of the main transcription factors which regulate GRP78. Furthermore, knockdown of ATF4 using siRNA also resulted in suppression of GRP78 levels in HEL and SET-2 cells. These results suggested that JAK2V617F induce GRP78 through activation of ATF4. We also confirmed that megakaryocytes from MF patients had statistically higher nuclear to cytoplasmic intensity of ATF4 compared to megakaryocytes found in control subjects. For the next step, we tried to find out the pathological action of GRP78 in MPN cells. Although the GRP78 usually works as chaperon protein in cytosol, some cancer cells secret GRP78 into tumor microenvironment and activates signal transduction molecules in surrounding cells. Therefore, we hypothesized that overexpressed GRP78 proteins in MPN cells may secret and modulate phenotype of bone marrow stroma cells. Initially, we confirmed the presence of GRP78 proteins in culture media from HEL and SET-2 cells. Next, we investigated whether GRP78 secreted by HEL and SET-2 cells affect phenotype of bone marrow stromal cells. For this purpose, we used human bone marrow stroma cell line, HS-5. HS-5 cells were co-cultured with HEL or SET-2 cells with trans-well system, and changes of several key molecules involved in development of fibrosis were analyzed. We found that co-culture with HEL or SET-2 cells clearly indued expression of lysyl oxidase (LOX) , which mediate cross-linking of collagen fiber and induce tissue fibrosis , in HS-5 cells. Anti-GRP78 neutralizing antibody abrogated LOX elevation, in contrast, recombinant form of GRP78 proteins induced LOX proteins in HS-5. According to the above results, we examined LOX expression in bone marrow specimens from MF patients. The areas of LOX expression cells were statistically higher in MF patients compared to control group. In addition, high power field observations detected not only in round cells but also in spindle like cells expressed LOX in MF patients, suggesting that bone marrow stromal cells in MF patients express LOX. In conclusion, we demonstrated that GRP78 was overexpressed both in MPN derived cell lines and also in primary megakaryocytes from MF patients. Our observations proposed that overexpressed GRP78 in MPN cells contribute development of myelofibrosis through secreted in microenvironment and induced expression of extracellular matrix modulating enzyme LOX in bone marrow stromal cells.

Circ_0001946 Promotes the Development of Acute Myeloid Leukemia by Upregulating PDL1

Circ_0001946 has been identified as an oncogenic factor, and the aim of this study was to explore the detailed roles and putative targets of circ_0001946 in acute myeloid leukemia (AML). Levels of circ_0001946 were examined in AML tissues and cells. Furthermore, the regulatory functions of circ_0001946 in AML were explored. The expression of circ_0001946 was evaluated in AML samples and a matched para-carcinoma control, as well as in AML cell lines and a human bone marrow stromal cell line using reverse transcription-quantitative polymerase chain reaction. Cell proliferation was examined using a CCK-8 kit, and migration/invasion was measured by transwell assay. Furthermore, interactions between associated molecules were assessed using RNA pulldown, and the mRNA stability of the relevant gene was examined by mRNA stability assay. Our data indicated that circ_0001946 was upregulated in AML specimens/cells. Additionally, overexpression of circ_0001946 promoted the proliferation, migration, and invasion of AML cells and, vice versa, these biological processes were suppressed by knockdown of circ_0001946. Furthermore, PDL1 is a potential downstream molecule of circ_0001946 in AML and its stability was improved by circ_0001946. The expression of PDL1 was increased in AML specimens and positively correlated with circ_0001946 expression. Moreover, biological behavioral alterations in AML cells induced by oe-circ_0001946 were abrogated by sh-PDL1 and the effects of sh-circ_0001946 were enhanced by treatment with sh-PDL1. Taken together, these data suggest that levels of circ_0001946 are elevated in AML and that circ_0001946 could promote the growth of AML cells. Furthermore, PDL1 is a novel downstream molecule of circ_0001946 in AML. Circ_0001946/PDL1 signaling may play crucial roles in tumor progression in AML and could be a novel candidate for targeted treatments for AML patients.

Effect of Yifei Qinghua Granule on the Tumor Microenvironment in Lung Cancer by Regulating the Inflammatory Pathway.

To investigate the mechanism of action of inflammatory molecules regulating the tumor microenvironment and anti-tumor through Yifei Qinghua granules and phloroglucinol-containing serum intervening in the changes of tumor microenvironment in vitro in the co-culture of lung cancer cells and bone marrow cells. A549 lung adenocarcinoma cell line and ST2 bone marrow stromal cell line were selected and a transwell chamber was used to establish the co-culture system of the two kinds of cells. They were divided into normal saline, phloroglucinol, Qifei Qinghua granule, and phloroglucinol + Yifei Qinghua granule groups. They were given drug-containing serum interventions respectively. A549 cells and ST2 cells cultured separately were used as control. Flow cytometry was used to detect the proportions of MDSCs and Tregs in bone marrow cells of ST2 cells. ELISA was used to detect the levels of inflammatory factors in the culture supernatant. Western blot was used to detect the expressions of inflammatory pathways in A549 and ST2 cells. ST2 cells and A549 cells were co-cultured. The ratio of MDSCs and Treg in ST2 cells was increased. The levels of some inflammatory factors in the culture supernatant were increased. The expression level of the inflammatory pathway in ST2 cells was increased. However, the expression level of the inflammatory pathway in A549 cells had no obvious change. While Yifei Qinghua granule and phloroglucinol could partially reverse these changes. The combination of the two was more effective than a single drug. The conversion of cells to MDSCs and Treg was accelerated after the co-culture of ST2 cells and A549 cells. The combination of Yifei Qinghua granules with phloroglucinol can reshape the tumor microenvironment, prevent this phenomenon from occurring, reduce inflammatory secretion and inhibit tumor cell growth. This may be related to the inhibition of the expressions of TNF-α/IL-1- and NF-κB/STAT3 inflammatory pathways.

Sustained release of a highly specific GSK3β inhibitor SB216763 in the PCL scaffold creates an osteogenic niche for osteogenesis, anti-adipogenesis, and potential angiogenesis.

The safe and effective use of Wnt signaling is a hot topic in developing osteogenic drugs. SB216763 (S33) is a widely used highly specific GSK3β inhibitor. Here, we show that S33 initiates canonical Wnt signaling by inhibiting GSK3β activity in the bone marrow stromal cell line ST2 and increases osteoblast marker alkaline phosphatase activity, osteoblast marker gene expression including Alpl, Col1α1, and Runx2, promoting osteogenic differentiation and mineralization of ST2 cells. In addition, S33 suppressed the expression of adipogenic transcription factors Pparg and Cebpa in ST2 cells to suppress adipogenesis. ICRT-14, a specific transcriptional inhibitor of Wnt signaling, reversed the effects of S33 on the differentiation of ST2 cells. S33 also increased the expression of osteoclast cytokines RANKL and Opg but decreased the RANKL/Opg ratio and had the potential to inhibit osteoclast differentiation. In addition, we printed the PSCI3D (polycaprolactone, S33, cell-integrated 3D) scaffolds using a newly established integrated 3D printing system for hard materials and cells. S33 sustained release in the hydrogel of the scaffold with 25.4% release on day 1% and 81.7% release over 7days. Cells in the scaffolds had good cell viability. The ratio of live/dead cells remained above 94% for 7days, while the cells in the scaffolds proliferated linearly, and the proliferative activity of the PSCI3D scaffold group increased 1.4-fold and 1.7-fold on days 4 and 7, respectively. Similarly, in PSCI3D scaffolds, osteogenic differentiation of st2 cells was increased. The alkaline phosphatase activity increased 1.4- and 4.0-fold on days 7 and 14, respectively, and mineralization increased 1.7-fold at 21days. In addition, PSCI3D conditioned medium promoted migration and tubulogenesis of HUVECs, and S33 upregulated the expression of Vegfa, a key factor in angiogenesis. In conclusion, our study suggests that S33 functions in osteogenesis, anti-adipogenesis, and potential inhibition of osteoclast differentiation. And the sustained release of S33 in PSCI3D scaffolds creates a safe osteogenic niche, which promotes cell proliferation, osteogenesis, and angiogenesis and has application prospects.

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia.

Transduced mouse immature thymocytes can be differentiated into T cellsin vitrousing the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

Abstract 2516: Analysis of CLL Celllular Environment and Response (ACCER) is a novel method to understand the microenvironment in CLL

Abstract Microenvironments such as lymph nodes and bone marrow allow chronic lymphocytic leukemia (CLL) cells to survive after drug treatments. There are limited methods to study the to study the contribution of the microenvironment. We have adapted a solid tumour microenvironment cell culture system that provides elements of the CLL microenvironment called Analysis of CLL Cellular Environment and Response (ACCER). We optimized the cell number for patient’s primary CLL cells and HS-5 human bone marrow stromal cell line that will give sufficient cell number and viability with the ACCER. We then determined the amount of collagen type 1 to give the best extracellular matrix to seed CLL cells to the membrane. Finally, we determined that ACCER provide CLL cell protection against cell death following treatment with fludarabine and ibrutinib compared to co-culture conditions. This describes novel microenvironment model to investigate factors that promote drug resistance and cell survival in CLL. In the future, this will further validate new treatment strategies to overcome microenvironmental CLL cell survival signals. Citation Format: Spencer Gibson, Tricia Choquette, Elizabeth S. Henson, Xioyan Yang, James B. Johnston. Analysis of CLL Celllular Environment and Response (ACCER) is a novel method to understand the microenvironment in CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2516.

A novel method to investigate drug resistance in the chronic lymphocytic leukemia (CLL) microenvironment: Analysis of CLL Cellular Environment and Response (ACCER)

Microenvironments such as lymph nodes allow chronic lymphocytic leukemia (CLL) cells to survive and become drug resistant. There are limited methods to study the to study the contribution of the stromal microenvironment. We have adapted a solid tumor microenvironment cell culture system that provides elements of the CLL microenvironment called Analysis of CLL Cellular Environment and Response (ACCER). We optimized the cell number for patient’s primary CLL cells and HS-5 human bone marrow stromal cell line that will give sufficient cell number and viability with the ACCER. We then determined the amount of collagen type 1 to give the best extracellular matrix to seed CLL cells to the membrane. Finally, we determined that ACCER provide CLL cell protection against cell death following treatment with fludarabine and ibrutinib compared to co-culture conditions. This describes novel microenvironment model to investigate factors that promote drug resistance in CLL.

SPARC is a decoy counterpart for c‑Fos and is associated with osteoblastic differentiation of bone marrow stromal cells by inhibiting adipogenesis.

Secreted protein acidic and rich in cysteine (SPARC), also called basement‑membrane protein 40 or osteonectin, is a matricellular protein that is abundant not only in bone tissue as a non‑collagenous protein but is also ubiquitously expressed in non‑calcified tissue. SPARC is located intracellularly and disruption of the Sparc gene has been reported to reduce bone formation and increase fat tissue; however, the mechanism by which SPARC inhibits adipogenesis remains unclear. The present study evaluated the intracellular function of SPARC in adipogenesis using the bone marrow stromal cell line ST2. When ST2 cells with low SPARC production were cloned, intrinsic activator protein‑1 (AP‑1) activity was markedly higher, mineralized nodule formation was significantly lower and lipid accumulation was significantly increased compared with in the parental ST2 cells. Forced expression of secreted SPARC with the signal peptide‑coding sequences of wild‑type Sparc or preprotrypsin in SPARC‑low ST2 cells significantly reduced AP‑1 transcription activity; however, these reductions were not observed in the absence of signal peptide sequences. Recombinant SPARC, produced using Brevibacillusbrevis, specifically bound to c‑Fos but not c‑Jun and inhibited the binding of c‑Fos/c‑Jun to a TPA‑response element sequence. These data suggested that SPARC was incorporated into the cells from the extracellular spaces and serves an intracellular role as a decoy counterpart for c‑Fos, as well as being associated with osteoblastogenesis through the inhibition of adipogenesis. These findings may provide new insights into regenerative medicine.

Improved Relapse Prediction in Pediatric Acute Myeloid Leukemia By Deconvolving Lineage-Specific and Cancer-Specific Features in Single-Cell Data

Introduction While most children with acute myeloid leukemia (AML) achieve first remission, nearly 40% will relapse. Of these children, few survive to a second remission even with highly-escalated treatment protocols. Recent studies have shown that many AML patients harbor rare, stem cell-like subpopulations that resist chemotherapy and drive relapse. However, the exact characteristics of these relapse-associated cells are a matter of contention, with reported phenotypes spanning the hematopoietic developmental continuum. In some patients, treatment-resistant cells can be detected as minimal residual disease (MRD), which is often used to predict relapse, albeit with limited accuracy and only after induction chemotherapy. Thus, the identity of treatment-resistant cells as well as their relationship to normal progenitors remain mysterious, thereby limiting the development of targeted therapies for pediatric AML. Here, we present a computational approach for decomposing high-dimensional single-cell measurements into two components: a lineage-specific component that can be used to align cancer cells with specific stages of myeloid development and a cancer-specific component that can be used to identify aberrant phenotypes unique to AML cells. We show that, together, these components can be used at the time of diagnosis to predict relapse more accurately than clinical information alone. Methods and Results Using mass cytometry, we analyzed paired diagnostic and post-induction samples collected from 19 (8 relapse, 11 non-relapse) pediatric patients who enrolled on the Children's Oncology Group trial AAML1031. All patients were treated on the control arm and consented to banking of tissue for research. We also included 5 bone marrow samples from healthy donors. After thawing, samples were divided in half and stimulated with conditioned medium from the human bone marrow stromal cell line HS-5 to activate relevant signaling pathways, or left unstimulated. An average of 5 x 105 cells per patient were analyzed for each condition. The mass cytometry panel included 31 antibodies to surface markers, 6 antibodies to intracellular signaling mediators, and 4 antibodies to intracellular proteins and transcription factors. Following data collection, the singular value decomposition was applied to the data matrix of healthy single-cell measurements to construct a linear subspace representing the predominant protein expression programs ("eigencells") within the healthy myeloid developmental continuum. By projecting AML single-cell measurements onto this subspace, we derived a healthy feature vector aligned with the healthy subspace and a cancer-specific feature vector orthogonal to the healthy subspace for each AML cell. These feature vectors-along with clinical metadata about each patient including age, blast percentage at diagnosis, and cytogenetic status-were used as the input to regularized Cox proportional hazards models predicting time-to-relapse for each patient. Using the relative risk scores from the proportional hazards model, patients were assigned to high-risk or low-risk groups according to the optimal log-rank test threshold. The baseline clinical model used only age, blast percentage at diagnosis, and cytogenetic status as predictors and predicted relapse status with an accuracy of 13/19 (68%). This baseline model was outperformed by the model constructed using the average value of the single-cell cancer-specific feature vectors for each patient, which predicted relapse status with an accuracy of 16/19 (84%). Interestingly, despite using only information available at diagnosis, the single-cell model also outperformed a clinical model incorporating patients' MRD status after induction chemotherapy, which predicted relapse with an accuracy of 15/19 (79%). Interrogation of the coefficients of the single-cell feature model revealed specific cellular signaling programs associated with relapse, including enhanced pCreb and pSTAT1 signaling as well as depleted pSTAT5 signaling relative to healthy lineage cells (Figure 1). Conclusions These results support the feasibility of predicting relapse in AML as early as diagnosis by leveraging a computational approach that compares cancer cells to the native lineage from which they arise. Validation of this approach in an independent cohort is ongoing and will be presented. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Induction of Human T Cell Development In Vitro with OP9-DL4-7FS Cells Expressing Human Cytokines.

For nearly a generation now, OP9-DL1 and OP9-DL4 cells have provided an efficient and reliable cell system to generate T cells from mouse and human hematopoietic stem cells (HSCs) and pluripotent stem cells. OP9-DL1 and OP9-DL4 were originally derived from the OP9 mouse bone marrow stromal cell line, which was transduced to ectopically express Delta-like 1 or 4 proteins, respectively. OP9-DL cells mimic the thymic microenvironment in that when cocultured with mouse or human (h) HSCs, they interact with and activate Notch receptors present on HSCs, required for T cell differentiation. The HSC/OP9-DL cocultures require additional cytokines that are necessary for survival and proliferation of hematopoietic cells. For hHSCs, these factors are interleukin-7 (IL-7), stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L) that are normally exogenously added to the cocultures. In this chapter, we describe methods for establishing a novel and improved version of OP9-DL4 cells, called OP9-DL4-7FS cells that circumvent the addition of these costly cytokines, by transducing OP9-DL4 cell line to express human IL-7, FLT3L, and SCF (7FS). Herein, we describe the protocol for the generation of OP9-DL4-7FS cells and the conditions for OP9-DL4-7FS/hHSC coculture to support T cell lineage initiation and expansion while comparing it to the now "classic" OP9-DL4 coculture. The use of OP9-DL4-7FS cell system will provide an improved and cost-effective method to the commonly used OP9-DL/HSC coculture for studying both mouse and human T cell development.

Doxorubicin induces prolonged DNA damage signal in cells overexpressing DEK isoform-2.

DEK has a short isoform (DEK isoform-2; DEK2) that lacks amino acid residues between 49-82. The full-length DEK (DEK isoform-1; DEK1) is ubiquitously expressed and plays a role in different cellular processes but whether DEK2 is involved in these processes remains elusive. We stably overexpressed DEK2 in human bone marrow stromal cell line HS-27A, in which endogenous DEKs were intact or suppressed via short hairpin RNA (sh-RNA). We have found that contrary to ectopic DEK1, DEK2 locates in the nucleus and nucleolus, causes persistent γH2AX signal upon doxorubicin treatment, and couldn't functionally compensate for the loss of DEK1. In addition, DEK2 overexpressing cells were more sensitive to doxorubicin than DEK1-cells. Expressions of DEK1 and DEK2 in cell lines and primary tumors exhibit tissue specificity. DEK1 is upregulated in cancers of the colon, liver, and lung compared to normal tissues while both DEK1 and DEK2 are downregulated in subsets of kidney, prostate, and thyroid carcinomas. Interestingly, only DEK2 was downregulated in a subset of breast tumors suggesting that DEK2 can be modulated differently than DEK1 in specific cancers. In summary, our findings show distinct expression patterns and subcellular location and suggest non-overlapping functions between the two DEK isoforms.

Life in an optical fiber: Monitoring of cell cultures with microcavity in-line Mach-Zehnder interferometer

Monitoring cell adhesion and growth are crucial for various applications involving drug screening, cytotoxicity, and cytocompatibility studies. However, acquiring accurate information about the growing state and responsiveness to a treatment of a cell system in a real-time and label-free manner is still a challenge. This work presents the first research on direct, real-time, and label-free adherent cell culture monitoring using a microcavity in-line Mach–Zehnder interferometer (μIMZI) fabricated in an optical fiber. The sensing solution based on μIMZI offers a great advantage over many other monitoring concepts tracking the changes taking place on the microcavity's bottom surface and within its volume, thus offering a greater penetration depth. In this study, we verified performance of the approach using a non-cancer bone marrow stromal cell line HS-5. The results demonstrate that the changes of the acquired signal are closely related to the different states of cells' adhesion, proliferation, morphology, and variation of mass. Thus, this label-free, real-time μIMZI-based monitoring technique gives a great promise to the analysis or monitoring of relevant new treatments in future scientific, as well as clinical applications.

Functionalized 3D-Printed ST2/Gelatin Methacryloyl/Polcaprolactone Scaffolds for Enhancing Bone Regeneration with Vascularization.

Growth factors were often used to improve the bioactivity of biomaterials in order to fabricate biofunctionalized bone grafts for bone defect repair. However, supraphysiological concentrations of growth factors for improving bioactivity could lead to serious side effects, such as ectopic bone formation, radiculitis, swelling of soft tissue in the neck, etc. Therefore, safely and effectively applying growth factors in bone repair biomaterials comes to be an urgent problem that needs to be addressed. In this study, an appropriate concentration (50 ng/mL) of Wnt3a was used to pretreat the 3D-bioprinting gelatin methacryloyl(GelMA)/polycaprolactone(PCL) scaffold loaded with bone marrow stromal cell line ST2 for 24 h. This pretreatment promoted the cell proliferation, osteogenic differentiation, and mineralization of ST2 in the scaffold in vitro, and enhanced angiogenesis and osteogenesis after being implanted in critical-sized mouse calvarial defects. On the contrary, the inhibition of Wnt/β-catenin signaling in ST2 cells reduced the bone repair effect of this scaffold. These results suggested that ST2/GelMA/PCL scaffolds pretreated with an appropriate concentration of Wnt3a in culture medium could effectively enhance the osteogenic and angiogenic activity of bone repair biomaterials both in vitro and in vivo. Moreover, it would avoid the side effects caused by the supraphysiological concentrations of growth factors. This functionalized scaffold with osteogenic and angiogenic activity might be used as an outstanding bone substitute for bone regeneration and repair.

RNA Polymerase II Is a Therapeutic Target to Overcome FLT3 Inhibitor Resistance Mediated By the Bone Marrow Microenvironment

Background: Gilteritinib is a clinically active FLT3 tyrosine kinase inhibitor (TKI) approved for relapsed/refractory FLT3-mutant AML, but nearly all patients treated with gilteritinib and other FLT3 TKIs eventually develop clinical resistance. Activating RAS/MAPK pathway mutations are a predominant non-FLT3 dependent resistance mechanism in patients treated with gilteritinib. AML blasts can also develop FLT3 TKI resistance secondary to paracrine MAPK activation stimulated by FLT3 Ligand, FGF2, or other protective cytokines within the bone marrow microenvironment (BME). To identify potential targets that sensitize AML cells to gilteritinib-induced apoptosis in a model of the BME, we performed a genome-wide CRISPR/Cas9 death screen in MOLM-14 FLT3-ITD+ human AML cells cultured in bone marrow stromal cell conditioned media. We hypothesize that identified genes represent promising combinatorial therapeutic targets that can enhance clinical efficacy of FLT3 TKIs in AML.Methods: To model stroma-mediated TKI resistance, we used the HS5 human bone marrow stromal cell line that secretes multiple cytokines (G-CSF, GM-CSF, FGF2) and supports myeloid progenitor proliferation in co-culture. MOLM-14 CRISPRi cells transduced with CRISPRi-v2 genome-wide sgRNA library were cultured in HS5 conditioned media for 24 hours and then treated with gilteritinib 250 nM. Cells were stained with a fluorogenic caspase 3/7 reagent and then fixed after 24 hours of drug treatment. Caspase-3 positive cells were sorted from the entire drug-treated cell population by FACS and guide RNAs enriched or depleted in this sample as compared to an untreated T0 sample were determined by NGS.Results: We identified several gene-level hits that were enriched in the apoptotic population (FDR <0.2). Among these, we identified multiple transcription factors or regulators of transcriptional activation. The latter included multiple components of RNA pol II machinery (POLR2G, RTF1) and multiple subunits of Mediator (MED12, MED30, MED21, MED11), a complex that regulates RNA pol II activity and has been shown to modulate super-enhancer-associated genes in AML cells. To validate select hits, we transduced MOLM-14 and MV411 CRISPRi cells with a tetracycline-inducible sgRNA expression vector. Using this system, we found that conditional knockdown of MED12, the top scoring Mediator subunit in our screen, significantly sensitized MOLM-14 (FgH1) cells to gilteritinib while modestly augmenting the cytotoxicity of gilteritinib in MV411 (FgH1) cells when compared to a non-targeting sgRNA. Functionally, MED12 associates with MED13, Cyclin C, and CDK8 to form the CDK8 kinase module of Mediator. MED12 knockdown, as expected, led to suppression of STAT1 S7272 and STAT5 S726 phosphorylation, known targets of CDK8. Based on these results, we hypothesized that CDK8 inhibition would augment apoptosis induced by gilteritinib in HS5 conditioned media. Using SEL120, a novel CDK8 inhibitor already in early phase AML clinical trials, we performed an 8 x 8 dose matrix drug synergy analysis of gilteritinib and SEL120 in multiple FLT3-mutant AML cell lines using Bliss independence modeling. We found that SEL120 was synergistic with gilteritinib in inducing apoptosis in MOLM-14 and MV411 cells in HS5 conditioned media (Bliss synergy scores of 2.44 and 11.45 with most synergistic area scores of 10.91 and 26.85, respectively). We also found combinatorial activity against MOLM-14 cells harboring secondary NRAS activating mutations (G12C and Q61K), suggesting the therapeutic combination could potentially overcome cell intrinsic and extrinsic MAPK-activating resistance mechanisms. Lastly, we found that gilteritinib and SEL120 combined to impart greater cytotoxicity than either drug alone in a primary sample (AML #1) from a patient with newly diagnosed AML possessing a FLT3-ITD mutation at high mutant allele ratio.Conclusions: The results and validation of our CRISPRi screen suggest that combined CDK8 and FLT3 inhibition is a novel strategy for augmenting gilteritinib cytotoxicity. Assessment of the activity of the combination in additional primary AML samples and in vivo murine models of AML is planned. Additional candidate targets already described and other Mediator and RNA pol II subunits from our screen are also being further evaluated to precisely define the transcriptional programs that influence FLT3 inhibitor resistance. DisclosuresLogan: Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding; Amgen, Pfizer, AbbVie: Consultancy. Gilbert: Denali Therapeutics: Ended employment in the past 24 months, Other: Spouse/Significant Other's Employment; GSK: Consultancy, Research Funding; AstraZeneca: Research Funding; Chroma Medicine: Consultancy, Other: Co-founder. Smith: Revolutions Medicine: Research Funding; AbbVie: Research Funding; Daiichi Sankyo: Consultancy; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Exosomal lncAY927529 enhances prostate cancer cell proliferation and invasion through regulating bone microenvironment

ABSTRACT Exosomes mediate the interaction between cancer cells and their microenvironment, and play a key role in tumor development. Although exosomes can package lncRNAs to mediate extracellular communication, the role of exosomal lncRNA AY927529 in prostate cancer (PCa) remains unclear. Exosomes were extracted from normal human prostatic epithelial cell lines (BPH-1 and RWPE-1) and PCa cell lines (VCaP and LNCaP, DU145, PC3) by ultrahigh speed centrifugation. Results of Western blot indicated that Alix, HSC70 and TSGl01 protein levels were upregulated in exosomes derived from PCa cells. LncAY927529 level was upregulated in PCa cells and exosomes derived from PCa patient serum and human PCa cells. CCK-8, Transwell and Flow cytometry assays demonstrated that bone marrow stromal cell line (ST2) conditioned medium (ST2-CM), treated with exosomes derived from PCa cells with high lncAY927529 level, promoted proliferation and invasion of PC3 and DU145 cells, and inhibited cell apoptosis. RT-qPCR assay indicated that lncAY927529 level was downregulated in PC3 and DU145 cells, exosomes derived from PCa cells (PCa-Exo) and ST2-CM treated with PCa-Exo with low expression of lncAY927529, and overexpression of lncAY927529 had the opposite results. In addition, Western blot assay showed that the autophagy related protein LC3II level was increased in ST2 cells treated with exosomes derived from DU145 cells with high expression of lncAY927529, and LC3I protein level was decreased. CXCL14 acted as a RNA-binding protein of lncAY927529, and exosome-mediated lncAY927529 positively regulated CXCL14 levels in ST2 cells. In general, exosome-mediated lncAY927529 could promote PCa cell proliferation and invasion by regulating bone microenvironment, suggesting that exosomal lncAY927529 may be a potential molecular diagnostic marker of PCa.

Protease-activated receptor-2 promotes osteogenesis in skeletal mesenchymal stem cells at the expense of adipogenesis: Involvement of interleukin-6

Bone marrow mesenchymal stem cells (MSCs) give rise to osteoblasts and adipocytes, with an inverse relationship between the two. The MSCs from protease-activated receptor-2 knockout (PAR2 KO) mice have a reduced capacity to generate osteoblasts. Here we describe the observation that PAR2 KO osteoblastic cultures generate more adipocytes than wildtype (WT) cultures. Osteoblasts from PAR2 KO mice expressed lower levels of osteoblastic genes (Runx2, Col1a1 and Bglap), and higher levels of the adipocytic gene Pparg than WT osteoblasts. Bone marrow stromal cells from PAR2 KO mice generated fewer osteoblastic colonies (assessed by staining for alkaline phosphatase activity and mineral deposition) and more adipocytic (Oil Red-O positive) colonies than cultures from WT mice. Similarly, cultures of the bone marrow stromal cell line (Kusa 4b10) in which PAR2 was knocked down (F2rl1 KD), were less osteoblastic and more adipocytic than vector control cells. Putative regulators of PAR2-mediated osteogenesis and suppression of adipogenesis were identified in an RNA-sequencing (RNA-seq) investigation; these include C1qtnf3, Gpr35, Grem1, Snorc and Tcea3, which were more highly expressed, and Cnr1, Enpep, Hmgn5, Il6 and Ramp3 which were expressed at lower levels, in control than in F2rl1 KD cells. Interleukin-6 (IL-6) levels were higher in medium harvested from F2rl1 KD cells than from control cells, and a neutralising anti-IL-6 antibody reduced the number of adipocytes in F2rl1 KD cultures to that of control cultures. Thus, PAR2 appears to be a mediator of the reciprocal relationship between osteogenesis and adipogenesis, with IL-6 having a regulatory role in these PAR2-mediated effects.

MicroRNA-107 promotes apoptosis of acute myelocytic leukemia cells by targeting RAD51.

IntroductionThis study aimed to investigate the role of microRNA (miRNA) that affects acute myelocytic leukemia (AML) and its potential molecular mechanism by constructing a miRNA–mRNA interaction network using bioinformatics methods.Material and methodsMicroRNA expression data of AML were retrieved from Gene Expression Omnibus (GEO) and analyzed by microarray analysis. Expression levels of miR-107 and RAD51 mRNA were detected by quantitative real time polymerase chain reaction (qRT-PCR). Protein expression of RAD51, pro-apoptotic protein Bax, apoptosis related protein CytC and anti-apoptotic protein Bcl-2 were determined by Western blot. The rate of cell apoptosis was detected by Annexin-V/PI. The predicted targeting relationship between miR-107 and the 3′UTR of RAD51 was first predicted by the online application TargetScan and then verified by dual-luciferase assay.ResultsAcute myelocytic leukemia-associated genes (n = 197) and miRNAs (n = 1701) were retrieved from the database, the interaction network of miRNA-mRNA was constructed and the core position was occupied by RAD51. miR-107 exhibited a regulatory effect on RAD51 in which the mRNA and protein expression of RAD51 were both significantly inhibited by miR-107 mimics in vitro. Additionally, down-regulated expression of miR107 as well as up-regulated expression of RAD51 were detected not only in the plasma of AML patients compared to healthy volunteers, but also in AML cell lines compared to the normal bone marrow stromal cell line. Further study found that increased expression of miR-107 and the consequent down-regulation of RAD51 could aggravate the apoptosis of AML cells in vitro.ConclusionsOur present results showed that the crucial role of RAD51 and miR-107 in the apoptosis of AML cells, i.e., miR-107 promotes the apoptosis of AML cells through down-regulating the expression of RAD51.