scholarly journals Mesenchymal Stem Cells With Cancer-Associated Fibroblast-Like Phenotype Stimulate SDF-1/CXCR4 Axis to Enhance the Growth and Invasion of B-Cell Acute Lymphoblastic Leukemia Cells Through Cell-to-Cell Communication

2021 ◽  
Vol 9 ◽  
Author(s):  
Chengyun Pan ◽  
Qin Fang ◽  
Ping Liu ◽  
Dan Ma ◽  
Shuyun Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Migration And Invasion ◽  
Integrin Β1 ◽  
Cancer Associated Fibroblast ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Cxcr4 Axis

Background: Bone marrow mesenchymal stem cells (BM-MSCs) are the stromal cells in the leukemia microenvironment, and can obtain cancer-associated fibroblast (CAF)-like phenotype under certain conditions to further promote leukemia progression. However, the mechanism of MSCs with CAF-like phenotype interacting with leukemia cells in B-cell acute lymphoblastic leukemia (B-ALL) and promoting the progression of B-ALL remains unclear.Methods: Mesenchymal stem cells with CAF-like phenotype were obtained by treating MSCs with recombinant human transforming growth factor-β (rhTGF-β), hereafter referred to as TGF-β conditioned MSCs. In vivo mouse model experiments, in vitro transwell chamber experiments, three-dimensional (3D) cell culture models, lentiviral transfection and other experimental methods were used to investigate the possible mechanism of the interaction between TGF-β conditioned MSCs and leukemia cells in promoting the growth, migration and invasion of B-ALL cells.Results: Compared with untreated MSCs, TGF-β conditioned MSCs significantly promoted the growth and proliferation of leukemia cells in mice, and increased the expression of CXCR4 in tumor tissues. In vitro cell experiments, TGF-β conditioned MSCs obviously promoted the migration and invasion of Nalm-6/RS4;11 cells, which were effectively blocked by the CXCR4 inhibitor AMD3100, thereby inhibiting the secretion of MMP-9 in TGF-β conditioned MSCs and inhibiting the activation of the PI3K/AKT signaling pathway in leukemia cells. Further, findings were made that the interaction between TGF-β conditioned MSCs and leukemia cells were mediated by the interaction between the integrin receptor α5β1 on the surface of leukemia cells and the increased expression of fibronectin on TGF-β conditioned MSCs. AMD3100 could weaken such effect by reducing the expression of integrin α5β1 on leukemia cells. Further regulation of integrin β1 could effectively interfere with the interaction between TGF-β conditioned MSCs and leukemia cells.Conclusion: Mesenchymal stem cells with CAF-like phenotype could be a key factor in promoting the growth and invasion of B-ALL cells, and the SDF-1/CXCR4 axis might be a significant factor in mediating the communication of MSCs with CAF-like phenotype and leukemia cells. To prevent the progression of B-ALL cells, blocking the SDF-1/CXCR4 axis with AMD3100 or targeting integrin β1 might be a potential therapeutic strategy.

scholarly journals Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Marc El Beaino ◽  
Jiayong Liu ◽  
Amanda R. Wasylishen ◽  
Rasoul Pourebrahim ◽  
Agata Migut ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Soft Agar ◽  
Tumor Formation ◽  
Cellular Migration ◽  
Migration And Invasion ◽  
Sequencing Studies ◽  
Genetic Perturbations ◽  
Lentiviral Infection

Abstract Background Ewing sarcoma is a malignancy of primitive cells, possibly of mesenchymal origin. It is probable that genetic perturbations other than EWS-FLI1 cooperate with it to produce the tumor. Sequencing studies identified STAG2 mutations in approximately 15% of cases in humans. In the present study, we hypothesize that loss of Stag2 cooperates with EWS-FLI1 in generating sarcomas derived from murine mesenchymal stem cells (MSCs). Methods Mice bearing an inducible EWS-FLI1 transgene were crossed to p53−/− mice in pure C57/Bl6 background. MSCs were derived from the bone marrow of the mice. EWS-FLI1 induction and Stag2 knockdown were achieved in vitro by adenovirus-Cre and shRNA-bearing pGIPZ lentiviral infection, respectively. The cells were then treated with ionizing radiation to 10 Gy. Anchorage independent growth in vitro was assessed by soft agar assays. Cellular migration and invasion were evaluated by transwell assays. Cells were injected with Matrigel intramuscularly into C57/Bl6 mice to test for tumor formation. Results Primary murine MSCs with the genotype EWS-FLI1 p53−/− were resistant to transformation and did not form tumors in syngeneic mice without irradiation. Stag2 inhibition increased the efficiency and speed of sarcoma formation significantly in irradiated EWS-FLI1 p53−/− MSCs. The efficiency of tumor formation was 91% for cells in mice injected with Stag2-repressed cells and 22% for mice receiving cells without Stag2 inhibition (p < .001). Stag2 knockdown reduced survival of mice in Kaplan-Meier analysis (p < .001). It also increased MSC migration and invasion in vitro but did not affect proliferation rate or aneuploidy. Conclusion Loss of Stag2 has a synergistic effect with EWS-FLI1 in the production of sarcomas from murine MSCs, but the mechanism may not relate to increased proliferation or chromosomal instability. Primary murine MSCs are resistant to transformation, and the combination of p53 null mutation, EWS-FLI1, and Stag2 inhibition does not confer immediate conversion of MSCs to sarcomas. Irradiation is necessary in this model, suggesting that perturbations of other genes beside Stag2 and p53 are likely to be essential in the development of EWS-FLI1-driven sarcomas from MSCs.

Mesenchymal stem cells require integrin β1 for directed migration induced by osteopontin in vitro

2010 ◽  
Vol 47 (3) ◽  
pp. 241-250 ◽  
Author(s):  
Chengyu Zou ◽  
Guanbin Song ◽  
Qing Luo ◽  
Lin Yuan ◽  
Li Yang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Integrin Β1 ◽  
Directed Migration

scholarly journals Exosomal transfer of miR-769-5p promotes osteosarcoma proliferation and metastasis by targeting DUSP16

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wanshun Liu ◽  
Binyu Wang ◽  
Ao Duan ◽  
Kai Shen ◽  
Qi Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Lines ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Clinical Specimens ◽  
Proliferation And Metastasis

Abstract Background Osteosarcoma (OS) is a malignant tumor originating from mesenchymal stem cells, and has an extremely high fatality rate and ability to metastasize. Although mounting evidence suggests that miR-769-5p is strongly associated with the malignant progression and poor prognosis of various tumors, the exact role of miR-769-5p in OS is still unclear. Therefore, this study aimed to explore the relationship between miR-769-5p and the malignant progression of OS, and its underlying mechanism of action. Methods miR-769-5p expression was analyzed in GSE28423 from the GEO database and measured in OS clinical specimens and cell lines. The effects of miR-769-5p on OS proliferation, migration and invasion were measured both in vivo and in vitro. In addition, bioinformatics analyses and luciferase reporter assays were used to explore the target genes of miR-769-5p. Rescue experiments were also conducted. Moreover, a co-culture model was used to test the cell interaction between bone mesenchymal stem cells (BMSC) and OS cells. Results We found that miR-769-5p is highly expressed in OS clinical specimens and cell lines. In vivo and in vitro experiments also showed that miR-769-5p significantly promoted the proliferation, migration and invasion of OS cells. Dual-specific phosphatase 16 (DUSP16) was negatively associated with miR-769-5p expression in OS cells and tissue samples and was validated as the downstream target by luciferase reporter assay and western blotting. Rescue experiments showed that DUSP16 reverses the effect of miR-769-5p on OS cells by negatively regulating the JNK/p38 MAPK signaling pathway. Additionally, the results of the co-culture of BMSCs and OS cells confirmed that miR-769-5p was transferred from BMSCs to OS cells through exosomes. Conclusions In summary, this study demonstrates for the first time that BMSC-derived exosomal miR-769-5p promotes OS proliferation and metastasis by targeting DUSP16 and activating the JNK/p38 MAPK signaling pathway, which could provide rationale for a new therapeutic strategy for OS.

Inhibitory Effects of Homoharringtonine on Leukemic Stem Cells and BCR-ABL Induced Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2912-2912 ◽  
Author(s):  
Yaoyu Chen ◽  
Yiguo Hu ◽  
Shawnya Michaels ◽  
Dennis Brown ◽  
Shaoguang Li
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Leukemic Stem Cells ◽  
Lymphoid Leukemia

Abstract The Abl tyrosine kinase inhibitors (TKIs) imatinib mesylate (IM) and dasatinib, targeting BCR-ABL for the treatment of Philadelphia-positive (Ph+) leukemia including chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL), have produced impressive results in terms of therapeutic outcome and safety for patients. However, clinical resistance to these TKIs likely at the level of leukemic stem cell negates curative results in Ph+ leukemia. At present, an anti-stem cell strategy has not been developed for treating these leukemia patients. Homoharringtonine (HHT) (omacetaxine mepesuccinate - USAN/INN designation) has shown significant clinical activity in CML in combination with IM or alone for patients failing IM. However, little is known about whether HHT has an inhibitory effect on leukemic stem cells. The purpose of this study is to determine whether HHT inhibits BCR-ABL-expressing leukemic stem cells (Lin-c-Kit+Sca-1+) that we identified previously (Hu et al. Proc Natl Acad Sci USA 103(45):16870–16875, 2007) and to evaluate therapeutic effects of HHT on CML and B-ALL in mice. We find that in our in vitro stem cell assay, greater than 90% of leukemic stem cells were killed after being treating with HHT (12.5, 25, and 50 nM) for 6 days, and in contrast, greater than 75% or 92% of leukemic stem cells survived the treatment with dasatinib (100 nM) or imatinib (2 mM). We next treated CML mice with HHT (0.5 mg/kg, i.p., once a day). 4 days after the treatment, FACS analysis detected only 2% GFP+Gr–1+ myeloid leukemia cells in peripheral blood of HHT -treated CML mice and in contrast, 41% GFP+Gr–1+ myeloid leukemia cells in placebo-treated mice. We also treated mice with BCR-ABL induced B-ALL with HHT, and found that only 0.78% GFP+B220+ lymphoid leukemia cells were detected in peripheral blood compared to 34% GFP+B220+ lymphoid leukemia cells in placebo-treated mice. Furthermore, HHT significantly inhibited in vitro proliferation of K562 and B-lymphoid leukemic cells isolated from mice with B-ALL induced by BCR-ABL wild type and BCR-ABL-T315I resistant to both imatinib and dasatinib. In sum, HHT has an inhibitory activity against CML stem cells, and is highly effective in treating CML and B-ALL induced by BCR-ABL in mice.

Class I/II Histone Deacetylase Inhibitors Are a Potential Therapeutic For Notch-1 Driven T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1435-1435
Author(s):  
Michaela Waibel ◽  
Edwin D Hawkins ◽  
Kelly M Ramsbottom ◽  
Benjamin P Martin ◽  
Ricky W Johnstone
Keyword(s):  
Stem Cells ◽  
Acute Lymphoblastic Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Notch 1 ◽  
Cell Counts ◽  
Blood Cell Counts ◽  
Cell Acute Lymphoblastic Leukemia ◽  
White Blood Cell Counts

Abstract Introduction Although T cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease, mutations resulting in activation of the Notch-1 signalling pathway are present in over 50% of patients, thus defining Notch signaling as a central player in T-ALL disease regulation. Furthermore, despite improvement in remission rates following conventional chemotherapeutics, the prognosis for T-ALL remains poor due to disease relapse, which is often refractory to the initial therapies. Inhibitors against the γ-secretase complex, which is part of intracellular Notch-1 (ICN1) signalling, have so far shown limited efficacy and are associated with severe toxicity. Therefore, alternative approaches to treat Notch-1-driven T-ALL are required. The gene expression signature of Notch-1-mutated T-ALLs has revealed downstream activation of numerous pathways, including Myc, PI3K-AKT-mTOR and NFκB, indicating potential sensitivity to small molecule histone deacetylase inhibitors (HDACi). Therefore, the aim of this study was to use a traceable pre-clinical mouse model of Notch-1 driven T-ALL to investigate the potential of HDACi as therapy. Methods Retroviral transduction of mouse hematopoietic stem cells with constructs encoding ICN, followed by transplantation into irradiated recipient mice, is an established model of T-ALL resulting from Notch-1 activating mutations, and closely mimics human disease. For the present study, we transduced foetal liver stem cells from day E14.5 embryos with constructs expressing either ICN1-GFP, or GFP only. Cohorts of recipient mice developed leukaemia as described previously, characterized by splenomegaly, lymphadenopathy, elevated peripheral white blood cell counts, and the presence of double positive CD4+CD8+GFP+ blasts in peripheral blood, with a small fraction of recipients presenting with single positive CD4+GFP+ or CD8+GFP+ blasts. T-ALL blasts were isolated from this primary leukemia colony and transplanted into sub-lethally irradiated recipient mice. Using this model, cohorts of mice rapidly developed onset of leukemia with significant engraftment of T-ALL blasts in bone marrow, spleen, thymus, liver and detectable blasts in peripheral blood 15 days post transplant. Results GFP+ T-ALL blasts were isolated from lymph nodes and spleen and cultured in vitro in the presence of αCD3 and interleukin 2 to induce robust proliferation. Treatment of these T-ALL blasts with the pan-HDACi Panobinostat (LBH-589) induced cell death and inhibited proliferation of remaining viable blasts in a dose-dependent manner. These results illustrate that LBH-589 is a potent inhibitor of survival and proliferation of Notch driven T-ALL at low nM concentrations in vitro. Furthermore, treatment of cohorts of mice transplanted with ICN1-T-ALL with LBH-589 significantly prevented expansion of disease in vivo as determined by white blood cell counts. Additionally, we monitored leukemic cells infiltration in secondary lymphatic tissues using non-invasive GFP imaging. Whilst high GFP readings were observed in vehicle treated groups, we observed very little signal in LBH-589 treated mice confirming prevention of disease expansion. Of most importance, the effect of LBH-589 on progression of disease was reflected in a significant increase in survival of treated cohorts compared to controls. Conclusions The considerably high relapse rates of T-ALL patients demands the development of novel therapies for disease that is refractory to initial therapies. Our results indicate that LBH-589 has strong potential for development of therapies for Notch driven T-ALL and may be a useful addition to current therapies, either during initial treatment or after relapse of chemoresistant T-ALL. Moreover, we have preliminary data suggesting that LBH-589 has the ability to target the predominant oncogenic “Myc signature” associated with constitutive Notch expression. We are currently performing gene and protein expression analysis to exactly determine the molecular effects of HDACi on ICN and associated downstream effector signalling. Disclosures: Johnstone: Novartis: Research Funding.

scholarly journals Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1322-1322
Author(s):  
Manabu Kusakabe ◽  
Ann Chong Sun ◽  
Kateryna Tyshchenko ◽  
Rachel Wong ◽  
Aastha Nanda ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Research Funding ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4295-4295
Author(s):  
Jae-Hung Shieh ◽  
Tsann-Long Su ◽  
Jason Shieh ◽  
Malcolm A.S. Moore
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Culture System ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4295 Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

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