Development of a Model for Evaluating the Interaction Between Human Pre-B Acute Lymphoblastic Leukemic Cells and the Bone Marrow Stromal Cell Microenvironment

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3817-3828 ◽  
Author(s):  
Nisha Shah ◽  
LeAnn Oseth ◽  
Tucker W. LeBien
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Cell Precursor ◽  
Survival And Growth ◽  
Allelic Deletion

Clonal expansion of B-cell precursor acute lymphoblastic leukemia (ALL) is potentially regulated by survival, growth, and death signals transduced by the bone marrow (BM) microenvironment. Using a human BM stromal cell culture that supports the growth of normal human B-cell precursors, we established a pre-B ALL cell line designated BLIN-2. BLIN-2 has a clonal rearrangement of the Ig heavy chain locus, a dic(9;20) chromosomal abnormality, and a bi-allelic deletion of thep16INK4a and p19ARF genes. The most interesting feature of BLIN-2 is an absolute dependence on adherent human BM stromal cells for sustained survival and growth. BLIN-2 cultured in the absence of BM stromal cells undergo apoptosis, and direct contact with viable BM stromal cells is essential for optimal growth. BLIN-2 cells also grow on vascular cell adhesion molecule-1 (VCAM-1)–negative human skin fibroblasts, making it unlikely that a very late antigen-4 (VLA-4)/VCAM-1 interaction is required for BLIN-2 growth. Western blot analysis of BLIN-2 cells cultured in the presence or absence of BM stromal cells demonstrates that contact of BLIN-2 with BM stromal cells induces hyperphosphorylation of Rb. In contrast, the pre-B ALL cell line BLIN-1, which has a bi-allelic deletion of p16INK4ap19ARF but does not require BM stromal cells for growth, does not undergo Rb phosphorylation after BM stromal cell contact. The BLIN-2 cell line will facilitate identification of ligand/receptor interactions at the B-cell precursor/BM stromal cell interface and may provide new insight into microenvironmental regulation of leukemic cell survival and growth.

Development of a Model for Evaluating the Interaction Between Human Pre-B Acute Lymphoblastic Leukemic Cells and the Bone Marrow Stromal Cell Microenvironment

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3817-3828 ◽  
Author(s):  
Nisha Shah ◽  
LeAnn Oseth ◽  
Tucker W. LeBien
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Cell Precursor ◽  
Survival And Growth ◽  
Allelic Deletion

Abstract Clonal expansion of B-cell precursor acute lymphoblastic leukemia (ALL) is potentially regulated by survival, growth, and death signals transduced by the bone marrow (BM) microenvironment. Using a human BM stromal cell culture that supports the growth of normal human B-cell precursors, we established a pre-B ALL cell line designated BLIN-2. BLIN-2 has a clonal rearrangement of the Ig heavy chain locus, a dic(9;20) chromosomal abnormality, and a bi-allelic deletion of thep16INK4a and p19ARF genes. The most interesting feature of BLIN-2 is an absolute dependence on adherent human BM stromal cells for sustained survival and growth. BLIN-2 cultured in the absence of BM stromal cells undergo apoptosis, and direct contact with viable BM stromal cells is essential for optimal growth. BLIN-2 cells also grow on vascular cell adhesion molecule-1 (VCAM-1)–negative human skin fibroblasts, making it unlikely that a very late antigen-4 (VLA-4)/VCAM-1 interaction is required for BLIN-2 growth. Western blot analysis of BLIN-2 cells cultured in the presence or absence of BM stromal cells demonstrates that contact of BLIN-2 with BM stromal cells induces hyperphosphorylation of Rb. In contrast, the pre-B ALL cell line BLIN-1, which has a bi-allelic deletion of p16INK4ap19ARF but does not require BM stromal cells for growth, does not undergo Rb phosphorylation after BM stromal cell contact. The BLIN-2 cell line will facilitate identification of ligand/receptor interactions at the B-cell precursor/BM stromal cell interface and may provide new insight into microenvironmental regulation of leukemic cell survival and growth.

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2328-2328
Author(s):  
Katja C. Weisel ◽  
Ying Gao ◽  
Jae-Hung Shieh ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stromal Cell Line ◽  
Mes Cells

Abstract The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

scholarly journals DNMT3A Low-Expression is Correlated to Poor Prognosis in Childhood B Cell Precursor Acute Lymphoblastic Leukemia and Confers Resistance to Daunorubicin on Leukemic Cells

2021 ◽  
Author(s):  
Weijing Li ◽  
Shuguang Liu ◽  
Chanjuan Wang ◽  
Lei Cui ◽  
Xiaoxi Zhao ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Line ◽  
Poor Prognosis ◽  
Lymphoblastic Leukemia ◽  
Prognostic Significance ◽  
Leukemic Cells ◽  
Cell Precursor ◽  
Knock Out ◽  
Low Expression

Abstract Background Little is known about DNMT3A expression and its prognostic significance in childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL). MethodsWe determined DNMT3A mRNA expression in 102 children with BCP-ALL. Correlations with relapse-free survival (RFS) and common clinical characteristics were analyzed. DNMT3A was stably knocked out by CRISPR/Cas9 gene editing technology in 697 cell line. Cell proliferation activity after treated with daunorubicin was determined by CCK8 assay in DNMT3A KO 697 cell line.Results DNMT3A expression in BCP-ALL patients who were in CCR was higher than in those who got relapse (P=0.0111). Receiver operating characteristic curve showed prognostic significance of DNMT3A expression (P=0.003). Low expression of DNMT3A (<0.197) was significantly correlated with poor RFS (P<0.001) in children with BCP-ALL. Knock-out of DNMT3A in 697 cell line significantly increased IC50 of daunorubicin (P=0.0057), indicating elevated resistance to daunorubicin. ConclusionsLow expression of DNMT3A associates with poor prognosis in children with BCP-ALL. Knock-out of DNMT3A confers resistance to daunorubicin on leukemic cells.

Evaluation of FOXP1 gene expression in pediatric B-cell precursor acute lymphoblastic leukemia patients at remission induction therapy

2020 ◽  
Author(s):  
Gholamhossein Tamaddon ◽  
Mehran Bahraini ◽  
Alieh Fazeli
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
Remission Induction ◽  
Control Group ◽  
Cell Precursor ◽  
New Diagnosis

Background: Transcription factors (TFs) play a key role in the development, therapy, and relapse of B-cell malignancies, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Given the essential function of Forkhead box protein P1 (FOXP1) transcription factor in the early development of B-cells, this study was designed to evaluate FOXP1 gene expression levels in pediatric BCP-ALL patients and NALM6 cell-line. Materials and Methods: This case-control study was done on the NALM6 cell-line and bone marrow specimens of 23 pediatric BCP-ALL patients (median age: 7.5 years; range: 2.0 – 15.0 years) at different clinical stages including new diagnosis, 15th day after the treatment, and relapse. Also, 10 healthy children were included as the control group. FOXP1 gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The correlation analysis was performed between the FOXP1 gene expression and patients’ demographic and laboratory characteristics. Results: The results showed that FOXP1 gene expression was significantly downregulated in the NALM-6 cell-line (median=0.05, P<0.001) and patients at new diagnosis (median=0.06, p<0.0001), and relapse (median=0.001, p<0.0001) phases, compared to the control group (median=0.08). FOXP1 gene expression on the 15th day of the treatment was significantly higher than its level at the new diagnosis stage (p<0.001). Moreover, FOXP1 gene was significantly downregulated in the relapse phase compared to the new diagnosis. Patients whose number of bone marrow blasts on the 15th day of the treatment was below 5% had higher FOXP1 gene expression at the diagnosis phase (Spearman’s correlation, P<0.05, r=-0.485) and higher ratio of diagnosis/day 15 (p<0.001, Mann-Whitney U test). Conclusions: FOXP1 levels could be a potential biomarker of therapy response in remission induction therapy for pediatric BCP-ALL patients.

Clonal Profile Analysis of Leukemic Progenitors and Diagnosis Blast Cells in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4879-4879
Author(s):  
Marinella Veltroni ◽  
Maddalena Paganin ◽  
Chiara Frasson ◽  
Giulia Fabbri ◽  
Antonio Marzollo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Gene Rearrangements ◽  
Healthy Children ◽  
Cell Precursor ◽  
Clonal Rearrangement

Abstract Recent studies suggest that the majority of malignant cells found in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) arise from a rare population of leukemic progenitors. Little information is available on the presence of clonal rearrangements in cells at the stage of early precursor. To address this issue we analyzed clonality profile of early leukemic precursors sorted by flow-cytometry. Leukemic cells were obtained from bone marrow samples collected at diagnosis from 6 patients with childhood BCP-ALL. Furthermore, bone marrow cells were collected from 3 healthy children who were harvested for bone marrow donation. Three subpopulations of leukemic cells were investigated: total unsorted blasts, the sorted CD34+/CD38−/CD19+, and the sorted CD34+/CD38−/CD19− cells. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements were screened by polymerase chain reaction (PCR) in the sorted populations and in the bulk leukemic cells in order to identify molecular markers of clonal evolution. Sequence analysis was then performed on the N-region. Overall, a total of 38 different Ig/TCR gene rearrangements were identified in the 3 cell populations under study (total blasts, CD34+/CD38−/CD19+, and CD34+/CD38−/CD19−). Of them, 13 (34%) were found in the three populations; 12 (31%) were found in two of the three populations: 7 in total blasts and CD19+ subset, 3 in total blasts and CD19−, 2 in CD19− and CD19+; finally, 13 were found only in one subpopulation: 4 in total blast cell, 5 in CD19+, 4 in CD19−. In all the six patients studied, BCP-ALL progenitors CD34+/CD38−/CD19− and CD19+ and the bulk tumor blasts shared at least one Ig/TCR gene clonal rearrangement with the same N-region. In 5 out of 6 patients at least one rearrangement detected in the BCP-ALL progenitors was undetectable in total blasts. Conversely, in 3 patients the clonal rearrangement observed in the bulk leukemic cells was not identified in any of the two sorted ALL precursor populations. Clonal rearrangement was never detected in the samples from healthy bone marrow donors. Our findings confirm that clonal rearrangement may be detected at the stage of early B-lineage precursor CD34+/CD38−/CD19−, suggesting that leukemic transformation may occur at this stage or even before in BCP-ALL. We plan to extend this observation by repopulating studies in NOD/SCID mice.

C/EBPβ expressed By Bone Marrow Mesenchymal Stromal Cells Is Indispensable For Precursor B-Cell Lymphopoiesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1213-1213
Author(s):  
Satoshi Yoshioka ◽  
Yasuo Miura ◽  
Hisayuki Yao ◽  
Masaki Iwasa ◽  
Atsushi Sato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Cells ◽  
Transcriptional Factor ◽  
Precursor B Cells

Abstract B-cell lymphopoiesis is critically dependent on the bone marrow microenvironment. Early B-cell lymphopoiesis is regulated by direct interaction with bone marrow stromal cells (BM-MSCs) and by soluble factors produced by BM-MSCs. The roles of transcription factors expressed by hematopoietic cells that involve early B-cell lymphopoiesis have been well investigated; those include PU.1, Ikaros, E2A, early B-cell factor, and paired box protein 5. By contrast, transcriptional factors expressed by BM-MSCs that are important for early B-cell lymphopoiesis remain unknown. We show that CCAAT-enhancer binding protein (C/EBP) β expressed by BM-MSCs contributes to the early B-cell lymphopoiesis. In addition, the proliferation of precursor B-cell acute lymphoblastic leukemia (B-ALL) cells is also associated with BM-MSCs, in which C/EBPβ is one of the regulatory transcription factors. When c-kit+ Sca-1+ lineage- (KSL) HSCs from Wild-type (WT) mice were co-cultured with C/EBPβ-deficient BM-MSCs in the presence of stem cell factor (SCF), Flt3-L, and interleukin (IL)-7 (Figure A), the generation of hematopoietic cells from KSL cells was significantly lower than when they were co-cultured with WT BM-MSCs. In addition, the generation of B220+ B-cells from KSL cells was also significantly lower when they were co-cultured with C/EBPβ-deficient BM-MSCs than when co-cultured with WT BM-MSCs. Detailed analysis of the generated B-cell subsets showed that differentiation of KSL cells into precursor B-cells was reduced and differentiation from pre-pro-B-cells to pro-B-cells/pre-BI cells was suppressed when cells were co-cultured with C/EBPβ-deficient BM-MSCs (Figure B). Therefore, C/EBPβ was an indispensable transcriptional factor expressed by BM-MSCs for supporting the differentiation of HSCs into precursor B-cells. We next examined the expression of B-cell lymphopoiesis-associated molecules in C/EBPβ-deficient BM-MSCs by quantitative real-time PCR analysis. Levels of IL-7 and SCF mRNA tended to be lower in C/EBPβ-deficient BM-MSCs than in WT BM-MSCs, although the difference was not statistically significant in our analysis. Levels of CXCL12/SDF-1 and Flt3-L mRNA were significantly lower in C/EBPβ-deficient BM-MSCs than in WT BM-MSCs. In addition, the protein concentration of CXCL12/SDF-1 was significantly lower in the culture supernatant of C/EBPβ-deficient BM-MSCs than that of WT BM-MSCs. The concentration of CXCL12/SDF-1 in the supernatant of BM-MSC co-cultures strongly correlated with the number of B220+ B-cells that differentiated from KSL cells. Thus, the impaired differentiation of HSCs into B-cells is associated, at least in part, with reduced production of CXCL12/SDF-1 by C/EBPβ-deficient BM-MSCs. Precursor B-ALL is a hematological disease characterized by malignant transformation of precursor B-cells. We examined whether C/EBPβ expressed by BM-MSCs has effects on the precursor B-ALL cells. When the precursor B-ALL cell line BaF3/Bcr-Abl cells was co-cultured with WT BM-MSCs, the number of BaF3/Bcr-Abl cells was significantly increased than when they were cultured alone. Interestingly, when the precursor B-ALL cell line BaF3/Bcr-Abl cells was co-cultured with C/EBPβ-deficient BM-MSCs, the number of BaF3/Bcr-Abl cells was reduced to the level similar to when they were cultured alone. This was true in co-cultures with BM-MSCs that were pharmacologically treated to down-regulate the C/EBPβ expression. As well as the correlation of SDF-1 concentration in co-cultures with precursor B-cell differentiation, the proliferation of BaF3/Bcr-Abl cells was associated with the correlation of SDF-1 concentration in co-cultures. In conclusion, this work demonstrates that C/EBPβ expressed by BM-MSCs is a critical transcriptional factor for both the differentiation of physiological precursor B-cells and pathological proliferation of leukemic precursor B-cells. Disclosures: No relevant conflicts of interest to declare.

B-Cell Precursor Acute Lymphoblastic Leukemia Cells Create a Self-Reinforcing Niche Independent of the CXCR4/CXCL12 Axis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1297-1297
Author(s):  
Bob de Rooij ◽  
Roel Polak ◽  
Rob Pieters ◽  
Monique L. Den Boer
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Cell Precursor ◽  
Cxcr4 Antagonist

Abstract Background Acute lymphoblastic leukemia (ALL) cells create a leukemic niche that protects malignant cells from the effects of cytostatic agents and immune cells by altering their bone marrow microenvironment. This malignant process can be counteracted by impairing the homing of leukemic cells towards the bone marrow. Hematopoietic cells express the chemokine receptor CXCR4 and migrate towards its ligand CXCL12, which is actively produced by MSCs in the bone marrow. Therefore clinical trials have been initiated using the CXCR4 antagonist AMD3100 (Plerixafor) during leukemia treatment. However, these trials, as well as priming of AML in more than 4000 patients using a CXCR4 dependent mechanism, have not resulted in improved overall survival rates. This suggests that CXCR4 inhibition is not sufficient to disrupt leukemic niches. Objectives In this study we investigated how leukemic cells regulate the chemoattractive properties of their microenvironment. Results Here we show, using an ex vivo niche model with primary MSCs, that B-cell precursor ALL (BCP-ALL) cells affect their healthy microenvironment without altering CXCL12 secretion. Using a transwell migration assay we studied the chemoattractive properties and chemokine secretion patterns of several cell types and co-cultures. We confirmed that BCP-ALL cells migrate towards a CXCL12 gradient produced by primary MSCs (11-fold more migrated cells compared to background, p < 0.001). Inhibition of CXCR4 by AMD3100 reduced migration towards MSCs by 80% (p < 0.01). BCP-ALL cells migrated even more towards co-cultures of BCP-ALL cells and primary MSCs (24-fold more migrated cells compared to background, p < 0.001). Strikingly, this ex vivo leukemic niche did not produce higher levels of CXCL12 compared to MSC mono-cultures. Moreover, the induced migration towards MSC-ALL co-cultures could not be inhibited by AMD3100 treatment, indicating that BCP-ALL cells enhance the chemoattractive properties of their microenvironment in a CXCL12-independent manner. In contrast to BCP-ALL cells, the migration of CD34+ progenitor cells towards co-cultures of BCP-ALL cells and MSCs was significantly reduced (0.8-fold compared to migration towards MSCs, p < 0.05). Similar results were observed when we studied the migratory behavior of MSCs. MSCs actively migrated towards BCP-ALL cells (1.7 fold compared to background, p < 0.001), while migration of MSCs was significantly reduced towards MSC-ALL co-cultures (0.4-fold compared to migration towards BCP-ALL, p < 0.001). To find candidate factors influencing this process, we quantified the secreted levels of 64 cytokines in co-cultures of patient-derived BCP-ALL cells and MSCs. We observed leukemia-driven cytokine secretion patterns that were not influenced by the source of primary MSCs. In contrast to unaltered levels of CXCL12, we observed significant inductions of MCP-1/CCL2 and MDC/CCL22 (CCR4-ligands), IL8 and GRO-1 (CXCR1/2-ligands) and IP10/CXCL10 (CXCR3-ligands). Conclusion Our data indicate that leukemic cells alter the chemoattractive properties of their microenvironment, resulting in the secretion of multiple chemokines into the leukemic niche. This leukemic niche is highly potent in attracting BCP-ALL cells and repels the influx of healthy hematopoietic cells and MSCs using a CXCL12-independent mechanism. Furthermore, our results identify candidate factors that might be valuable future therapeutic targets. Disclosures No relevant conflicts of interest to declare.

scholarly journals B-Cell Precursor Acute Lymphoblastic Leukemia Instructs Mesenchymal Stromal Cells to Alter Interferon-Related Gene Expression and Cyto/Chemokine Secretion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Mandy W.E. Smeets ◽  
Femke Stalpers ◽  
Myrthe M.P. Vermeeren ◽  
Alex Q. Hoogkamer ◽  
Stefan Nierkens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Stromal Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Sequencing Data ◽  
Cell Precursor ◽  
And Migration ◽  
Pro Inflammatory Cytokines

Introduction: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells that are present in the bone marrow microenvironment are able to hijack the normal hematopoietic stem cell niches to create a leukemic niche. The importance of this microenvironment for leukemic cells is demonstrated by the protection that the niche provides against chemotherapeutic agents. Patient-derived mesenchymal stromal cells (MSCs) mimic this protective effect in vitro. Unraveling the mechanism of protection is important to provide potential novel options for therapeutic intervention. Therefore, our research is focused on the interaction between BCP-ALL cells and MSCs. We aimed to determine gene expression changes in BCP-ALL cells and MSCs after co-culture compared to mono-culture, and to investigate which cyto- and chemokines are differentially secreted upon contact between BCP-ALL cells and MSCs. Methods: We performed co-cultures of primary MSCs and BCP-ALL cells, and mono-cultures of MSCs or BCP-ALL cells for 40 hours to determine gene expression changes. Viable cells were sorted by FACS and RNA was isolated. Total-RNA sequencing data (Illumina) were analyzed using R. Supernatant was saved to determine cyto/chemokine profiles by Luminex technology, and to investigate the effect of these cyto/chemokines on the survival and migration of the leukemic cells. Moreover, migration experiments using transwells (3.0µm pore size) were performed to determine the level of BCP-ALL migration towards cyto/chemokines of interest. Results: RNA sequencing data from 15 independent co-culture experiments revealed that interferon (IFN)-related genes, such as IFI6, MX1, IFI27, and OAS1, were 2.5 to 3.1-fold upregulated in the MSCs after co-culture with BCP-ALL compared to MSC mono-culture. The type of upregulated pro-inflammatory genes and amount of upregulation varied between BCP-ALL patients. However, the observed changes were always similar when an ALL case was co-cultured with different MSC samples. This suggests that the observed changes are induced by the leukemic cells and that leukemic cells manipulate MSCs. Survival benefit (0.3 - 37.9%) was observed in BCP-ALL cells after co-culture with MSCs compared to BCP-ALL mono-culture. Moreover, pro-inflammatory cytokines, and several migration-related chemokines such as CCL2, CXCL8, and CXCL10/IP-10 were upregulated in 5 out of 15 co-cultures compared to the sum of the separate mono-cultures of BCP-ALL and MSCs. A gradient of CXCL10/IP-10 in transwell experiments showed that this chemokine did not enhance the migration of primary BCP-ALL cells, suggesting that the ALL-induced secretion of this chemokine serves a different role in BCP-ALL. The role of CXCL10/IP-10 and other cyto/chemokines in immune regulation at the time of overt leukemia is part of ongoing studies. Conclusion: Our data show that IFN-related genes, pro-inflammatory cytokines and migration-related chemokines become upregulated in bone marrow stromal cells upon exposure to BCP-ALL. These induced changes may be important for BCP-ALL cell survival, affecting the mobility of other immune cells, and/or ensure that leukemic cells remain in close contact with MSCs. We postulate that interference with these affected genes and cyto/chemokines may disrupt the direct contact between leukemic cells and their niche, and may provide an alternative way to eliminate leukemic cells more efficaciously. Functional studies addressing this concept are currently being executed and the results will be presented during the meeting. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential roles of stromal cells, interleukin-7, and kit-ligand in the regulation of B lymphopoiesis

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1185-1192 ◽  
Author(s):  
LG Billips ◽  
D Petitte ◽  
K Dorshkind ◽  
R Narayanan ◽  
CP Chiu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Kit Ligand ◽  
Interleukin 7 ◽  
Stromal Cell Line ◽  
Differentiation And Proliferation

Abstract Newly formed B lymphocytes are a population of rapidly renewed cells in the bone marrow of mammals and their steady state production presumably depends on a cascade of regulatory cells and cytokines. Although considerable information has been forthcoming about the role of interleukin-7 (IL-7) in potentiating pre-B-cell proliferation, few studies have addressed the possibility that multiple cytokines are involved in the progression of early events in cellular differentiation and proliferation in this hematopoietic lineage. Our laboratory previously described pre-B-cell differentiation mediated by the bone marrow stromal cell line S17. In this study, we further delineate the role of stromal cells in differentiation and proliferation of pre-B cells. These experiments show that the stromal cell line S17 potentiates the proliferative effect of IL-7 on B-lineage cells and that this S17-derived potentiator can be replaced with recombinant kit- ligand (KL). Our results further show that pre-B-cell formation from B220-, Ig- progenitor cells and expression of mu heavy chain of immunoglobulin is uniquely dependent on the presence of S17 stromal cells and cannot be reproduced with IL-7, KL, or costimulation with both IL-7 and KL. These data contribute to a rapidly evolving model of stromal cell regulation of B-cell production in the marrow and suggest unique roles for IL-7, KL, and as yet uncharacterized stromal cell- derived lymphokines in this process.

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