2.42 Mimicking the Tumor Microenvironment: Three Different Co-culture Systems Induce a Similar Phenotype but Different Proliferative Signals in Primary Chronic Lymphocytic Leukemia Cells

Department of Haematology, King's College London, London, UK.
British Journal of Haematology (Impact Factor: 4.71). 06/2012; 158(5):589-99. DOI: 10.1111/j.1365-2141.2012.09191.x
Source: PubMed


Interactions in the tumour microenvironment can promote chronic lymphocytic leukaemia (CLL) cell survival, proliferation and drug resistance. A detailed comparison of three co-culture systems designed to mimic the CLL lymph node and vascular microenvironments were performed; two were mouse fibroblast cell lines transfected with human CD40LG or CD31 and the third was a human microvascular endothelial cell line, HMEC-1. All three co-culture systems markedly enhanced CLL cell survival and induced a consistent change in CLL cell phenotype, characterized by increased expression of CD38, CD69, CD44 and ITGA4 (CD49d); this phenotype was absent following co-culture on untransfected mouse fibroblasts. In contrast to HMEC-1 cells, the CD40LG and CD31-expressing fibroblasts also induced ZAP70 expression and marked CLL cell proliferation as evidenced by carboxyfluorescein succinimidyl ester labelling and increased Ki-67 expression. Taken together, our data show that co-culture on different stroma induced a remarkably similar activation phenotype in CLL cells but only the CD40LG and CD31-expressing fibroblasts increased ZAP70 expression and CLL cell proliferation, indicating that ZAP70 may play a critical role in this process. This comparative study reveals a number of striking similarities between the co-culture systems tested but also highlights important differences that should be considered when selecting which system to use for in-vitro investigations.

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    • "Adhesion to microvascular endothelial cells promotes CLL survival, activation and drug resistance (Badoux et al., 2011; Cols et al., 2012; Hamilton et al., 2012; Maffei et al., 2012, 2014). CLL cells bind to β1 and β2 integrins (Maffei et al., 2012) and to BAFF and APRIL on the surface of microvascular endothelial cells (Cols et al., 2012). "
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    ABSTRACT: Chronic Lymphocytic Leukemia (CLL) is a prototype microenvironment-dependent B-cell malignancy, in which the neoplastic B cells co-evolve together with a supportive tissue microenvironment, which promotes leukemia cell survival, growth, and drug-resistance. Chemo-immunotherapy is an established treatment modality for CLL patients, resulting in high rates of responses and improved survival, especially in low-risk CLL. New, alternative treatments target B-cell receptor (BCR) signaling and the Chemokine (C-X-C motif) Receptor 4 (CXCR4)- Chemokine (C-X-C motif) Ligand 12 (CXCL12) axis, which are key pathways of CLL-microenvironment cross talk. The remarkable clinical efficacy of inhibitors targeting the BCR-associated kinases bruton’s tyrosine kinase (BTK) and phosphoinositide 3-kinase delta (PI3Kδ) challenge established therapeutic paradigms and corroborate the central role of BCR signaling in CLL pathogenesis. In this review, we discuss the cellular and molecular components of the CLL microenvironment. We also describe the emerging therapeutic options for CLL patients, with a focus on inhibitors of CXCR4-CXCL12 and BCR signaling.
    Full-text · Article · Dec 2014 · Pharmacology [?] Therapeutics
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    • "We and others recently demonstrated that the contact with endothelial cells rescues CLL from spontaneous and drug-induced apoptosis, induces activation and proliferation and generates a peculiar gene expression profile on leukemic cells [6], [8], [10], [32]. ET-1 was observed among the most up-regulated genes in CLL after co-culture and is also secreted at high amount by activated endothelium. "
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    ABSTRACT: The endothelin axis, comprising endothelins (ET-1, ET-2 and ET-3) and their receptors (ETAR and ETBR), has emerged as relevant player in tumor growth and metastasis. Here, we investigated the involvement of ET-1/ETAR axis in chronic lymphocytic leukemia (CLL). CLL cells expressed higher levels of ET-1 and ETA receptor as compared to normal B cells. ET-1 peptide stimulated phosphoinositide-3-kinase and mitogen-activated protein kinase signaling pathways, improved survival and promoted proliferation of leukemic cells throughout ETAR triggering. Moreover, the blockade of ETAR by the selective antagonist BQ-123 inhibited the survival advantage acquired by CLL cells in contact with endothelial layers. We also found that blocking ETAR via BQ-123 interferes with ERK phosphorylation and CLL pro-survival effect mediated by B-cell receptor (BCR) activation. The pro-apoptotic effect of phosphoinositide-3-kinase δ inhibitor idelalisib and mitogen-activated protein kinase inhibitor PD98059 was decreased by the addition of ET-1 peptide. Then, ET-1 also reduced the cytotoxic effect of fludarabine on CLL cells cultured alone or co-cultured on endothelial layers. ETAR blockade by BQ-123 inhibited the ET-1-mediated protection against drug-induced apoptosis. Lastly, higher plasma levels of big ET-1 were detected in patients (n = 151) with unfavourable prognostic factors and shorter time to first treatment. In conclusion, our data describe for the first time a role of ET-1/ETAR signaling in CLL pathobiology. ET-1 mediates survival, drug-resistance, and growth signals in CLL cells that can be blocked by ETAR inhibition.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "Subsequently, 1×106/ml cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS), penicillin (50U/ml), streptomycin (50μg/ml) and recombinant human IL-4 (R and D Systems, Abingdon, UK) (5ng/ml). Normal Mouse embryonic fibroblast L-cells, either non-transfected (NTL) or L-cells expressing CD40 ligand (CD40L)[37] were used where indicated as feeder layers. "
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    ABSTRACT: Cdk9 is a key elongation factor for RNA transcription and functions by phosphorylating the C-terminal domain of RNA polymerase II. Here we present direct evidence that cdk9 is important for cancer cell survival and describe the characterization of the potent cdk9 inhibitor CDKI-73 in primary human leukemia cells. CDKI-73 induced caspase-dependent apoptosis that was preceded by dephosphorylation of cdk9 and serine 2 of RNA polymerase II. CDKI-73 was more potent than the pan-cdk inhibitor flavopiridol and showed >200-fold selectivity against primary leukemia cells when compared with normal CD34+ cells. Furthermore, CDKI-73 was equipotent in poor prognostic sub-groups of leukemia patients and showed cytotoxic synergy with the nucleoside analog fludarabine. The Mechanism of synergy was associated with CDKI-73-mediated transcriptional inhibition of MCL1 and XIAP that was maintained when used in combination with fludarabine. Our data present a strong rationale for the development of cdk9 inhibitors such as CDKI-73 as anticancer therapeutics.
    Full-text · Article · Dec 2013 · Oncotarget
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