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F. Portale et al.
though the cure rate exceeds 80% in children, BCP-ALL is the leading cause of cancer-related death in children and young adults.2 In spite of the notable improvements in dis- ease management, the emergence of chemoresistance decreases the probability that therapy will be successful, and leads to relapse in more than 20% of treated patients.3 BCP-ALL cells critically depend on interactions with the bone marrow (BM) microenvironment, which provides essential regulatory cues for proliferation, survival and drug resistance, and such interactions contribute to treat- ment failure and disease relapse.4 In particular, mesenchy- mal stromal cells (MSCs) have been recognized as an essential supportive element of the leukemic hematopoi- etic microenvironment because of their ability to define exclusive BM niches that sustain leukemic cells to the detriment of normal hematopoiesis and resist chemother- apy.5 In this complex network, it has been shown that chemokines could contribute to BCP-ALL development by driving the migration of leukemic cells toward protective BM niches, as well as by providing anti-apoptotic signals.6
ActivinA is a pleiotropic cytokine that belongs to the TGF-b superfamily. It has a broad tissue distribution, being involved in multiple physiological and pathological processes, including inflammation, metabolism, immune response, and endocrine function. Recent studies have demonstrated that ActivinA is an important regulator of carcinogenesis. Indeed, it can directly modulate cancer cell proliferation and migration. It can also enhance tumor progression by regulating the tumor microenvironment.7 ActivinA sends signals through its transmembrane serine/threonine kinase receptors. It binds to type II Activin receptors (ACVR2A or ACVR2B), causing recruit- ment, phosphorylation and activation of type I Activin receptors (ALK2 or ALK4). ActivinA signaling is inhibited by Inhibins, through competitive binding for Activin receptors, and by Follistatin (FST) and Follistatin like-3 (FSTL3), which act as trap molecules.8 The Activin recep- tor II ligand trap ACE-011 is currently under investigation in a Phase II clinical trial on multiple myeloma.9
The aim of the current study was to explore the role of ActivinA in the leukemic BM niche, with a particular focus on its supportive role for BCP-ALL cells to the detri- ment of healthy hematopoiesis.
Methods
Patients’ and healthy donors’ samples
Bone marrow plasma samples were collected from 125 BCP- ALL patients at diagnosis and from 56 healthy donors (HDs). Primary BCP-ALL cells were isolated at diagnosis from 22 BM aspirates and used for in vitro assays. Details of the study cohort are shown in the Online Supplementary Appendix. The study was approved by the Institutional Review Board (AIEOP-BFM ALL 2009 protocol; EudraCT-2007-004270-43).
Culture of B-cell precursor-acute lymphoblastic leukemia cell lines
The leukemic cell lines 697, NALM-6, RS4;11, SUP-B15 and REH were cultured as described in the Online Supplementary Appendix.
Isolation of bone marrow-mesenchymal stromal cells
Bone marrow-mesenchymal stromal cells (BM-MSCs) from 15
BCP-ALL patients (ALL-MSCs) and 15 age-matched HDs (HD- MSCs) (Online Supplementary Table S1) were cultured as described in the Online Supplementary Appendix.
Cord blood-CD34+ and bone marrow-CD34+ cell isolation
CD34+ cells were isolated from cord blood (CB) units and HD BM aspirates, as described in the Online Supplementary Appendix.
Co-culture of primary leukemic cells with bone marrow-mesenchymal stromal cells
Bone marrow-mesenchymal stromal cells were co-cultured with primary leukemic cells at an MSC:leukemia ratio of 1:30, either in the presence or in the absence of 0.4 mm Transwell inserts (Costar Transwell® Permable Supports, Corning Inc., MA, USA) in DMEM 2% FBS. After 72 hours (h), supernatants were collected and cryopreserved at -80°C for further analyses.
ELISA assay for quantification of ActivinA, CXCL12 and pro-inflammatory cytokines
The levels of ActivinA, pro-inflammatory cytokines (IL-1b, IL-6 and TNF-α) and CXCL12 were assessed in BM plasma samples and culture supernatants using commercially available ELISA kits (R&D Systems, USA), according to the manufacturer’s instruc- tions.
Quantitative RT-PCR
qRT-PCR was performed using LightCycler® 480 (Roche, Basel, Switzerland), as reported in the Online Supplementary Appendix.
Gene expression profile analysis
Gene expression profile analysis of 697 cells treated or not with ActivinA (50 ng/mL) for 6 h and 24 h was evaluated by GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix Inc., Santa Clara, CA, USA). Details of the procedure are described in the Online Supplementary Appendix.
Time-lapse microscopy
Leukemic cells were seeded in Gelatin B-coated wells of an 8- well chamber slide (Ibidi, Martinsried, Germany). Cell tracks were recorded as described in the Online Supplementary Appendix.
Chemotaxis assays
Both leukemic and healthy CD34+ cells, pretreated or not with ActivinA (24 h stimulation), were tested for chemotaxis in Transwell-based assays (Costar Transwell® Permable Supports, Corning, MA, USA). Selected experiments were performed using the ALK4 blocker SB431542 (SigmaAldrich, St Louis, MO, USA). Details are described in the Online Supplementary Appendix.
Invasion assays
The invasive capacity of leukemic cells was evaluated using Matrigel-coated Transwells, as described in the Online Supplementary Appendix.
Activin receptor analyses
697 leukemic cell line and primary blasts were analyzed for ALK4, ACVR2A and ACVR2B expression by flow cytometry and qRT-PCR and for ALK2 expression by western blot, as described in the Online Supplementary Appendix.
CXCR4 and CXCR7 staining
Expression of CXCR4 and CXCR7 was analyzed by flow cytometry in 697 cells, pretreated or not with ActivinA (Online Supplementary Appendix).
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haematologica | 2019; 104(3)