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ness.1,7 Hence, deciphering the complex biology of inflam- mation in the CLL microenvironment is paramount for the successful "bench-to-bedside" transition of novel thera- peutics that will drastically improve the outcomes of CLL patients.
Recently, the mitogenic cytokine stem cell factor (SCF, KIT ligand, encoded by the KITL gene) was found to be increased in the serum of CLL patients and also expressed by CLL cells infiltrating the bone marrow and lymph nodes.7-9 SCF is a pro-inflammatory glycoprotein that, upon homo-dimerization, binds to the c-kit receptor (KIT gene, CD117), a class III receptor tyrosine kinase that is primarily localized on the cell surface but can also be found in the cytoplasm and the nucleus.10-12 There are two major alternative splice variants of the KITL gene, distin- guished based on the presence or not of a specific exon (exon 6) that encodes a proteolytic cleavage site. The vari- ant of the KITL gene carrying exon 6 encodes a transiently transmembrane isoform that is swiftly released after pro- teolytic cleavage from metalloproteases such as MMP-9 (soluble SCF, KL-1). The variant lacking exon 6 encodes a predominantly transmembrane isoform that can also be processed by metalloproteases albeit with less affinity (membrane SCF, KL-2). The KL-2 isoform elicits a more durable activation of c-kit whereas the KL-1 isoform elicits a more rapid but transient activation due to ensuing recep- tor internalization.13,14 Both isoforms induce cell prolifera- tion and differentiation albeit with distinct efficacy depending on the tissue involved, the available synergistic growth factors and the developmental stage of the c-kit+ cells.12,15 Furthermore, the KL-1 isoform serves as a chemoattractant whereas the KL-2 isoform is involved in cellular adhesion.16,17 Both SCF isoforms increase in inflamed tissues due to reactive oxygen species (ROS) and potentiate the alleviation of excessive oxidative stress, thus functioning as compensatory survival factors.18,19
SCF is considered an adverse prognostic marker in can- cer, correlating with shorter overall and relapse-free sur- vival as well as increased angiogenesis.20 Furthermore, activation of c-kit by SCF binding promotes survival, pro- liferation, stemness, immune evasion and drug resistance of malignant cells while it potentiates their metastatic dis- semination.15,21-23 SCF overproduction by cancer cells leads to a plethora of interactions with surrounding c-kit+ cells which underpins the prevalence of the tumor microenvi- ronment at the expense of the neighboring normal cellular niches.24
There is scant information regarding the role of SCF in the pathobiology of CLL. Realizing this knowledge gap, here we sought to investigate the expression profile of SCF in CLL and also explore how SCF could be modulated by the CLL microenvironment. We report that SCF overex- pression in CLL is associated with adverse prognosis and a shorter time-to-first treatment. Moreover, we provide evi- dence that immune and redox signaling induce SCF expres- sion in CLL cells, which affects mitochondrial dynamics and cell proliferation, highlighting a crucial role for this inflammatory growth factor in CLL cell homeostasis.
Methods
Chronic lymphocytic leukemia patients
Blood samples were collected with informed consent from 75 patients diagnosed with CLL according to the guidelines of the
International Workshop on Chronic Lymphocytic Leukemia/National Cancer Institute (iwCLL/NCI).The patients were either untreated or off therapy for at least 6 months prior to sampling.25 The study was approved by the local Ethics Review Committee of the participating institutions. Demographic, clinical and biological data for the entire cohort of patients are given in Online Supplementary Table S1 and specifically for the ibrutinib cohort in Online Supplementary Table S2.
Cell purification, cultures and stimuli
CD19+ B cells were negatively selected from whole blood and cultured in the presence or absence of specific ligands for certain periods, depending on the assay. A detailed description of the cell culture conditions and stimuli is provided in the Online Supplementary Material.
Quantification of KITLG and KIT mRNA expression
Total cellular RNA was isolated from purified B cells with the Qiagen RNAeasy mini kit (QIAGEN), including a DNAse incuba- tion step. KITLG and KIT mRNA levels were quantified by real- time quantitative polymerase chain reaction (RQ-PCR) (for more
details please see the Online Supplementary Methods).
Western blotting and flow cytometry studies
Protein expression of SCF and hypoxia-inducible factor-1 alpha (HIF-1a) was assessed by western blotting and flow cytometry while cell viability, apoptosis, mitochondrial mass, membrane potential and intracellular expression of Ki-67 were analyzed by flow cytometry. A detailed description of these studies is provided in the Online Supplementary Material and Online Supplementary TableS3.
Detection of soluble stem cell factor in chronic lymphocytic leukemia cell cultures by enzyme-linked immunosorbent assay
Cell-free supernatants of primary CLL cells stimulated with CpG for 24 h or not (control) were harvested and analyzed using the Human Stem Cell Factor ELISA Kit (Abcam) following the manufacturer’s instructions.
Gene silencing by siRNA methodology
Purified CD19+ B cells from MEC1 CLL cells were transfected with 100 nM of FlexiTube siRNA directed against human SCF or 40 nM of AllStars Negative Control siRNA (Qiagen), using the Amaxa Human B Cell Nucleofector Kit (Lonza) as previously reported.26 MEC1 cells were collected and processed for flow cytometry analysis at the indicated time points.
Statistical analysis
The statistical significance of bivariate relationships between variables normally distributed was assessed with the use of the Student t-test. When the underlying distribution was not normal, the Mann–Whitney or Wilcoxon test was applied. For all compar- isons, the level of statistical significance was set at P=0.05. Survival analysis was conducted to assess the impact of SCF on overall sur- vival and time-to-first treatment (TTFT). All statistical analyses
haematologica | 2021; 106(3)
Immunohistochemistry
Aberrant expression of SCF in CLL
Immunohistochemistry was performed on 5 mm thick sections of analyzed samples (for details see Online Supplementary Methods) by using SCF antibody (Abcam). Six lymph node samples, three bone marrow biopsies with CLL infiltration along with biopsy specimens of reactive lymph nodes were also stained with an anti- CD117 antibody (Dako). For immunostaining a Bond MaxTM autostainer (Leica Microsystems, Wetzlar, Germany) was used.
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