Efficient RNA interference in patients' acute lymphoblastic leukemia cells amplified as xenografts in mice
© Höfig et al; licensee BioMed Central Ltd. 2012
Received: 7 November 2011
Accepted: 26 March 2012
Published: 26 March 2012
Signaling studies in cell lines are hampered by non-physiological alterations obtained in vitro. Physiologic primary tumor cells from patients with leukemia require passaging through immune-compromised mice for amplification. The aim was to enable molecular work in patients' ALL cells by establishing siRNA transfection into cells amplified in mice.
We established delivering siRNA into these cells without affecting cell viability. Knockdown of single or multiple genes reduced constitutive or induced protein expression accompanied by marked signaling alterations.
Our novel technique allows using patient-derived tumor cells instead of cell lines for signaling studies in leukemia.
Characterization of intracellular signaling mechanisms is crucial for the understanding of tumor development and for the design of novel strategies in anti-tumor therapy. For practical reasons, signaling studies are mainly performed in cell lines established from human tumors decades ago and are prone to non-representative mutations [1, 2]. Example given, more than 50% of ALL cell lines inherit mutations of p53, while less than 5% of primary pediatric samples at initial diagnosis do [3–5].
To overcome these limitations, several groups had successfully transfected primary leukemia cells. Best results were obtained in samples from adult patients with chronic leukemias [6–8]. In contrast, in acute leukemias, rapid onset of therapy impedes repetitive sampling and reliable results of molecular experiments in primary cells. In pediatric leukemias, small sample volumes generally disable molecular work on these primary cells. For the transfection of childhood ALL samples by nucleofection, so far transgene expression was studied in detail rendering varying results between 1% and 62.3% .
Acute leukemia samples can be amplified in severely immuno-compromised mice. The xenograft mouse model of acute leukemia has been characterized to enable frequent engraftment with little genetic and phenotypic alterations upon passaging through mice . The regular detection of CD surface markers revealed stable phenotypes over several passages .
Here, we describe a new method which will routinely allow repetitive and reliable molecular signaling studies on patient-derived childhood ALL cells amplified in NOD/SCID mice. The aim was to establish a suitable transfection technique to perform knockdown experiments in patient-derived acute lymphoblastic leukemia (ALL) cells, instead of ALL cell lines.
Patient characteristics of leukemia samples xenografted in NOD/SCID mice
Optimization of the transfection of patient-derived childhood ALL cells
The siRNA delivery by nucleofection leads to stable and reproducible results for a cohort of xenografted ALL samples.
Efficient knockdown of target genes in patient-derived ALL cells
Beyond single targets, successful double knockdown was obtained by targeting two proteins and yielded a simultaneously diminished expression of both proteins (Figure 2E and Additional file 1: Figure S1B). In selected samples with high viability over prolonged periods of time in cell culture (> 70% at 120 h), stability of knockdown was detected for up to 120 h (Figure 2F and data not shown).
Knockdown of target genes altered signaling in patient-derived ALL cells
Knockdown of target genes alters protein regulation and function in patient-derived ALL cells
So far, the efficiency of our technique was evaluated for basal protein expression. In a next step, we evaluated the effect of siRNA-mediated knockdown on protein regulation in patient-derived ALL cells.
On a broader level, these data prove that efficient siRNA delivery in patient-derived childhood ALL cells induces significant functional alterations which can be achieved either upon knockdown of constitutively expressed genes or by inhibition of protein regulation. Taken together, we have successfully established a method allowing reliable RNAi-based signaling studies in patient-derived pediatric ALL cells.
Applicability of the described technique to primary ALL cells
The data obtained in this primary ALL sample suggest that the described technique is well applicable not only to xenografted primary ALL cells but although to at least some primary ALL samples.
We present a novel method allowing repetitive molecular signaling studies in pediatric ALL cells from individual children. The focus of our method are studies of signaling pathways which are not adequately modeled in ALL cell lines and require patient-derived tumor cells, such as the p53 network. The combination of amplification of these cells in immune-incompetent mice and the presented transfection technique allows molecular signaling studies (i) in patient-derived pediatric samples, where small volumes are notoriously restrictive; (ii) in cells which resemble primary tumor cells substantially better than established cell lines do [11, 12]; (iii) repetitively on highly viable cells allowing reliable results. To return to the discussed difficulty of p53 signaling studies in leukemia cell lines, the presented technique will enable routine investigation of primary childhood ALL cells that have been performed so far mainly in childhood ALL cell lines with restricted relevance to the clinical setting due to inherited mutations i.e. in CEM and JURKAT leukemia cells [13, 16].
Upon isolation from spleen or bone marrow of mice, patient-derived cells can be used freshly for repetitive experiments and show better viability compared to primary samples due to more favorable handling and shipping. To our experience, cell viability is sufficient for transfection experiments in the majority of patient-derived samples freshly isolated from mice. The data obtained in the primary childhood ALL sample suggest that the described technique is as well applicable to freshly isolated primary ALL samples. It remains to be tested, whether our method generally facilitates transfection of freshly isolated, primary ALL cells, e.g. from adult patients and other types of primary leukemia cells which were not studied so far using this technique.
TRAIL was obtained from PeproTech (Hamburg, Germany), all further reagents were obtained from Sigma-Aldrich (St. Louis, MO). For Western Blot analysis, the following antibodies were used: anti NFκBp65 and anti p53 from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti PUMA from Cell Signaling Technology Inc. (Danvers, MA); anti GAPDH from Thermo Fisher (Waltham, MA); anti NOXA from Calbiochem (San Diego, CA); anti Caspase-8 from Alexis Corp. (Lausen, Switzerland).
For flow cytometric analysis of CD surface marker stability, the following antibodies were used: anti CD4-PE from DAKO (Hamburg, Germany), anti CD5-PeCy5.5, anti CD7-APC, anti CD10-APC, anti CD19-PeCy5.5, anti CD22-PE and anti HLA-DR-PeCy5.5 from Life Technologies (Darmstadt, Germany), anti CD19-FITC, anti CD20-FITC, anti CD34-PE and anti CD38-PE from BD Biosciences (San Jose, CA).
Amplification of primary childhood ALL cells in NOD/SCID mice
Informed consent was obtained from all patients and experiments were performed according to the declaration of Helsinki as approved in written form by the ethical committee of the medical faculty of the Ludwig Maximilians University Munich (LMU 068-08) and the Children's Hospital of the TU Munich (TU 2115/08). Primary ALL blasts were obtained from children treated at the Ludwig Maximilians University Children's Hospital or the children's hospital of the TU Munich. Tumor cells were isolated from blood or bone marrow samples.
Animal work was approved by the Regierung von Oberbayern (55.2-1-54-2531-2-07). The xenograft NOD/SCID mouse model was performed as described by others [17–19]. Fresh primary childhood ALL cells were isolated by Ficoll gradient centrifugation for 30 min at 500 g from peripheral blood or bone marrow aspirates that had been obtained from leftovers of clinical routine sampling followed by two washing steps in PBS before resuspension in cell culture medium. 10 million ALL cells were injected into 6-8 weeks old NOD/SCID mice via the tail vain. Engraftment was monitored by flow cytometry and measurement of human cells in peripheral blood. Engrafted human ALL cells were isolated from spleens of diseased mice by pressing through a cell strainer (BD Biosciences) and Ficoll gradient centrifugation. Cells were separated and simultaneously injected into next generation of mice and subjected to in vitro experiments. Regular detection of CD surface markers revealed stable phenotypes of the samples over all passages .
Transfection and stimulation of patient-derived ALL cells
For nucleofection (AmaxaNucleofector, Lonza, Basel, Switzerland), 5 million cells were used per reaction. Patient-derived ALL cells were resuspended in 100 μl pre-warmed buffer from the nucleofector kit for human B/T-cells plus 5 μlsiRNA oligonucleotides (20 μM). The following siRNAs were used: silencer validated siRNA against p53 (5'-GGGUUAGUUUACAAUCAGC-3', Ambion, Austin, TX), siRNA against NOXA (5'-GUCGAGUGUGCUACUCAACU-3'); siRNA against PUMA (5'-UCUCAUCAUGGGACUCCUG-3'; siRNA against Caspase-8 (5'-GCUCUUCC GAAUUAAUAGATT-3', second siRNA against Caspase 8 (siCasp8_2; 5'-GCUCUUCCGAAUUAAUAGATT-3') and siRNA against NFκBp65 (all from MWG Biotech, Ebersberg, Germany) and All Star negative control siRNA conjugated with Alexa-Fluor-488 or Alexa-Fluor-647 (Qiagen, Hilden, Germany). For double transfections, siRNA against lamin conjugated to Alexa-Fluor-488 (analyzed in FITC channel) and negative control siRNA conjugated with Alexa-Fluor-647 (APC-channel) was analyzed using a LSR II flow cytometer (BD Biosciences) and FlowJo (TreeStar, Ashland, OR) software version 8.3. After transfection, cells were incubated 5 min at room temperature in the nucleofection cuvette, then transferred to 5 ml pre-warmed RPMI medium supplemented with 20% FCS, 1% penicillin/streptomycin, 1% gentamycin, 6 μl/ml mixture of insulin, transferrin and selenium (Invitrogen, Carlsbad, CA), 1 mM sodium pyruvate and 50 μM 1-thioglycerole (Sigma-Aldrich, St. Louis, MO). After transfection with fluorochrome-conjugated negative control siRNA, cells were washed twice in PBS prior to flow cytometric determination of transfection efficiency. Stimulation experiments were started 6 or 48 h after transfection.
Western blot and measurement of apoptosis
Western Blot analysis was performed out of total cellular lysates using the following total cell lysis buffer: 20 mMTris-HCl (pH 7.5), 150 mMNaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4 supplemented with 10 μg/ml protease inhibitor cocktail set I (Calbiochem). Lysates were separated by SDS-page gel electrophoresis followed by transfer onto a nitrocellulose membrane and detection of the primary antibody by a HRP-conjugated secondary antibody. Apoptosis was measured by forward side scatter analysis and precision of this technique confirmed by Annexin V and propidium iodide double staining according to the manufacturers instructions using FACscan or LSR II flow cytometry and Cell Quest Pro (BD Biosciences) software version 3.2.1.
Specific apoptosis was calculated as [(apoptosis of stimulated cells at end point minus apoptosis of unstimulated cells at end point) divided by (100 minus apoptosis of unstimulated cells at end point) times 100]. Whenever indicated, paired t-test was performed to detect statistically significant differences out of at least three independent experiments with two technical replicates. Statistical significance was accepted with p-values < 0.05.
The skilled technical work of L. Mura and U. Borgmeier is kindly appreciated. We thank the animal facility for caring for the mice and K. Schneider and V. Groiß for isolating patient-derived ALL cells. This work was supported by Else Kroener Fresenius Stiftung P45/05//A19/05/F0 (to HE and IJ).
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