3. Results
3.1 Evaluation of Stem Cell Marker Expression in Canine B-Cell Lymphoma Cell
and Primary Samples
Wen Liu, Feyza Selçuk, Barbara C. Rütgen, Mohammed Moulay, Saskia Wil-lenbrock, Sabine E. Hammer, Katharina Anna Sterenczak, Christian Junghanss, Marion Hewicker-Trautwein, Ingo Nolte, Hugo Murua Escobar
Anticancer Reserch, 2015, 35: 2805-2816
Contribution to this study:
Wen Liu performed all the experiments, data analyses and wrote partially the man-uscript.
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Abstract:
Background: Canine lymphoma has lately been focused on as a model of human non-Hodgkin’s lymphoma due to its spontaneous occurrence and similar biological behavior. Cells with stem cell-like characteristics are believed to play a key role in therapeutic failure. Thus, an initial characterization and the possibility of specific de-tection of such cells could bear significant value. Materials and Methods: Expressions of 12 stem cell markers were analyzed in two canine B-cell lymphoma cell lines, their generated spheres, and in primary lymphoma samples by quantitative real-time pol-ymerase chain reaction and partially by flow cytometry and immunocytochemistry.
Results: Expression of maternal embryonic leucine zipper kinase (Melk) was signifi-cant higher in CLBL-1, CLBL-1M and the primary B-cell lymphoma samples com-pared to non-neoplastic lymph nodes. Spheres displayed higher expression of v-myc myelocytomatosis viral oncogene homolog (Myc) and lower expression of Cd44 compared to original cell lines and primary B-cell lymphoma samples. Conclusion:
The results suggest a potential interesting role of Melk in canine B-cell lymphoma.
Furthermore, the up-regulation of Myc in serum-free generated spheres offers inter-esting possibilities for functional assays characterizing the specific generated sub-population.
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Supplementary data
Material and methods
1. Conventional reverse-transcription PCR analyses
The stem cell marker expression patterns of CLBL-1, CLBL-1M and 14 primary samples were examined initially by conventional PCR in order to obtain a qualitative expression status. Following respective amplification, the generated PCR products were separated by gel electrophoresis and extracted by QIAquick Gel Extraction Kit (Qiagen). Subsequently, the fragments were cloned into the pGEM-T easy Vector System (Promega) and transformed into thermocompetent E. coli DH5α cells. Speci-ficity of the cloned DNA fragments was verified by sequencing (GATC Biotech).
2. Doxorubicin resistant analyses of CLBL-1S and CLBL-1MS
In 96 well plates, 5x104 CLBL-1 and CLBL-1M cells and their generated sphere cells CLBL-1S and CLBL-1MS were plated in 150 μl medium with different concentra-tions of doxorubicin (1.0 - 10 nM). Control cells were cultured in growth medium.
Each concentration was performed in triplicate. Metabolic activities were analysed by using WST-1 reagent (Roche) after 72h treatment with doxorubicin. Absorbance at 450 nm and the reference wavelength at 750 nm were determined by GloMax®-Multi Detection System (Promega GmbH). Experiments were repeated three times inde-pendently. Values are presented as the percentage of metabolic activity normalized with untreated cells.
3. Long-term doxorubicin selection
5.0 × 106 CLBL-1M cells were cultured in 5 ml growth medium containing 2.5 nM of doxorubicin. Cells were counted twice per week and passaged depending on the number of viable cells. Stepwise increase doxorubicin from 2.5 nM to 130 nM in 6 months. The generated CLBL-1M subline was named CLBL-1MDoxoR130.
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total RNA was used in a total volume of 20 µl. The qPCR reactions were performed using the ViiA™ 7 Real-Time PCR System (Life Technologies) and QuantiTect SYBR green qPCR Kit (Qiagen). ß-actin (ACTB) was used as reference gene. The qPCR results were analysed using the delta delta CT (ΔΔCT) method relative to CLBL-1M cells. Three samples of different passages were used. All samples were analysed in triplicates including non-template and non-reverse transcriptase controls for each reaction. Significant differences were calculated using Student’s t-test, where a p-value of less than 0.05 was considered to be statistically significant.
Results
1. Conventional reverse-transcription PCR analyses
The stem cell marker expression in the screened two lymphoma cell lines and 14 primary lymphoma samples revealed positive expression of Cd44, Itga6, Myc, Ddx5 and Melk. Oct4 and Nanog showed a faint expression in the two cell lines. Cd34, Cd133, c-Kit, Klf4 and Sox2 could not be detected by conventional PCR. In all 14 primary lymphoma samples, Nanog expression was not detectable. The primary lym-phoma samples showed mixed PCR positivities for the different genes: Klf4 10/14, Cd34 8/14, Oct4 7/14, c-Kit 2/14, Sox2 2/14 and Cd133 1/12, respectively (Table 2).
2. CLBL-1S and CLBL-1MS cells did not reveal doxorubicin resistance
Doxorubicin resistant assays were performed on CLBL-1S and CLBL-1MS cells after 9 days culture in serum-free medium. CLBL-1S and CLBL-1MS cell did not dis-play a doxorubicin resistant capability. CLBL-1S even showed a slight decrease in the resistance. At the concentration of 1.0 and 2.5 nM, CLBL-1MS revealed a higher resistant to doxorubicin but not significant (Figure 3).
3. Stem cell marker genes expression of doxorubicin resistant cells
Ddx5, Melk and Myc expressions were significantly decreased in CLBL-1MDoxoR130 cells. Expressions of Cd44 and Itga6 remained comparable to CLBL-1M cells. Nanog was the only gene that up-regulated in CLBL-1MDoxoR130 cells (Figure 4).
Table 2. Results of conventional PCR detection.
Sample name
Genes
Cd34 Cd133 c-Kit Cd44 Itga6 Oct4 Nanog Klf4 Sox2 Myc Melk Ddx5
CLBL-1 - - - + + ± ± ± - + + +
CLBL-1M - - - + + ± ± ± - + + +
Lymphoma 8/14 1/14 2/14 all all 7/14 0/14 10/14 2/14 all all all ++: High expression; ±: very weak; -: no expression.
Figure 3. Doxorubicin resistant assay. Metabolic activities were measured by WST-1 assay after 72h treatment with doxorubicin. Values are presented as the percentage of met-abolic activity normalized with untreated cells. CLBL-1 and CLBL-1M are the native cells.
CLBL-1S and CLBL-1MS are serum-free generated sphere cells.
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Figure 4. Stem cell marker genes expression of doxorubicin resistant cells.
Relative real-time PCR were performed to analyze stem cell marker genes expression levels of doxorubicin resistant cells. CLBL-1M cells were used as control. β-Actin was used as ref-erence gene. The Student’s t-test were performed and *p<0.05 was assigned as significantly different.