General information of the patients
Newly diagnosed patients with DLBCL in our hospital from January 2016 to January 2018 were included in this study. Exclusion criteria: age > 18 years, other complicated malignancies, viral infections with HIV, cytomegalovirus, or hepatitis B, leukemia, aplastic anemia, and autoimmune diseases. DLBCL patients were divided into two groups according to the presence of anemia: DLBCL without anemia and DLBCL with anemia; each group consisted of thirty patients. Statistical analysis showed that there was no significant difference of the age, sex, weight, and duration of disease between the above two groups. Meanwhile, contemporaneous healthy volunteers coming to our hospital for physical examination were selected to be the control group.
Lymphoma tissues in each group were collected for paraffin sectioning. A portion of the tissues was stained with hematoxylin & eosin dyes (H&E) (ZsBio, Beijing, China) to examine the pathological changes, and others were subjected to immunohistochemical staining. Briefly, 3% H2O2 and sheep serum blocking solution (ZsBio, Beijing, China) were applied to the dewaxed paraffin sections which then were washed three times with PBS. Subsequently, these sections were incubated with primary antibodies including Ki-67, CD31, P-AMPK and CPT1A (antibodies were purchased from Abcam, USA) overnight at 4 ˚C and washed three times with PBS. Afterwards, Biotin-labeled secondary antibody and streptavidin working solution (ZsBio, Beijing, China) were sequentially added. Finally, sections were counterstained with hematoxylin (ZsBio, Beijing, China) for 10 min after colored staining agents were developed and observed under a microscope.
Identification and sorting of CD45+ EPCs
Peripheral blood from patients of each group was collected and slowly added into the upper layer of human lymphocyte separation medium (TBD Company, Tianjin, China). Samples were centrifuged at 2000 g for 20 min and white membrane median layer was aspirated. After washing three times with PBS, each sample was incubated with flow cytometric antibodies for 15 minutes at room temperature in the dark, including CD45-PE, CD71-APC, and TER119-PerCP/Cy5.5, respectively (antibodies were purchased from BD, USA). After washing three times with PBS, CD45+ EPC proportions were analyzed on a flow cytometer. In the meantime, CD45+ EPCs were sorted out by flow cytometry and then cultured in RPMI1640 medium (Hyclone, USA) containing 10% fetal bovine serum (Hyclone, USA) for subsequent in vitro experiments.
Transwell chamber was used for co-culture. Lymphocyte was in the lower chamber and CD45 + EPC was in the upper chamber. This experiment consisted of three groups: control group (lymphocytes only), CD45+ EPCs group (co-culture of lymphocytes and CD45+ EPCs), and ROS inhibition group (co-culture of lymphocytes and CD45+ EPCs in combination with ROS inhibitor apocynin). After co-culturing for 48 hours, cells were harvested in each group and incubated with flow cytometric antibodies including CD3-PE and CD8-APC (BD Biosciences, USA) for 15 min at room temperature in the dark. Cells were washed three times with PBS. Cell proportions in each group were quantified by flow cytometry.
The DLBCL cell line U2932 was cultured in RPMI1640 medium containing 10% fetal calf serum. DLBCL cells in exponential growth phase were cultured adherently for 48 hours, and divided into four groups: control group (adding the same volume of PBS), lymphocyte group (adding peripheral blood lymphocytes), CD45+ EPCs group (adding lymphocytes in CD45+ EPCs group), and ROS inhibitior group (adding lymphocytes in CD45+ EPCs group and ROS inhibitor apocynin). After co-culturing for 48 hours, DLBCL cells were collected and stained with Annexin V-FITC and PI (BD Biosciences, USA). The proportion of apoptotic cells was detected by flow cytometry.
U2932 cells were seeded into 96-well plates with 3,000 in each well. Cells were treated with 0.1 mmol/L oleic acid (Sigma, USA) for 48 h under physiological condition (atmospheric O2, 5% CO2, 37 ˚C) or hypoxic condition (3% O2, 5% CO2, 37 ˚C). Thereafter, cells were incubated for another 4 h in the presence of 5 g/L MTT solution (Sigma, USA). At last, the supernatant was removed and 150 mL of DMSO (Sigma, USA) was added into each well. The absorbance of each well was measured using a microtiter plate reader following shaking for 10 min.
RNA extraction and real-time quantitative PCR
After co-culturing for 48 hours, each group of U2932 cells was collected. Total RNAs in the cells were extracted. Reverse transcription of total RNAs was performed using the First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The expressions of FFA-transporter-related genes (Cd36, Fabp1, Slc27a2, Slc27a5, and Fabp4) , ACC and CPT1A were assessed by western blotting.
In order to develop the adipose depots, 1 ´ 106 U2932 cells in 30 mL PBS were slowly transplanted into inguinal white adipose tissue (iWAT) of C57BL/6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd) . There were four groups: lymphoma group (transplantation of U2932 cells), anti-VEGF group (transplantation of U2932 cells followed by tail-vein injection of anti-VEGF), CD45+ EPC group (combined transplantation of U2932 cells and CD45+ EPCs followed by tail-vein injection of anti-VEGF) and ROS inhibition group (combined transplantation of U2932 cells and CD45+ EPCs followed by injection of anti-VEGF and ROS inhibitor apocynin). Two weeks after implantation, mice were anesthetized with 0.1% sodium pentobarbital and euthanized by cervical dislocation. Tumor tissues were obtained and fixed in 4% paraformaldehyde. After paraffin sectioning, a part of the tissues was stained with H&E, and some tissues were subjected to immunohistochemistry to detect the expressions of Ki-67, CD31, Caspase 3, P-AMPK, and CPT1A.
Statistical analyses were carried out using SPSS 19.0. Data values were presented as mean ± standard deviations. Multiple data comparisons were analyzed via ANOVA and Bonferroni post hoc test. Correlation analyses were performed via Spearman 's rank correlation. A P < 0.05 is considered statistically significant.