br BD cytometric bead array CBA for cytokine analysis
2.10. BD™ cytometric bead array (CBA) for cytokine analysis
Eﬀector T 1174018-99-5 and ovarian cancer cells were co-cultured as de-scribed previously to determine CAR T cell cytotoxicity at an E/T ratio of 10:1 for 10 h. The supernatant was then collected and the level of cytokines in the supernatant was estimated using a BD™ CBA Human Th1/Th2 cytokine kit (Becton Dickinson, Singapore) with a BD Accuri flow cytometer according to the manufacturer’s instructions. Serial di-lutions of IFN-γ, TNF, IL-2, IL-4, IL-5, and IL-10 were prepared as cy-tokine standards. Capture beads were added to each tube with samples, standards, and a negative control. The tubes were incubated for 30 min in dark, the flow cytometer was calibrated using cytometer setup beads, and the assay was performed.
2.11. Enzyme-linked immune-sorbent assay (ELISA) and granzyme B secretion
The levels of uPA and uPAR in the serum samples were determined using an ELISA kit (Yuanye Bio-Technology, Shanghai, China) ac-cording to the manufacturer’s instructions. Eﬀector T cells were co-cultured with each of the four groups of ovarian cancer cells (vector A2780 + CT, vector A2780+ATF-CAR T,uPAR-A2780 + CT, uPAR- Biomedicine & Pharmacotherapy 117 (2019) 109173
2.12. Statistical analysis
Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Data were expressed as mean ± SD. Data were determined by an F-test, normal distribution subsequent p-values were calculated using the two-tailed independent samples t-test and abnormal distribution of data were tested using Mann-Whitney U test For the assays, each experimental group was as-sayed at least twice in triplicate. A p value of ≤ 0.05 was considered statistically significant.
3.1. UPA and soluble uPAR elevated expression in patients with ovarian cancer
45 patients with ovarian cancer and 42 age-matched patients with benign ovarian tumor as controls were enrolled. Median age at diag-nosis was 48 years (range: 28–72 years). All patients underwent sur-gery. Demographic and clinical characteristic of patients and controls are shown (Table 1). UPA and soluble uPAR levels were significantly higher in patients than in the controls (P > 0.05) showed (Table 2).
3.2. Expression of uPAR in human ovarian cancer cell lines
Flow cytometry was used to detect the expression of uPAR on human ovarian cancer cell lines. The expression of uPAR on the surface of cancer cells was stained by PE-labeled monoclonal antibody. RH30 human rhabdomyosarcoma cells were used as positive controls . It was found that ES-2, HO8910, C13 K and SKOV3 all expressed uPAR in ovarian cancer cell lines, and uPAR was barely expressed in A2780 with expression rate 0.7% (Fig. 1). Among them, ES-2 has the highest ex-pression rate 95.2% (Fig. 1).
3.3. Structure of ATF-CAR lentivirus
The ATF mRNA sequence is 471 bp in length from the human uPA (NM_002658.5). ATF was used to replace in most CAR framework with scFv as the extracellular recognition region, referring to the sequence of the Synthetic construct FMC63-28Z receptor . Since the vector carried eGFP gene, it was convenient to observe after transduction. A schematic diagram of ATF-CAR is shown in Fig. 2. The control lentivirus carried only the eGFP gene, and the other aspects were identical to ATF-CAR. After the construction of the host plasmid was completed, the gene was sequenced and the sequencing was successful.
Demographic and clinical characteristics of patients.
Characteristic Malignant Benign ovarian
ovarian cancer cancer
cell tumor/ sex cord-stromal tumor
Urokinase plasminogen activator and Urokinase plasminogen activator receptor levels in malignant cancer and benign control groups.
Variable Malignant ovarian cancer Benign ovarian cancer P value
3.4. Detection of T lymphocytes from peripheral blood of humans with lentivirus infection, expression and phenotype
After three days of culturing the human peripheral blood T lym-phocytes in vitro, the proportion of CD3+ cells was detected by flow cytometry as ≥ 99%, demonstrating that the cells prepared for trans-duction with the lentivirus were T lymphocytes without any con-tamination from other peripheral blood cells (Fig. 3A). As shown in Fig.3B, there was no obvious diﬀerence in the CD4+/CD8 + T cell ratio after the three-day culture described above, prior to lentivirus transduction. CD8 + T cells are known as cytotoxic T lymphocytes (CTLs) and are regarded to be the main classical cytotoxic eﬀector cells in the anticancer immune mechanism, although some CD4 + T cells do possess cytotoxic activity. Seven days after ATF-CAR and the control lentivirus had been transfected into T cells, eGFP expression was ob-served using a fluorescence inverted microscope. The eGFP expression rates in the CTs and ATF-CAR T cells were 68.4% and 61.0%, respec-tively, confirming the eﬃcient lentiviral transduction (Fig. 3C). Transfected ATF-CAR T cells are shown in Fig. 3D. The cells were