br The generation of up expression vector
2.7. The generation of up-expression vector and small hairpin RNA (shRNA) lentivirus for uPAR gene (PLAUR)
Since A2780 is a uPAR-negatively expressed cell line, the uPAR-positive A2780 cell line was constructed by overexpression of plasmid transfection. The uPAR sequence was derived from the NCBI database (NM_002659.4) and human PLAUR over-expression vector and
scramble vector were purchased from VectorBuilder Inc. (Shenandoah, Texas). The plasmid was extracted using the TIANGEN Plasmid Mini Kit (Beijing, China) and stored at −20 °C. Transfected into logarithmic phase A2780 using the Lipofectamine™ 3000 Transfection Reagent (Invitrogen, USA) according to the manufacturer’s instructions. Over-expressed stably transfected cell line and control vector transfected cell line were obtained by puromycin selection (80 ng/ml) 3 days after transduction. Mock A2780, vector A2780 and uPAR-overexpressing A2780 were cultured and tested by q-RT PCR for uPAR expression. On the contrary, ES-2 is a uPAR strong positive cell line, so it was knocked down using the shRNA lentiviral system. The low sequence was: CAC TCTCCTCTGGACCTAAAC. The connection and packaging of the viral vector was completed by Beijing Saiye Company.
2.8. Quantitative real-time polymerase chain reaction
Total RNA was prepared from cultured Stattic using Trizol reagent (Invitrogen) following the manufacturer’s instructions. RNA was re-verse transcribed to cDNA using the TransScript All‐in-One first‐strand cDNA synthesis SuperMix (Transgen Biotech, Beijing, China). Real‐time PCR was performed using the TransStart Top Green qPCR SuperMix (Transgen Biotech) and the ABI 7300 Real‐Time qPCR system. All PCR experiments were performed in triplicate. The primers were synthesized by Comate Bioscience (Changchun, China). Sequences for primers used for RT-qPCR are as follows: uPAR (Forward: 5′- TGTAAGACCAACGG GGATTGC-3′, Reverse: 5′-AGCCAGTCCGATAGCTCAGG-3′) and b-actin (Forward: 5′-GGATTACAGCTCACCATGGA -3′, Reverse: 5′- AATCCTT CTGACCCATGCC -3′). Relative quantification of the expression of each gene was calculated by 2− Ct method. r> 2.9. Detection of CAR t cytotoxicity
On day 7 after the lentiviral transduction of T cells, A2780, vector A2780, uPAR-A2780, ES-2, vector ES-2, and shRNA ES-2 cells in 96-well U-bottom microplates were treated with ATF-CAR T cells and control T (CT) cells at diﬀerent eﬀector-target (E/T) ratios (1:1, 5:1, 10:1, and 20:1). The lactic dehydrogenase (LDH) released from the cancer cells was measured to ensure CAR T cell cytotoxicity. Before co-culture, T cells were cultured with a complete medium without IL-2 for 6 h and cancer cells were plated in 96-well plates for 4 h. T cells were then added to the ovarian cancer cells at the ratios mentioned above and co-incubated at 37 °C in 5% CO2 for 4 h. LDH activity was detected by the manufacturer’s protocol (Dojindong, Japan).
2.10. BD™ cytometric bead array (CBA) for cytokine analysis
Eﬀector T cells 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.