Docetaxel

Lx2-32c is prepared by the State Key Laboratory of Bioactive Substances and Functions of Natural Medicines at Institute of Materia Medica (Chinese Academy of Medical Sciences) following the previous protocol with a purity of 98% checked by HPLC⁸ (link). Docetaxel was purchased from Beijing Shongshuo Pharmaceutical Technology Development Co., Ltd. (Beijing, China) with a purity of 99.5%. Lecithin was provided by Avanti Polar Lipids Inc., (Alabaster, AL, USA). Cholesterol was purchased from Hubei KangBaoTai Fine Chemicals Co., Ltd. (Wuhan, China). In experiments in vitro, Lx2-32c and Docetaxel were both dissolved in DMSO and stored at −20 °C for less than 1 month before use. The vehicle (DMSO) was used as a control in all experiments at a maximum concentration of 0.1%. In the animal model, Lx2-32c liposomes were prepared according to our previously published protocol¹¹ (link) and the Docetaxel injection solution was obtained from Qilu Pharmaceutical Co., Ltd. (Jinan, China).

Thirty-three patients were selected for this study between January 12, 2016 and February 16, 2018, at the First Affiliated Hospital of Zhengzhou University, 18 in the chemotherapy group, which received docetaxel (Qilu Pharmaceutical Co., Jiangsu, People’s Republic of China) plus S-1 (Hengrui Pharmaceutical Co.), and 15 in the apatinib combination group, which received apatinib combined with docetaxel. Treatment continued until disease progression or intolerable toxicities were observed. Progression-free survival (PFS) was measured from the first day of administration to disease progression or death from any cause. Follow-up was carried out for all patients. The study endpoints were PFS, disease control rate (DCR), objective response rate (ORR), and the incidence and severity of adverse events (AEs). It is necessary to note that all 33 patients were previously treated with one of the following: cisplatin, carboplatin, oxaliplatin or other platinum and fluorouracil as first-line chemotherapy. Apart from the 33 patients who were included in the analysis, other patients were excluded from the study due to poor treatment compliance or economic hardship.

shRNA targeting the human LACTB gene (shLACTB) was designed and synthesized, and the negative control was shNC. The target sequences are listed in Table II. Then 40-80 pmol of shLACTB or shNC was used to transfect them into the tumor cells with Lipo2000 agent (Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h. Then fresh medium was added into each well and cultured for another 48 h followed by subsequent experiments.
A lentiviral expression plasmid carrying the coding sequence of the human LACTB gene (pLACTB) was constructed, confirmed by sequencing and the lentivirus-expressing LACTB was prepared by Shanghai GeneChem. Then, 2 µl of the plasmid was added into the cancer cells. After culture for 72 h, subsequent experiments were carried out.
Cells were treated with the designed dosage of docetaxel (10, 50,100 and 200 nmol/l) (QILU Pharmaceutical Co.,Ltd.) in combination with shNC, shLACTB or empty vector pCDH (Ctrl) or pLACTB for 48 h followed by subsequent experiments.

Intravenous infusion of sintilimab injection (sintilimab, IBI308, Innovent Biologics, Inc., Suzhou, China) at a dose of 200 mg once every 3 weeks or docetaxel (Qilu Pharmaceutical Co., Ltd., Haikou, China) at a dose of 75 mg/m² once every 3 weeks was administered. The treatment was continued until disease progression, death, intolerable toxicity, withdrawal of consent, initiation of a new anti‐cancer therapy, or other protocol‐specified reasons. Sintilimab treatment beyond initial radiographic disease progression was allowed if the investigators deemed the patient was clinically stable and might benefit from the treatment.