Cfpac

The BxPC3, Sw1990, AsPC-1, and CFPAC pancreatic cancer cell lines and the SKBR3 breast cancer cell line were from ATCC (Rockville, MD) and were cultured following the ATCC recommendations. The two PDXs (P4604 and P2846) were generated from resected hepatic and peritoneum metastatic samples, respectively, from two patients with pancreatic cancer treated with gemcitabine (P4604) or untreated at surgery time (P2846) (PDX Platform, Institut de Recherche en Cancérologie de Montpellier) (58 (link)). The PDX cell line C-PDX P4604 was derived from the PDX P4604. The gemcitabine-resistant cell line BxPC3-GR was generated by exposure to progressively increasing doses of gemcitabine, as previously described (58 (link)). All cell lines and the PDXs express EGFR at high level (between 150,000 and 350,000 receptors/cell), HER2 at intermediate level (between 6,000 and 78,000 receptors/cell), and HER3 at low level (between 5,000 and 20,000 receptors/cell) (26 (link), 58 (link), 59 (link)), except the SKBR3 cell line that is HER2^high (>1 million receptor/cell) (59 (link)). The CD16/FcγRIII (158V allotype)-transfected human NK92 cell line was cultured in complete RPMI medium supplemented with 100 IU/ml interleukin 2, as previously described (60 (link)).

The pancreatic cell line CFPAC was acquired from ATCC company (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, United States) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, United States) were utilized for the culture of CFPAC cells at 37°C in 5% CO₂ atmosphere.
The 4 miRNA mimics were designed and synthesized by GenePharma (Shanghai, China). The miRNA mimics (Supplementary Table 4) were transfected into CFPAC cells using lipofectamine RNAiMAX reagent (Invitrogen, United States) according to the manufacturer’s protocols. Then, qRT-PCR was used to evaluate transfection efficiency after 24h.

Pancreatic cancer cell lines (T4- and T3-KPC cells) were derived from KPC mice as previously described (43 (link)) and were a gift from David Tuveson (Cold Spring Harbor Laboratory, Long Island, New York, USA). MS1, a mouse endothelial pancreas cell line, and CFPAC were obtained from ATCC. The Kras^LA1/+p53^R172H/Δg/+ lung adenocarcinoma cell line (393P) was generated as previously described (44 (link)). All cell lines were cultured in DMEM (Thermo Fisher Scientific) with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin, and incubated at 37°C in a humidified incubator with 5% CO₂. C2C12 myoblasts were purchased from ATCC and cultured in DMEM with 10% FBS until confluent. After reaching confluency, the myoblasts were differentiated in DMEM with 2% horse serum and 1 μg/mL insulin for 72 hours, as previously described (22 (link)). 3-MA (item no. 13242) for in vitro and in vivo studies was purchased from Cayman Chemical.

PC cell lines SW1990, and Miapaca2 were obtained from the Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of the National Ministry of Education (Shanghai Jiao Tong University School of Medicine, Shanghai, 200,025, China). The remaining cell lines (including PANC1, Bxpc-3, Aspc-1, su86.86, CFPAC, and HPAC) were obtained from ATCC (Manassas, VA). Short tandem repeat (STR) DNA was used to identify the above cell lines. All mycoplasma tests represented negative outcomes. Cell lines were cultured in RPMI 1640 medium supplemented with 0.10% fetal calf serum, 1% penicillin, and streptomycin under a 5% carbon dioxide atmosphere at 37℃ .

The human PC cell lines (BXPC-3, CFPAC, MIAPACA and PANC-1) and a normal human pancreatic ductal cell line (HPNE) that we used in this paper were obtained from ATCC. Conditions including Dulbecco’s modified Eagle’s medium (DMEM) (Wisent, Quebec, Canada) contained with 10% fetal bovine serum (Wisent, Quebec, Canada) at 37 °C in a 5% CO2 in humidified air.

The human pancreas cancer cell lines CFPAC and PANC-1 (American Type Culture Collection, Rockville, MD) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 5% penicillin-streptomycin (5 mg/mL penicillin, 5 mg/mL streptomycin; Thermo Fisher Scientific, Waltham, MA). The gastric cancer cell line MKN45 (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan), esophagus cancer cell lines TE4 and TE9 (Japanese Cell Resource Center for Biomedical Research, Sendai, Japan), and pancreas cancer cell line PK9 (Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan) were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FBS and 5% penicillin-streptomycin.

Pancreatic cancer tissues and normal tissues adjacent to pancreatic cancer were harvested from patients who underwent surgery in Nanjing Drum Tower Hospital from July 2017 to July 2019. All samples were histologically reviewed by two pathologists. None of the patients received radiotherapy or chemotherapy before surgery. All specimens were immediately frozen in liquid nitrogen within 5 min after resection and then stably sorted at −80 °C. Clinical samples were collected from patients after written informed consent was obtained. Human pancreatic cancer cells (HPAC, Bxpc-3, Panc1, CFPAC) were obtained from the American Type Culture Collection (ATCC), and HPDE cells were a gift from the Technical University of Munich, Germany. All cell lines were grown in the recommended culture media and maintained at 37 °C in 5% CO₂.
Quantitative real-time RT-PCR (qRT-PCR) was used to validate relative mRNA levels. Total RNA was extracted from the cultured cells by the TRIzol method (Invitrogen, Waltham, MA, USA). According to the manufacturer’s protocols (Vazyme, Nanjing, China), cDNA was synthesized by reverse transcription and then used as a substrate of qRT-PCR. GAPDH expression was employed as an internal control for mRNA. The primers used for qRT-PCR are given in Table S1.