Hpaf 2

Pancreatic cancer cell lines, HPAF-II (CRL-1997) and CFPAC (CRL-1918), T lymphocyte cell line, Jurkat (TIB-152), and breast cancer cell like, MDA-MB-231 (HTB-26), were obtained from American Type Culture Collection (ATCC), and grown using EMEM (HPAF-II), IMDM (CFPAC), RPMI 1640 (Jurkat) and Leibovitz’s L-15 (MDA-MB-231) medium (ATCC) supplemented with 10% fetal bovine serum (ATCC) and 100 U/mL penicillin-streptomycin (ATCC). All cell lines were grown at 37°C with 5% CO₂ and were routinely passaged at 80% confluence using an iso-osmotic sodium citrate solution for cell release (Thermo). When preparing cells for laser microdissection, cells were released from the culture plates using the same sodium citrate solution. Following a wash with the culture medium, each cell line was diluted to a density of 1000 cells per 100 μL. Approximately 1000 cells (100 μL) were smeared on PEN membrane slides (Leica), air-dried for 10 minutes, and fixed with 100 μL of 100% Ethanol. Cells were then isolated by laser microdissection as outlined below. DNA was extracted using the Qiagen Blood and Cell Culture DNA Mini Kit according to the manufacturer’s protocol. For extraction of DNA from < 100 cells, cell lysis solution from Qiagen RepliG Single Cell kit was used according to the manufacturer’s protocol.

H1975, A549, H1299, H2030, H2009, HPAC and HPAF-II cells were purchased from ATCC. H1975, A549, H1299, H2030, and H2009 cells were maintained in RPMI1640 (Gibco) supplemented with 10% v/v FBS (Gibco) and 1% penicillin-streptomycin (P/S, Gibco). HPAC cells were cultured in F12K (ATCC) with 10% FBS and 1% P/S. HPAF-II cells were maintained in EMEM (ATCC) with 10% FBS and 1% P/S. H1975-CEACAM5, H2009-CEACAM5, A549-CEACAM5, and H1299-CEACAM5, stably expressing CEACAM5, were generated by stable infection with lentivirus from the CEACAM5 lentiviral plasmid (Origene) using a commonly used protocol (36 (link)). Stably transfected cells were selected in RPMI1640 supplemented with 10% FBS, 1% P/S and 1 μg/ml (for A549-CEACAM5) or 2 μg/ml (for H1975-CEACAM5, H2009-CEACAM5, and H1299-CEACAM5) puromycin (Gibco). The cell surface CEACAM5 expression of the stably transfected cells was then assessed with PE-conjugated anti-CEACAM5 IgG1 (Miltenyi Biotec, 130-114-217) by flow cytometry. Anti-CEACAM5 CAR-T cells were generated as previously described (35 (link)) and expanded in the T cell media (RPMI1640 supplemented with extra 2mM glutamine, 10% human serum, and 1% P/S) in the presence of hIL-2 (fed every 2 days, 50 IU/ml, Miltenyi Biotec).

The pancreatic cancer cell lines AsPC-1, Capan-1, HPAF-II, MIA PaCa-2, and PANC-1 were received from ATCC (Manassas, VA). These cell lines were propagated, expanded, and frozen immediately upon receipt. The cells revived from the frozen stock were used within 10-20 passages, not exceeding a period of 2-3 months. The ATCC uses morphological, cytogenetic, and DNA profile analyses for characterization of cell lines. Human pancreatic ductal epithelial (HPDE) cells were kindly received from Dr. Ming Tsao of the Ontario Cancer Institute (Toronto, Canada). The L3.6pl cell line was kindly received from Dr. Isiah D. Fidler at The University of Texas MD Anderson Cancer Center (Houston, TX). Both HPDE and L3.6pl cell lines were handled as other cell lines and were genotyped by DNA fingerprinting (PowerPlex 16; Promega, Madison, WI) as per the manufacturer’s instructions. The growth conditions of all cell lines were performed as described previously [36 (link)].

Panc1(CRL-1469), MiaPaCa2(CRL-1420), Capan2 (HTB-80), and HPAFII (CRL-1997) cell lines were purchased from the ATCC. Panc1 and MiaPaCa2 cell lines were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS). The Capan2and HPAFII cell lines were maintained in 10% (v/v) FBS supplemented McCoy's 5Aor MEM medium, respectively. In some experiments, cells grown in normal medium were treated daily with 5-aza-2′-deoxycytidine (5-aza) or Trichostatin-A (EMD Biosciences). MiaPaCa2 cell lines were transfected with Firefly-luciferase under zeocin selection. Resultant luciferase-expressing MiaPaCa2 cells were subsequently transfected with plasmids encoding either eGFP or human CXCL12, and clones grown under neomycin selection conditions [26] (link). Cell lines were de-identified of all identification parameters from individual consenting patients with pancreatic cancer and were confirmed to be of PDAC origin following DNA karyotype and protein expression analysis.