Human recombinant insulin

Cell lines used were MCF7 (ATCC HTB-22) human ER+ breast cancer cells, T47D (ATCC HTB-133) human ER+ breast cancer cells, and E0771 (CH3 Biosystems) murine TNBC cells. T47D cells were cultured in RPMI-1640 (Corning) supplemented with 10% fetal bovine sera (FBS) (Gemini), 1% penicillin-streptomycin (P/S) (Corning), and 0.2 units/mL human recombinant insulin (Santa Cruz). MCF7 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) (ATCC) supplemented with 10% FBS (Gemini), 1% P/S (Corning), and 0.01 mg/mL human recombinant insulin (Santa Cruz). MCF7 and T47D cells were transduced with plasmids for the lentiviral control or CCT2-FLAG as previously described (40 (link)). For selection, cells were maintained with 0.5 μg/mL puromycin dihydrochloride (ThermoFisher) and microscopically observed for GFP expression. E0771 TNBC (CH3 Biosystems) were cultured in RPMI-1640 (Corning) supplemented with 10% FBS (Gemini) and 1% P/S (Corning). E0771 were transduced with lentiviral-based inducible small hairpin RNA (shRNA) to deplete CCT2 as previously reported (40 (link)). To induce shRNA expression, 0.5 µg/mL doxycycline was added to the media for 24-72 hours.

MDA-MB-231 cells (ATCC^® HTB-26^™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning) and supplemented with 10% fetal bovine serum (FBS) (Gemini) and 1% Penicillin-Streptomycin (P/S) (Corning). T47D cells (ATCC^® HTB-133^™) were transfected with a lentiviral plasmid to express CCT2 with a FLAG tag (DYKDDDDK) hereafter referred to as T47D-CCT2, as previously described [13 (link)]. T47D cells were cultured in Roswell Park Memorial Institute Medium (RPMI-1640) (Corning) supplemented with 10% FBS (Gemini), 1% P/S (Corning), and 0.2 units/mL human recombinant insulin (Santa Cruz). 0.5 μg/mL puromycin dihydrochloride (ThermoFisher) was added to maintain plasmids.

We used PDAC cell lines (derived from primary tumors) Panc02.03 (ATCC® CRL-2553), Panc08.13 (ATCC® CRL-2551), Panc10.05 (ATCC® CRL-2547) and Panc-1 (ATCC® CRL-1469). Panc02.03, Panc08.13 and Panc10.05 were cultured in RPMI-1640 (Gibco) supplemented with 15% FBS (Gibco), cyprofloxacine, antibiotic/antimycotic mix, glutamine (Sigma) and 10 U/ml human recombinant insulin (Santa-Cruz). Panc-1 cells were cultured in DMEM with 10% FBS (Gibco) and antibiotics. The control immortalized Human Pancreatic Duct Epithelial Cell Line (H6c7, HPDEC, Kerafast) was maintained in keratinocyte serum-free medium that contained EGF and bovine pituitary extract (Thermo Fisher, 17005042), antibiotic/antimycotic mix and glutamine (Sigma). Cells were tested for mycoplasma contamination with Hoechst staining and only negative cultures < 9 passage numbers were used in our experiments. Cells were washed three times with phosphate buffered saline (PBS) 2 days after culturing them in serum-free medium, the medium was then replaced and EVs were collected after 2 days. For 3D cultures, cells were removed from culture dishes (Eppendorf) with TrypLE (Gibco), embedded into Matrigel with 20,000 cells/droplet (25 μl) and cultured in 2.5% EV-free FBS (exosome depleted One-Shot FBS, Gibco) prior change to serum-free medium 2 days before starting EV collection.