Crl 5822

MCF10A, a non-tumorigenic breast cell line (ATCC, CRL-10317) and MCF10A CDH1^−/− cells (CLLS1042, Sigma-Aldrich, St Louis, MO, USA) were cultured in DMEM/F12/Glutamax (10565042, Thermo Fisher Scientific, Waltham, MA, USA) with 5% horse serum (16050, Thermo Fisher Scientific, Waltham, MA, USA), 10μg/μL neutral insulin (797139, Dunedin hospital, Dunedin, New Zealand), 500 ng/mL hydrocortisone (H0888, Sigma-Aldrich, St Louis, MO, USA), 100 ng/mL cholera toxin (C8052, Sigma-Aldrich, St Louis, MO, USA) and 20 ng/mL human epidermal growth factor (E9644, Sigma-Aldrich, St Louis, MO, USA). A CDH1-null strain of the gastric cancer cell line NCI-N87 (ATCC, CRL-5822) was generated using CRISPR-Cas9 [9 (link)]. Briefly, the sequence 5′-ATTCACATCCAGCACATCCA-3′ was used to target exon 10 of CDH1 and induce a single base frameshift, followed by a stop codon. Isogenic NCI-N87 cells were cultured in DMEM/F12/Glutamax with 10% fetal bovine serum (10091148, Thermo Fisher Scientific, Waltham, MA, USA). Both cell lines were grown at 37 °C with 5% CO₂.

All cancer cell lines used in this study were obtained from the American Type Culture Collection (ATCC) (LGC Standards, Germany). The HER2-positive invasive ductal carcinoma cell line BT-474 (ATCC^® HTB-20^™) and the HER2-negative adenocarcinoma MDA-MB-231 (ATCC^® HTB-26^™) were maintained in high glucose Dulbecco's Modified Eagle's Medium (DMEM 4.5 g/l glucose L-Glutamine; Lonza Benelux BV, Breda, the Netherlands), while the HER2-positive gastric carcinoma NCI-N87 cells (ATCC^® CRL-5822^™) were cultured in RPMI-1640 medium (L-Glutamine, 25 mM HEPES, Gibco^™; Thermo Fisher Scientific Inc, Breda, the Netherlands). All media were supplemented with 10% (v/v) fetal bovine serum (FBS; GE Healthcare Europe GmbH, Eindhoven the Netherlands), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich BV, Zwijndrecht, the Netherlands). Cells were kept at 37 °C in a humidified atmosphere containing 5% CO₂ and were constantly tested negative for Mycoplasma.

The studies were performed on two cancer cell lines: gastric epithelial cell line NCI-N87 (ATCC^® CRL5822™) and prostate cancer line VCaP (ATCC^® CRL-2876™). Healthy prostate epithelial cells HPrECs (ATCC^® PCS-440-010™) were used as a control. All cell lines were purchased from the American Type Culture Collection (ATCC), University Boulevard, Manassas, VI, USA.

Gastric adenocarcinoma (AGS; ATCC CRL-1739) and carcinoma (NCI-N87; ATCC CRL-5822) cells, colorectal adenocarcinoma (HT-29; ATCC HTB-38) and carcinoma (HCT-116; ATCC CCL-247) cells were purchased from ATCC (Manassas, VA, USA). AGS cells were maintained in F-12K medium (ATCC), NCI-N87 were maintained in RPMI-1640 medium, HT-29 and HCT 116 cells were maintained in McCoy’s 5A modified medium. All the culture media were complexed with 10% fetal bovine serum (ATCC). All the cells tested were placed at 37 °C in a humidified atmosphere of 5% CO₂ to retain the proliferating condition.

The HER2-positive/CAV1-positive gastric NCI-N87 and HER2-negative/CAV1-positive breast MDA-MB-231 human cancer cell lines were purchased from American Type Culture Collection (CRL 5822 and HTB-26) in 2014 and authenticated by the Memorial Sloan Kettering Cancer Center (MSK) Integrated Genomics Operation Core using short tandem repeat analysis. NCI-N87 and MDA-MB-231 cells were used within 15 passages and were confirmed to be Mycoplasma-free. NCI-N87 cells were maintained at 37°C in a humidified atmosphere at 5% CO₂ in RPMI-1640 growth medium supplemented with 10% fetal calf serum, 2 mM l-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1 mM sodium pyruvate, a 4.5 g/L solution of glucose, a 1.5 g/L solution of NaHCO₃, and a 100 unit/mL concentration of both penicillin and streptomycin. MDA-MB-231 cells were cultured in American Type Culture Collection–formulated Leibovitz L-15 medium supplemented with 10% fetal bovine serum.
NCI-N87 cells were incubated in medium containing 25 μM of the active form of lovastatin (Millipore) in medium for 4 h before addition of pertuzumab. Control experiments were performed by incubating NCI-N87 cancer cells in medium for 4 h before addition of pertuzumab.