Trastuzumab

Trastuzumab sensitive cell line SK-BR-3 was obtained from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 medium (Gibco, USA) with 10% fetal bovine serum (FBS). Cells in the logarithmic growth phase were used to establish Trastuzumab resistant cell line. The cells were first grown in complete medium containing 0.5 μg/ml Trastuzumab (Roche, Shanghai, 10 times of 50% inhibition concentration), and then grown and subcultured in increasing Trastuzumab concentrations from 0.5, 1, 2, 4, 6 to 8 μg/ml. The cells that stably grew in medium containing 8 μg/ml Trastuzumab for one month was designated SK-BR-3-TR (Trastuzumab-resistant cell line), and maintained in medium containing 4 μg/ml Trastuzumab for subsequent experiments.

The basic reagents used in our experiments and their sources are: HRG1-β1 10 μM (R&D Systems), pertuzumab (P) 30 μM (Genentech), U3 inhibitor 30 μM (U3 Pharma) and trastuzumab (T) 30 μM (Roche). Primary antibodies anti-EGFR (HER-1) mAb (ab30), anti-EGFR (HER-1) mAb (103575) and anti-HER-3 mAb ([2F9] 91084) were purchased from Abcam (Abcam, USA). Anti-HER-2 ([H-200] 134481) and anti-HER-4 ([L-20] 31149) mAbs were obtained from Santa Cruz (SCBT, USA). The DUOLINK In Situ Ligation Kit was purchased from Olink (DUO92002-Duolink In Situ PLA Probe Anti-Rabbit PLUS, DUO92004 - Duolink In Situ PLA Probe Anti-Mouse MINUS, DUO92006 - Duolink In Situ PLA Probe Anti-Goat MINUS, DUO92007 - Duolink In Situ Detection Reagents Orange) (Olink Bioscience, Uppsala, Sweden). Nuclei were stained with TOPRO3 (T3605) from Life Technologies (Thermo Fisher Scientific).

For sphere cultures, single dissociated OE19, N87, OE33 and GTR0455 cells were plated at a concentration of 10.000 cells/ml onto low-attachment dishes (6 wells) with an ultralow attachment surface (Corning, Corning, NY, USA). Cells were cultured in epithelial basal medium supplemented as described in [13 (link)], treated on Day 0 with trastuzumab (T) (20 μg/ml; F. Hoffmann-La Roche AG, Basel, Switzerland), pertuzumab (P) (10 μg/ml; F. Hoffmann-La Roche AG) and lapatinib (L) (10 nM; Selleck Chemical, Houston, TX, USA), and TVB3166 at appropriate dilution (Merck Life Science, Darmstadt, Germany), in monotherapy or combination, with NaCl 0.9% or DMSO as distinct diluents, and collected after 7 days. The spheres were counted microscopically on day 7, and representative images were acquired using an EVOS XL Core Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). The gastrosphere forming efficiency (GFE) was calculated as the ratio of the number of gastrospheres to the number of single cells that were initially seeded.

OE19 and OE33 (ATCC, Manassas, VA) were maintained in RPMI with 10% fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 units/mL), and streptomycin (500 μg/mL) (Lonza, Basel, Switzerland) according to routine cell culture procedures. HEK293T cells were maintained in high-glucose DMEM medium and supplemented as mentioned above. Long-term treated cells were continuously cultured with 1μg/ml trastuzumab for a duration of 1-6 months (Roche, Grenzach-Wyhlen, Germany). trastuzumab, panitumumab (Amgen, London, England), and pertuzumab (Roche) were all kindly provided by the Academic Medical Center pharmacy. Anti-HER3 antibody (H3.105.5) was purchased from Millipore (Temecula, CA). ADAM10 inhibitor GI 254023X and recombinant hNRG-1β were purchased from R&D systems (Oxon, United Kingdom). NRG-1β was used at 2ng/ml.

Paclitaxel (≥99.9% purity) was purchased from Qilu Antibiotic Pharmaceutical Co Ltd China. Poly lactic acid co-glycolic acid (75:25, Resomer^® RG 756 H, MW 76000–115000 Da) from Evonik Germany, trastuzumab from Roche Pharmaceuticals United Kingdom, poloxamer 407 and sodium lauryl sulfate (SLS) from Sigma-Aldrich Germany, disodium hydrogen phosphate (Na₂HPO₄), dialysis tubing-Dia 27/32”-21.5 mm 30 M MWCO ∼12,000–14,000 Da from Sigma-Aldrich Germany, acetonitrile (purity ≥ 99.9%), and other solvents used were of HPLC grade. The water used for solvent preparation was ultrapure.

To assess drug resistance due to CD44, cytotoxicity was measured using bortezomib or cisplatin. The cytotoxicity was determined using the alamarBlue® assay (Thermo Fisher Scientific, Waltham, MA) following the manufacturer's instructions. Briefly, cells were exposed to PS for 4 weeks. The cells were then seeded in 96-well culture plates at a density of 1 × 10⁵ cells/mL in medium (supplemented with 10% FBS) containing 8.61 × 10⁵ particles/mL of PS. The cells were incubated overnight and then treated with: bortezomib (Takeda Oncology, USA, 10 nM in PBS); cisplatin (United states Pharmacopeia, USA, 1.5 μg/mL in dimethyl sulfoxide [DMSO]); paclitaxel (Sigma-Aldrich, USA, 300 nM in DMSO); gefitinib (Sigma-Aldrich, USA, 4 mM in DMSO); lapatinib (Sigma-Aldrich, USA, 1 μM in DMSO); sorafenib (Sigma-Aldrich, USA, 10 μM in DMSO); or trastuzumab (Roche, Switzerland. 10 μg/mL in PBS). All treatment incubations were performed in RPMI-1640 medium supplemented with 5% FBS and/or 8.61 × 10⁵ particles/mL of PS. Cytotoxicity was measured at an emission of 590 nm using a microplate reader. The emission values are reported as a percentage of vehicle control, yielding a percentage cell cytotoxicity after 72 h of treatment. For the calculation of drug resistance, Δcytotoxicity was calculated by followings; Δcytotoxicity = %cytotoxicity w/ PS - % cytotoxicity w/o PS.