Cell lines and cell culture
HEK293, HCT116 (EGFR+), and SW620 (EGFR-) cell lines (Bioresource Collection and Research Center, Hsinchu 300193, Taiwan) were cultured in Dulbecco's Modified Eagle Medium (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% bovine calf serum (Cytiva, Washington, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific Inc., Waltham, MA, USA). The cells were maintained in an incubator with 5% CO2 at 37 °C.
Establishment of extracellular vesicles with membrane-expressed cetuximab and oxaliplatin encapsulation
The light and heavy chains of cetuximab (NCBI, US) fused to the immunoglobulin C2-type extracellular transmembrane–cytosolic domains of the mouse B7-1 antigen (eB7) [20] were cloned into the pLKO_AS2 vector (National Core Facility for Biopharmaceuticals, Taipei, Taiwan) using NheI, AscI, BglII, and BstxI restriction enzymes. The plasmid was then transformed into TOP 10 cells (Thermo Fisher Scientific Inc.) for amplification and extraction. The lentiviral vector was produced by co-transfecting pMD.G, pCMVΔR8.91 (National Core Facility for Biopharmaceuticals), and pLKO_AS2 cetuximab into HEK293 cells. Stable HEK293 cell lines that can secrete EVs with cetuximab-expressing membranes were selected using 1 mg/ml of neomycin sulfate (Thermo Fisher Scientific Inc.) after infection with virus. The HEK293 cells were cultured in serum-free medium and treated with 5mg/ml of oxaliplatin (Merck, Darmstadt, Germany) and irradiated with UVB once at a dose of 30 mJ/cm2 for 24 hours The EVs with cetuximab-expressing membranes were collected from the cells.
Collection of extracellular vesicles with cetuximab-expressing membranes and Western blot analysis
Collecting EVs from drug-treated or untreated cell lines involved a series of centrifugation steps. The conditioned medium from the cells was first centrifuged at 300 × g for 10 min at 4 °C to remove cellular debris. The supernatant was then subjected to further centrifugation at 2000 × g for 10 min at 4 °C to remove larger microvesicles. Next, the supernatant was centrifuged at 10,000 × g for 30 min at 4 °C to pellet smaller EVs. Finally, the pellet was resuspended in physiological saline and subjected to ultracentrifugation at 100,000 × g for 1.5 hours at 4 °C to collect the extracellular vesicle pellet. For Western blot analysis, the collected EVs with cetuximab-expressing membranes, at 10 ug/well, were separated by SDS-PAGE and transferred onto a nitrocellulose NC membrane (Schleicher and Schuell, Einbeck, Germany). The NC membranes were probed with Mouse Anti-Human IgG Fab Antibody [HRP] (GenScript Biotech, Piscataway, NJ, USA), monoclonal HSP70 antibody (3A3, MA3-006; Thermo Fisher Scientific Inc.), anti-CD81 antibody (ab155760; abcam, Cambridge, UK), CD9 (D8O1A) Rabbit mAb #13174 (abcam), peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG, Fc Fragment Specific (115-035-008, 1:1000; Jackson ImmunoResearch, West Grove, PA, USA), or Rabbit Anti-Goat IgG Antibody, HRP conjugate, (rabbit IgG, AP106P, 1:1000; Sigma-Aldrich) and visualized with the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Merck, Rahway, NJ, USA), according to the manufacturer’s protocol.
Transmission electron microscopy and nanoparticle tracking analysis
Two micrograms of EVs were pipetted (5 ml) onto formvar-coated copper grids (FF200-Cu; Electron Microscopy Sciences, Hatfield, PA, USA) and allowed to settle for 20 min at room temperature. Excess phosphate-buffered saline (PBS) was removed by wicking with filter paper before fixation using a 2% paraformaldehyde, 2% glutaraldehyde, and 0.05 M phosphate solution for 2 min. Grids were washed three times with distilled water prior to application of 1% phosphotungstic acid counterstain for 1 min. Excess liquid was removed by wicking with filter paper, and the grids were allowed to dry overnight at room temperature. Grids were analyzed using a transmission electron microscope (Technai G2 20; FEI, Hillsboro, OR, USA). EVs were visualized and quantified using a nanoparticle tracking analyzer (Nanosight NS300; Nanosight Ltd., Amesbury, UK) with 70 mW laser with a wavelength of 405 nm. Urine samples were diluted in PBS at a ratio of 1:100, while serum samples were diluted at 1:1000. Duplicate measurements were recorded for each sample.
Rhodamine B packaging by extracellular vesicles with cetuximab-expressing membranes and fluorescent staining
After mixing 5 mg/ml of rhodamine B (TCI, Tokyo, Japan) with 2 mg/ml of EVs with cetuximab-expressing membranes, the reaction was allowed to proceed for 1 hour. The mixture was then washed with PBS to separate the free rhodamine B and EVs by centrifugation at 100,000 × g for 10 min at 4 °C in an ultracentrifuge (Optima™ XPN; Beckman Coulter, Brea, California, USA). The pellet was collected and resuspended in PBS for further use. Then, 2 × 103 SW620 and HCT116 cells were separately seeded on glass slides and 5 μM of rhodamine B-loaded EVs with cetuximab-expressing membranes was added to each cell culture and incubated for 90 min. The supernatant was removed and cells were washed with PBS. The cells were then fixed with 10% formalin (KINGFEX CO., LTD., Taipei, Taiwan) and stained with CellTracker™ Green CMFDA Dye (Thermo Fisher Scientific Inc.) and DAPI (Merck, Darmstadt, Germany) and mounted with mounting media (Thermo Fisher Scientific Inc.). Fluorescent images were acquired with a confocal fluorescence microscope (FV1000; Olympus, Tokyo, Japan).
Cell viability assay
Cells were plated in 96-well plates (2000 cells/well) overnight and serially diluted EVs, oxaliplatin-encapsulating EVs, EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximab-expressing membranes were added.Cell viability was detected using the ATPlite kit (510-17281; PerkinElmer, Chennai, India) and the luminesce value was measured using a multimode plate reader (VICTORTM X2; PerkinElmer).
Establishment of an ectopic cancer mouse model and analysis of the accumulation of rhodamine B-loaded extracellular vesicles with cetuximab-expressing membranes
Eight-week-old male nude mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl) obtained from the National Laboratory Animal Center, Taiwan, were used in this study. The HCT116 and SW620 cell suspensions (2 × 106 in PBS) were subcutaneously inoculated into the right hind leg of the mice. Tumors were allowed to grow until they reached approximately 50 mm3 in size. Rhodamine B-loaded EVs were injected into the tail vein at a dose of 5 mg/kg, and the accumulation of tumor fluorescence was measured at different time points using IVIS (PerkinElmer, Inc., Waltham, MA, USA).
Oxaliplatin-encapsulating extracellular vesicles with cetuximab-expressing membranes enhance the therapeutic effect of oxaliplatin on EGFR+ cancer cells
When the tumors reached approximately 50 mm3 in size, mice were treated with PBS, oxaliplatin, EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximab-expressing membranes at a dose of 5 mg/kg by intraperitoneal injection every three days. Tumor size was measured every two days until the end of the experiment on day 30. The mice were then sacrificed and the tumors were collected for observation. The tumor volume was calculated using the formula, tumor volume [mm3] = (length [2]) × (width [2])2 × 0.52. The Kaohsiung Medical University Institutional Animal Care and Use Committee approved this study (approval no. 112056). All experimental procedures were conducted in accordance with regulations.