Htb 26tm

All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The A549 lung adenocarcinoma (ATCC CCL‐185TM), MDA‐MB‐231 triple‐negative breast adenocarcinoma (ATCC HTB‐26TM), and SH‐SY5Y neuroblastoma (ATCC CRL‐2266TM) cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM)‐high glucose (Gibco, Thermo Fisher Scientific, MA, USA). HT29 colon adenocarcinoma (ATCC HTB‐38) cell lines were maintained in DMEM/Nutrient Mixture F‐12 (Gibco). K562 chronic myeloid leukemia (ATCC CRL‐3344) were grown in Roswell Park Memorial Institute Medium 1640 (Gibco). The cells were cultured in medium supplemented with 10% fetal bovine serum (Gibco) and 1× penicillin/streptomycin (Gibco) and incubated in a 5% CO₂ incubator with saturated humidity.

The cell culture media used were: high glucose Dulbecco’s Modified Eagle’s Medium (DMEM), Eagle’s Minimum Essential Medium (EMEM), and Dulbecco’s Modified Eagle’s Medium and Ham’s F12 medium (1:1 mixture; DMEM:F12), acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). Of the cell lines tested in this study, two genotypically distinctive breast adenocarcinoma cell lines (MDA-MB-231—ATCC^® HTB-26 TM, MCF-7—ATCC^® HTB-22 TM) and one nontumorigenic cell line (MCF-10—ATCC^® CRL-10317) were obtained from ATCC (Manassas, VA, USA) as frozen items.

The immortalized human monocyte cell line THP-1 (ATCC^® TIB-202^TM, VA, Gaithersburg, MD, USA) was used for cell labeling (passage of <20). The cells were maintained in culture as described previously [31 (link)].
The human adenocarcinoma cell line (MDA-MB-231, passage 46, ATCC^® HTB-26^TM, Gaithersburg, MD, USA) was cultured under the same conditions.

SK-MEL-28 human melanoma cells (ATCC # HTB-72™) and MDA-MB-231 human breast adenocarcinoma cells (ATCC # HTB-26^TM) were grown in a monolayer in DMEM, supplemented with 10% (v/v) heat-inactivated FBS and 1% penicillin–streptomycin. Cell cultivation was carried out in humidified atmosphere containing 5% CO₂.

MDA-MB-231 cell line (HTB-26^TM, American Type Culture Collection^®, USA) [62 (link), 63 (link)] was used, which was originally derived from a 51-year-old white American woman with metastatic breast cancer [64 (link)]. MDA-MB-231 cells are spindle-like in shape with a mean diameter of 18–20 µm [65 (link)], as shown in Figure 2A and B. They were grown in the Dulbecco’s Modified Eagle Media (DMEM, 11965084, Thermo Fisher, USA) containing 10% fetal bovine serum (‎FBS, A5256801, Thermo Fisher, USA) and 1% penicillin-streptomycin (PS, 15070063, Thermo Fisher, USA) [66 (link)] to a final concentration of 10⁶ cells/mL.

A Boyden chamber assay was performed using the CytoSelect™ 96-well cell migration assay kit (8 µm, Fluorometric Format, Cat. No. CBA-106; Cell Biolabs, Inc., San Diego, CA, USA). Human breast cancer MDA-MB-231 cells (HTB-26TM; American Type Culture Collection, Manassas, VA, USA) were suspended in serum-free D-MEM medium (1.85 × 10⁵ cells/mL) containing 100 nM CD81-BP, AAAA or CD81 antibody (5A6) at 37 °C and 5% CO₂ for 1 h. Then, within the Boyden chamber, 100 µL of the serum-free cell suspension was added to the upper chamber, while 150 µL of 10% FBS-containing medium was placed in the lower chamber. After incubation at 37 °C and 5% CO₂ for 24 h, the cells that migrated to the lower chamber were detached from the membrane by cell detachment buffer, lysed by lysis buffer, and stained with CyQuant^® GR dye. The fluorescence intensity of the cell lysate was analyzed at 480/520 nm by a microplate reader (Biomedical Solutions, Inc., Stafford, TX, USA).

MDA-MB-231, Panc-1 and HeLa cells were purchased from the American Type Culture Collection (ATCC®) (VA) (HTB-26 TM, CRL-1469TM and CCL-2 TM respectively). Cells were cultured in low-glucose Dulbecco's Modified Medium (DMEM) (Merck, Germany, D6046) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, 10270). MDA-MB-231 cells were cultured at 5% CO₂ concentration and 37 °C and Panc-1 and HeLa were cultured at 10% CO₂ and 37 °C.