Cell culture
The breast cancer cell line, MDA-MB-231 (ATCC, VA, USA) was cultured in DMEM (Lonza, Switzerland). The medium was supplemented with 10% foetal bovine serum (FBS) (Thermo Fisher Scientific, MA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin. Human MSCs were from ATCC (VA, USA) and cultured in MesenPro RS medium (Thermo Fisher Scientific, MA, USA).
Cell labelling with MPs
Human MSCs were labelled with 1 mm MPs (Chemicell, Germany). For this, adherent cell populations were starved for 6 h before MPs (0.6 –10 mg/ml) were applied in serum-free medium for 24 h. The next day, the cells were thoroughly washed 5 times with phosphate-buffered saline (PBS) in order to remove excess particles.
Prussian blue staining
Cells were fixed with 4% paraformaldehyde (PFA) for 15 min and subsequently rinsed with deionised water. Freshly prepared, 20% aqueous solution of hydrochloric acid and 10% aqueous solution of potassium ferrocyanide were mixed in equal parts and added to the cells for 20 min. The staining solution was removed, and the cells were washed three times with deionised water. The stained cells were imaged in 200 ml deionised water using an Evos M5000 microscope (Thermo Fisher Scientific, MA, USA).
Cell viability assay
Impact on membrane integrity was assessed using a live/dead cell imaging kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. As a positive control for the dead stain, cells were killed and fixed with ethanol. The stained cells were imaged using an Evos M5000 microscope (Thermo Fisher Scientific, MA, USA).
DNA-hypodiploidy assays
Apoptosis was measured according to Nicoletti et al. (Nicoletti et al., 1991). Briefly, 24 h post-treatment, cells were harvested and washed once with PBS. Pelleted cells were resuspended in hypotonic fluorochrome solution containing 50 mg/ml propidium iodide, 0.1% sodium citrate, and 0.1% Triton-X100. After incubation at 4° C for 2 h, cells were analysed by flow cytometry.
ELISA
Cellular supernatants were removed and cleared by centrifugation. TRAIL and PD1 protein levels were determined by DuoSet ELISA (Biotechne, MN, USA), according to manufacturer’s instructions.
Adenoviral transduction
Adenoviral vectors expressing luciferase (Ad.Luc) and sPD1HAC (Ad.sPD1HAC) (Maute et al., 2015; Pascolutti et al., 2016) were purchased from Vector Biolabs (PA, USA). Ad.sTRAIL was generated as previously described (Mohr et al., 2019). For adenoviral transduction, the normal growth medium was changed to medium supplemented with 2% FBS. The cells were transduced with virus at 200 pfu/cell for 6 h and then washed off.
Surface marker stain
The surface marker expression was measured by incubating MSCs with PE- conjugated mouse anti-human antibodies for CD44 (BJ18), CD90 (5E10), CD105 (43A3) or APC-conjugated mouse anti-human antibodies for CD45 (HI30). PE-conjugated mouse IgG1κ and APC-conjugated mouse IgG1κ served as isotype controls. All antibodies were purchased from Biolegend (CA, USA).
Migration assay
Migration of BHM-MSCs and MSCs was measured with a Cell Migration/Chemotaxis Assay Kit (Abcam UK), according to the manufacturer’s instructions. Briefly, transgene expressing BHM-MSCs were cultured in serum free medium for 24 h before being trypsinised and resuspended in serum-free medium. BHM-MSCs were plated in the wells containing the insert chambers at a density of 50,000 cells per well in serum free medium. 10% FBS-containing medium was then added to the lower chamber to serve as a chemoattractant. Cells were allowed 24 h to migrate across the 8 mm polycarbonate membrane. Cell migration was analysed by reading fluorescence (Ex/Em = 530/590 nm) in a plate reader.
Luciferase assay
Luciferase expressing BHM-MSCs were washed five times with PBS before the addition of 1x passive lysis buffer (Promega, WI, USA). The culture vessel was gently rocked for 15 min at room temperature before the lysate was transferred to a microcentrifuge. 5 ml of the lysate was added into a black 96-well plate with a clear bottom (Corning, NY, USA) containing Luciferase Assay Reagent II (Promega, WI, USA). Luminescence was measured in a luminometer.
Crystal violet staining
Cells were seeded at a density of 1 × 104 cells in a 24-well plate. After 24 h the cells were treated with different concentrations of sTRAIL derived from MSC.sTRAIL. After 7 days, resulting colonies were fixed in 2% PFA and visualised with a 0.04% crystal violet solution. The crystal violet dye was solubilised in methanol and the optical density of each well was measured at 570 nm. The average OD570 of non-stimulated cells was set to 100%.
Magnetic actuation
The experimental setup consists of a MFG100 system (Magnebotix, Switzerland), optical camera (Basler, puA1280-54uc). The magnetic field generator can generate a controlled 3D magnetic field (<20 mT). Different field frequencies (<20 Hz) and intensities (<15 mT) were used to steer BHM-MSCs and study the steering performance. The MATLAB image processing toolbox was used to characterise the biohybrid microswarm performance. In all experiments, the same region of interest was considered for image processing.
Statistical Analysis
Experimental values are expressed as mean value ± standard error (SE). For significance analyses, analysis of variance (ANOVA) between groups was used and *p≤0.05 was considered significant, **p≤0.01 as highly significant and ***p≤0.001 as extremely significant.