Magneto-mechanical destruction of cancer-associated fibroblasts using ultra-small iron oxide nanoparticles and low frequency rotating magnetic fields

Abstract

The destruction of cells using the mechanical activation of magnetic nanoparticles with low-frequency magnetic fields constitutes a recent and interesting approach in cancer therapy. Here, we showed that superparamagnetic iron oxide nanoparticles as small as 6 nm were able to induce the death of pancreatic cancer-associated fibroblasts, chosen as a model. An exhaustive screening of the amplitude, frequency, and type (alternating vs. rotating) of magnetic field demonstrated that the best efficacy was obtained for a rotating low-amplitude low-frequency magnetic field (1 Hz and 40 mT), reaching a 34% ratio in cell death induction; interestingly, the cell death was not maximized for the largest amplitudes of the magnetic field. State-of-the-art kinetic Monte-Carlo simulations able to calculate the torque undergone by assemblies of magnetic nanoparticles explained these features and were in agreement with cell death experiments. Simulations showed that the force generated by the nanoparticles once internalized inside the lysosome was around 3 pN, which is in principle not large enough to induce direct membrane disruption. Other biological mechanisms were explored to explain cell death: the mechanical activation of magnetic nanoparticles induced lysosome membrane permeabilization and the release of the lysosome content and cell death was mediated through a lysosomal pathway depending on cathepsin-B activity. Finally, we showed that repeated rotating magnetic field exposure halted drastically the cell proliferation. This study established a proof-of-concept that ultra-small nanoparticles can disrupt the tumor microenvironment through mechanical forces generated by mechanical activation of magnetic nanoparticles upon low-frequency rotating magnetic field exposure, opening new opportunities for cancer therapy.

Fig. 1. (a) TEM image of 6 nm USPION@PO-PEG; (inset) particle size distribution deduced from TEM observations (bars) and fit of the size distribution (line); (b) hydrodynamic particle size distribution before (dashed black) and after functionalization with gastrin (full red) measured in volume by dynamic light scattering; (c) hysteresis loop of 6 nm USPION@PO–PEG measured at 300 K.

Fig. 2. USPION@gastrin specifically bind to CAF cells expressing the CCK2R, subsequently internalized and accumulated into lysosomes. (a) Kinetics of USPION@gastrin uptake by CAF1-CCK2 and CAF2-CCK2. Cells were incubated with 16 μg ml⁻¹ of USPION@gastrin for 2, 4, 6, 24 h, 48 h and 72 h. Fluorescence was measured by flow cytometry, and the results are expressed as fluorescence associated with the cells and are the mean ± SEM of at least three separate experiments. (b) CAF1-CCK2 and CAF2-CCK2 cells were incubated with 16 μg ml⁻¹ of USPION@gastrin for 24 h. Fe contained in superparamagnetic MNPs was measured by using a vibrating sample magnetometer. (c) and (d) USPION@gastrin uptake by CAF1 and CAF1-CCK2 (c) or CAF2 and CAF2-CCK2 (d) cells expressing or not the CCK2R. Cells were incubated with increased concentrations of USPION@gastrin for 24 h. Cell-associated fluorescence was measured by flow cytometry, and the results are expressed as fluorescence associated with the cells and are the mean ± SEM of at least three separate experiments.

Fig. 3. Subcellular colocalization of USPION@gastrin. (a–d) Electron microscopy analysis of USPION@gastrin accumulation in CAF1-CCK2 and CAF2-CCK2 cells. CAF1-CCK2 and CAF2-CCK2 cells were incubated with 16 μg ml⁻¹ of USPION@gastrin for 24 h. Image representative of USPION@gastrin accumulated in cells (a) and (c), and in lysosomes (b) and (d). (e and f) Analysis of USPION@gastrin localization by confocal microscopy. CAF1, CAF1-CCK2, CAF2 and CAF2-CCK2 cells were incubated with 16 μg ml⁻¹ of USPION@gastrin for 24 h, and then observed by confocal microscopy (e). Quantification of USPION@gastrin uptake by the analysis of confocal microscopy images (f). CAF1-CCK2 and CAF2-CCK2 cells were incubated with 16 μg ml⁻¹ of USPION@gastrin for 24 h, and then with 10 nM Lysotracker Green for 15 minutes and observed by confocal microscopy (g). Quantification of USPION@gastrin accumulation in lysosomes by the analysis of confocal microscopy images (h).

Fig. 4. USPION@gastrin generates a torque inducing the death of CAF-CCK2 cells under a low frequency rotating magnetic field. (a) Theoretical force generated by USPION@gastrin under AMF or RMF, compared to the evolution of CAF1-CCK2 cell death after RMF exposure experimentally obtained, for different amplitudes of the magnetic field. The force is calculated using monodisperse USPIONs of diameter d = 6 nm, with a uniaxial anisotropy K_eff = 13 kJ m⁻³ and is the one generated by the 3000 nanoparticle assembly at a volume concentration of 15% corresponding to the average number of particles in the lysosomes and their volume concentration. (b) Quantification of CAF1-CCK2 cell death labeled with Annexin V/propidium iodide (AnnV/PI) according to the RMF amplitude (mT) and frequency (Hz). CAF1-CCK2 cells were incubated for 24 h with USPION@gastrin at [Fe] = 16 μg ml⁻¹, washed and exposed to RMF for 2 h. Dead cells were labeled with iFluor488-tagged AnnV/PI, 4 h after magnetic field exposure, and counted by flow cytometry. (c) CAF1-CCK2 and CAF2-CCK2 cells were incubated with USPION@gastrin for 24 h, washed, and exposed or not to RMF (40 mT and 1 Hz) for 2 h. Dead cells were labeled with AnnV/PI and counted 4 h after RMF exposure by flow cytometry. (d) CAF1-CCK2 cells were incubated with USPION@gastrin for 24 h, washed, and exposed or not to static (SMF: 40 mT), alternating (AMF: 40 mT and 1 Hz) or rotating (RMF: 40 mT and 1 Hz) for 2 h. Dead cells were labeled with AnnV/PI and counted 4 h after magnetic field exposure by flow cytometry. (e) and (f) CAF1-CCK2 € and CAF2-CCK2 (f) cells were incubated with USPION@gastrin for 72 h, washed, and exposed or not to RMF (40 mT and 1 Hz) for 2 h, once a day for 6 days. Cell proliferation was evaluated by counting cells 24 h after RMF exposure, for 6 days. Results are expressed as mean ± SEM of at least three separate experiments. Statistical analysis was performed using a one- or two-way ANOVA test.

Fig. 5. RMF exposure caused the lysosomal rupture. CAF1-CCK2 cells were incubated with USPION@gastrin at [Fe] = 16 μg ml⁻¹ for 24 h, washed, exposed or not to RMF (40 mT and 1 Hz) for 2 h, and then incubated with 10 nM LysoTracker Green. Lysosome integrity was determined by analyzing Lysotracker fluorescence intensity by flow cytometry. (a) Representative peak diagrams of Lysotracker fluorescence intensity determined by flow cytometry. Bars indicate the percentage rate of pale cells with lysosomal rupture revealed by a decreased Lysotracker fluorescence intensity. (b) Quantification of lysosomal rupture was performed by counting the percentage of pale with a decreased Lysotracker fluorescence intensity by flow cytometry images and expressed as a fold change of fluorescence intensity over control cells (in the absence of USPION@gastrin and RMF). Significant difference compared to the control conditions corresponding to cells devoid of USPION@gastrin in the absence of RMF exposure was indicated above the histogram bar. Statistical significances between other conditions are also indicated by lines/brackets. Results are the mean ± SEM of three separate experiments. Statistical analysis was performed using a one-way ANOVA test.

Fig. 6. CAF death induced by USPION@gastrin torque depends on lysosomal cathepsin-B activity. CAF1-CCK2 cells were incubated with USPION@gastrin at [Fe] = 16 μg ml⁻¹ for 24 h, washed, exposed or not to RMF (40 mT and 1 Hz) for 2 h in the absence or presence of CA-074-Me (a) or pepstatin A and (b) inhibitors. Dead cells were labeled with AnnV/PI, 4 h after magnetic field exposure, and counted by flow cytometry. Results are expressed as mean ± SEM of at least three separate experiments. Statistical analysis was performed using a one-way ANOVA test.