Fluorescence Lifetime Imaging Microscopy (FLIM) reveals spatial-metabolic changes in 3D breast cancer spheroids.

Cancer cells are mechanically sensitive to physical properties of the microenvironment, which can affect downstream signaling to promote malignancy, in part through the modulation of metabolic pathways. Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure the fluorescence lifetime of endogenous fluorophores, such as the metabolic co-factors NAD(P)H and FAD, in live samples. We used multiphoton FLIM to investigate the changes in cellular metabolism of 3D breast spheroids derived from MCF-10A and MD-MB-231 cell lines embedded in collagen with varying densities (1 vs. 4 mg/ml) over time (Day 0 vs. Day 3). MCF-10A spheroids demonstrated spatial gradients, with the cells closest to the spheroid edge exhibiting FLIM changes consistent with a shift towards oxidative phosphorylation (OXPHOS) while the spheroid core had changes consistent with a shift towards glycolysis. The MDA-MB-231 spheroids had a large shift consistent with increased OXPHOS with a more pronounced change at the higher collagen concentration. The MDA-MB-231 spheroids invaded into the collagen gel over time and cells that traveled the farthest had the largest changes consistent with a shift towards OXPHOS. Overall, these results suggest that the cells in contact with the extracellular matrix (ECM) and those that migrated the farthest had changes consistent with a metabolic shift towards OXPHOS. More generally, these results demonstrate the ability of multiphoton FLIM to characterize how spheroids metabolism and spatial metabolic gradients are modified by physical properties of the 3D ECM.

Theranostic Calcium Phosphate Nanoparticles With Potential for Multimodal Imaging and Drug Delivery.

Calcium phosphate (CaP) bioceramics closely resemble the natural human bone, which is a main reason for their popularity as bone substitutes. However, this compositional similarity makes it difficult to distinguish CaPs, especially in particulate form, from native bone by imaging modalities such as X-ray radiography, computed tomography (CT), and magnetic resonance imaging (MRI) to monitor the healing progress. External contrast agents can improve the imaging contrast of CaPs but can affect their physicochemical properties and can produce artifacts. In this work, we have attempted to improve the contrast of CaP nanoparticles via ion substitutions for multimodal imaging. Calcium-deficient hydroxyapatite (CDHA) nanoparticles with silver (Ag), gadolinium (Gd), and iron (Fe) substitution were prepared by a microwave-accelerated wet chemical process to improve the contrast in CT, T1 (spin-lattice), and T2 (spin-spin) MRI relaxation modes, respectively. Ag, Gd, and Fe were substituted at 0.25, 0.5, and 0.25 at.%, respectively. The ion-substituted CDHA (ICDHA) was found to be phase pure by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). Transmission electron microscopy (TEM) images showed that the ICDHA nanoparticles were platelet shaped and of 52 ± 2 nm length and 6 ± 1 nm width. The ICDHA showed high contrast in X-ray and CT compared to CDHA. The vibrating sample magnetometry (VSM) studies showed the ICDHA to exhibit paramagnetic behavior compared to diamagnetic CDHA, which was further confirmed by improved contrast in T1 and T2 MRI mode. In addition, the in vitro tetracycline drug loading and release was studied to investigate the capability of these nanoparticles for antibiotic drug delivery. It was found that a burst release profile was observed for 24 h with 47-52% tetracycline drug release. The ICDHA nanoparticles also showed in vitro antibacterial activity against Staphylococcus aureus and Escherichia coli due to Ag, which was further enhanced by antibiotic loading. In vitro biocompatibility studies showed that the triple-ion-substituted ICDHA nanoparticles were cytocompatible. Thus, the ion-substituted CDHA nanoparticles can have potential theranostic applications due to their multimodal image contrast, antibacterial activity, and drug delivery potential. Future work will be conducted with actual bone samples in vitro or in animal models.