Wu, T.-D. | bioRxiv Science

bioRxiv2022

Discovery of a druggable copper-signaling pathway that drives cell plasticity and inflammation

Inflammation is a complex physiological process triggered in response to harmful stimuli. It involves specialized cells of the immune system able to clear sources of cell injury and damaged tissues to promote repair. Excessive inflammation can occur as a result of infections and is a hallmark of several diseases. The molecular basis underlying inflammatory responses are not fully understood. Here, we show that the cell surface marker CD44, which characterizes activated immune cells, acts as a metal transporter that promotes copper uptake. We identified a chemically reactive pool of copper(II) in mitochondria of inflammatory macrophages that catalyzes NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with a rationally-designed dimer of metformin triggers distinct metabolic and epigenetic states that oppose macrophage activation. This drug reduces inflammation in mouse models of bacterial and viral (SARS-CoV-2) infections, improves well-being and increases survival. Identifying mechanisms that regulate the plasticity of immune cells provides the means to develop next-generation medicine. Our work illuminates the central role of copper as a regulator of cell plasticity and unveils a new therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

cell biology↗

bioRxiv2021

Quantifying nanotherapeutics penetration using hydrogel based microsystem as a new 3D in-vitro platform

The huge gap between 2D in vitro assays used for drug screening, and the in vivo 3D-physiological environment hampered reliable predictions for the route and accumulation of nanotherapeutics in vivo. For such nanotherapeutics, Multi-Cellular Tumour Spheroids (MCTS) is emerging as a good alternative in vitro model. However, the classical approaches to produce MCTS suffer from low yield, slow process, difficulties in MCTS manipulation and compatibility with high-magnification fluorescent optical microscopy. On the other hand, spheroid-on-chip set-ups developed so far require a microfluidic practical knowledge difficult to transfer to a cell biology laboratory. We present here a simple yet highly flexible 3D-model microsystem consisting of agarose-based microwells. Fully compatible with the multi-well plates format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging including live high-resolution optical microscopy can be performed in-situ, with no manipulation of spheroids. As a proof-of-principle of the relevance of such in vitro platform for nanotherapeutics evaluation, this study investigates the kinetic and localization of nanoparticles within colorectal cancer MCTS cells (HCT-116). The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI (AGuIX(R), currently implicated in clinical trials as effective radiosensitizers for radiotherapy) and confocal microscopy after addition of Cy 5.5. We show that the amount of AGuIX(R) nanoparticles within cells is largely different in 2D and 3D. Using our flexible agarose-based microsystems, we are able to resolve spatially and temporally the penetration and distribution of AGuIX(R) nanoparticles within MCTS. The nanoparticles are first found in both extracellular and intracellular space of MCTS. While the extracellular part is washed away after few days, we evidenced intracellular localisation of AGuIX(R), mainly within lysosomes compartment, but also occasionally within mitochondria. Our agarose-based microsystem appears hence as a promising 3D in vitro user-friendly platform for investigation of nanotherapeutics transport, ahead of in vivo studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/427020v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@bd48adorg.highwire.dtl.DTLVardef@ee7502org.highwire.dtl.DTLVardef@143ee60org.highwire.dtl.DTLVardef@d6f5ca_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

bioengineering↗