<em>In vivo</em> identification of apoptotic and extracellular vesicle-bound live cells using image-based deep learning.

The in vivo detection of dead cells remains a major challenge due to technical hurdles. Here, we present a novel method, where injection of fluorescent milk fat globule-EGF factor 8 protein (MFG-E8) in vivo combined with imaging flow cytometry and deep learning allows the identification of dead cells based on their surface exposure of phosphatidylserine (PS) and other image parameters. A convolutional autoencoder (CAE) was trained on defined pictures and successfully used to identify apoptotic cells in vivo. However, unexpectedly, these analyses also revealed that the great majority of PS+ cells were not apoptotic, but rather live cells associated with PS+ extracellular vesicles (EVs). During acute viral infection apoptotic cells increased slightly, while up to 30% of lymphocytes were decorated with PS+ EVs of antigen-presenting cell (APC) exosomal origin. The combination of recombinant fluorescent MFG-E8 and the CAE-method will greatly facilitate analyses of cell death and EVs in vivo

Polymeric prodrug combination to exploit the therapeutic potential of antimicrobial peptides against cancer cells.

Antimicrobial Peptides (AMPs) have unique anticancer properties, but their clinical application is currently limited by an inadequate margin of safety. A prodrug strategy associated with a combination therapy approach could address this limitation by increasing their therapeutic index and their efficacy. Accordingly, the first targeted anticancer polymeric prodrug candidates of AMPs, intended for combination therapy with another polymeric prodrug of an approved antineoplastic agent (doxorubicin), were synthesized as either a PEG-based dual-release prodrug or two individual pegylated prodrugs. The latter are based on a cathepsin B-labile peptide linker and an acid-sensitive acyl hydrazone bond for the AMP and doxorubicin prodrugs, respectively. Anticancer activities and toxicity differentials achieved with the free peptide and its polymer conjugates against ovarian, cancer and non-malignant, cells, indicate that protease-dependent reversible pegylation could be implemented to increase the therapeutic indices of AMPs in cancer therapy. The results obtained also show that this approach can be developed if the releasable PEG linker can be optimised to conciliate the attributes and restrictions of pegylation against proteases. In addition, combination of the polymeric prodrugs of the AMP and of doxorubicin provides additive antitumor effects which could be exploited to enhance the efficacy of the AMP candidate

Polymeric prodrug combination to exploit the therapeutic potential of antimicrobial peptides against cancer cells

Antimicrobial Peptides (AMPs) have unique anticancer properties, but their clinical application is currently limited by an inadequate margin of safety. A prodrug strategy associated with a combination therapy approach could address this limitation by increasing their therapeutic index and their efficacy. Accordingly, the first targeted anticancer polymeric prodrug candidates of AMPs, intended for combination therapy with another polymeric prodrug of an approved antineoplastic agent (doxorubicin), were synthesized as either a PEG-based dual-release prodrug or two individual pegylated prodrugs. The latter are based on a cathepsin B-labile peptide linker and an acid-sensitive acyl hydrazone bond for the AMP and doxorubicin prodrugs, respectively. Anticancer activities and toxicity differentials achieved with the free peptide and its polymer conjugates against ovarian, cancer and non-malignant, cells, indicate that protease-dependent reversible pegylation could be implemented to increase the therapeutic indices of AMPs in cancer therapy. The results obtained also show that this approach can be developed if the releasable PEG linker can be optimised to conciliate the attributes and restrictions of pegylation against proteases. In addition, combination of the polymeric prodrugs of the AMP and of doxorubicin provides additive antitumor effects which could be exploited to enhance the efficacy of the AMP candidate