An immunogenic cell injury module for the single-cell multiplexed activity metabolomics platform to identify promising anti-cancer natural products.

Natural products constitute and significantly impact many current anti-cancer medical interventions. A subset of natural products induces injury processes in malignant cells that recruit and activate host immune cells to produce an adaptive anti-cancer immune response, a process known as immunogenic cell death. However, a challenge in the field is to delineate forms of cell death and injury that best promote durable antitumor immunity. Addressing this with a single-cell chemical biology natural product discovery platform, like multiplex activity metabolomics, would be especially valuable in human leukemia, where cancer cells are heterogeneous and may react differently to the same compounds. Herein, a new ten-color, fluorescent cell barcoding-compatible module measuring six immunogenic cell injury signaling readouts are as follows: DNA damage response (γH2AX), apoptosis (cCAS3), necroptosis (p-MLKL), mitosis (p-Histone H3), autophagy (LC3), and the unfolded protein response (p-EIF2α). A proof-of-concept screen was performed to validate functional changes in single cells induced by secondary metabolites with known mechanisms within bacterial extracts. This assay was then applied in multiplexed activity metabolomics to reveal an unexpected mammalian cell injury profile induced by the natural product narbomycin. Finally, the functional consequences of injury pathways on immunogenicity were compared with three canonical assays for immunogenic hallmarks, ATP, HMGB1, and calreticulin, to correlate secondary metabolite-induced cell injury profiles with canonical markers of immunogenic cell death. In total, this work demonstrated a new phenotypic screen for discovery of natural products that modulate injury response pathways that can contribute to cancer immunogenicity.

Electrospinning Drug-Loaded Alginate-Based Nanofibers towards Developing a Drug Release Rate Catalog.

Electrospinning natural polymers represents a developing interest in the field of biomaterials. Electrospun nanofibers have been shown to facilitate tissue regeneration and emulate body tissue, making them ideal for modern biomedical applications. These water-soluble natural polymers including alginate, have also shown promise as drug delivery vehicles. However, many biopolymers including alginate are inherently charged, making the formation of nanofibers difficult. To better understand the potential of natural polymer-based fibers in drug delivery applications, fiber formulations and drug loading concentrations of alginate-based scaffolds were investigated. It was found electrospinning poly(vinyl alcohol) with alginate facilitated fiber formation while the co-polymer agarose showed minor improvement in terms of alginate electrospinnability. Once uniform fibers were formed, the antibiotic ciprofloxacin was added into the polymer electrospinning solution to yield drug-loaded nanofibers. These optimized parameters coupled with small molecule release rate data from the drug-loaded, alginate-based fibers have been used to establish a catalog of small molecule release profiles. In the future, this catalog will be further expanded to include drug release rate data from other innately charged natural polymer-based fibers such as chitosan. It is anticipated that the cataloged profiles can be applied in the further development of biomaterials used in drug delivery.