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Ferroptosis—an iron-dependent, non-apoptotic cell death process—is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers 1 . The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions 2 – 5 . However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR–Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome–ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis. The cellular organelles peroxisomes contribute to the sensitivity of cells to ferroptosis by synthesizing polyunsaturated ether phospholipids, and changes in the abundances of these lipids are associated with altered sensitivity to ferroptosis during cell-state transitions.

The majority of patients with high grade serous ovarian cancer (HGSOC) develop recurrent disease and chemotherapy resistance. To identify drug combinations that would be effective in treatment of chemotherapy resistant disease, we examined the efficacy of drug combinations that target the three antiapoptotic proteins most commonly expressed in HGSOC—BCL2, BCL‐XL, and MCL1. Co‐inhibition of BCL2 and BCL‐XL (ABT‐263) with inhibition of MCL1 (S63845) induces potent synergistic cytotoxicity in multiple HGSOC models. Since this drug combination is predicted to be toxic to patients due to the known clinical morbidities of each drug, we developed layer‐by‐layer nanoparticles (LbL NPs) that co‐encapsulate these inhibitors in order to target HGSOC tumor cells and reduce systemic toxicities. We show that the LbL NPs can be designed to have high association with specific ovarian tumor cell types targeted in these studies, thus enabling a more selective uptake when delivered via intraperitoneal injection. Treatment with these LbL NPs displayed better potency than free drugs in vitro and resulted in near‐complete elimination of solid tumor metastases of ovarian cancer xenografts. Thus, these results support the exploration of LbL NPs as a strategy to deliver potent drug combinations to recurrent HGSOC. While these findings are described for co‐encapsulation of a BCL2/XL and a MCL1 inhibitor, the modular nature of LbL assembly provides flexibility in the range of therapies that can be incorporated, making LbL NPs an adaptable vehicle for delivery of additional combinations of pathway inhibitors and other oncology drugs.

In addition to systemic inequity, the lack of adequate parental leave conditions at many US institutions can negatively affect career trajectories for women in STEM. Support structures are needed at the institutional, departmental and funding agency level to promote the success of pregnant women and parents with childcare responsibilities in academia.In addition to systemic inequity, the lack of adequate parental leave conditions at many US institutions can negatively affect career trajectories for women in STEM. Support structures are needed at the institutional, departmental and funding agency level to promote the success of pregnant women and parents with childcare responsibilities in academia.

The blood–brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood–brain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood–brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood–brain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.

Degradable polymeric materials such as hydrogels are extensively utilized as delivery vehicles due to their biocompatibility and tunable properties. Encapsulating therapeutic agents inside hydrogels stabilizes the cargo by preventing degradation, extending circulation time, and also allows for targeted release and delivery. Due to their small size and tunable properties, nano-scale hydrogels, or nanogels, are frequently utilized to deliver therapeutics to areas difficult to reach, such as tumors and the cytoplasm, through traditional means. To control hydro- and nanogel function, degradable cross-links can be installed, allowing for cargo release in response to specific stimuli, such as hydrolysis or reduction. This dissertation offers three degradable strategies that can be applied to synthesize hydrogels and nanogels for the stabilization and release of therapeutic cargo. In the first example, mixed imine cross-linking chemistry was applied to synthesize poly(ethylene glycol) (PEG)-based hydrogels with tunable degradability to encapsulate and deliver cells. Time to degradation of the gels could be controlled from 24 hours to more than 7 days by varying the hydrazone structure and the ratio of hydrazone and oxime cross-links. Encapsulated cells exhibited high viability up to at least 7 days, suggesting this system may be useful for cell delivery applications. In the second example, disulfide cross-links were utilized to form redox-responsive nanogels comprised of trehalose copolymers. The synthesis of a methacrylate trehalose monomer (TrMA) was optimized, improving the overall yield from 14% to 42%. TrMA was subsequently copolymerized with pyridyl disulfide ethyl methacrylate (PDSMA) using free radical polymerization conditions to form copolymers with two monomer ratios (1:1 and 2:1) which were cross-linked with 1 kDa PEG-dithiol via disulfide exchange to form uniform nanogels approximately 9 nm in diameter. The addition of a cross-linker eliminated the need to add reducing agent to facilitate cross-linking and nanogel formation, making this approach ideal for the encapsulation of sensitive therapeutic agents. Next, PDSMA-co-TrMA nanogels were utilized to encapsulate, stabilize, and release glucagon, an unstable peptide hormone used to treat hypoglycemia. The amines on glucagon were modified with thiol groups while retaining their positive charges for reversible conjugation and cross-linking. Glucagon-nanogel conjugates were synthesized with >80% conjugation yield, and the reversible disulfide linkage between peptide and polymer allowed for efficient cargo release under mild reducing conditions. The nanogels stabilized glucagon against aggregation in solution up to five days as well as solubilized the peptide at neutral pH. In vitro bioactivity of the modified peptide was found to be comparable to native glucagon, suggesting this may be a promising formulation strategy for further in vivo study. Finally, a series of dual-enzyme responsive peptides was synthesized by masking the ϵ-amine of lysine with protease substrates. After unmasking the amine by enzymatic cleavage, a second enzyme was able to cleave at the C terminus of lysine, which was monitored colorimetrically. Three different dual-enzyme responsive peptides were prepared, (AcAAF)K-pNA, (AcFG)K-pNA, and (AcDEVD)K-pNA, for chymotrypsin, papain, and caspase 3 sensitivity, respectively, followed by trypsin sensitivity after cleavage by the first enzyme. This modular peptide design could be useful for selective drug delivery, studies on dual enzyme activity, as well as for diagnostic enzyme screening.

We report the development of a small molecule-based barcoding platform for pooled screening of nanoparticle delivery. Using aryl halide-based tags (halocodes), we achieve high-sensitivity detection via gas chromatography coupled with mass spectrometry or electron capture. This enables barcoding and tracking of nanoparticles with minimal halocode concentrations and without altering their physicochemical properties. To demonstrate the utility of our platform for pooled screening, we synthesized a halocoded library of polylactide-co-glycolide (PLGA) nanoparticles and quantified uptake in ovarian cancer cells in a pooled manner. Our findings correlate with conventional fluorescence-based assays. Additionally, we demonstrate the potential of halocodes for spatial mapping of nanoparticles using mass spectrometry imaging (MSI). Halocoding presents an accessible and modular nanoparticle screening platform capable of quantifying delivery of pooled nanocarrier libraries in a range of biological settings.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, resulting in significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids from plasma membranes also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

The blood-brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood-brain barrier vasculature. Here, we report a vascularized human glioblastoma (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood-brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood-brain barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics. Competing Interest Statement RDK is a co-founder of AIM Biotech that markets microfluidic systems for 3-dimensional culture and receives research funding from Amgen and Biogen. PTH is a co-founder and member of the board of LayerBio, a member of the Board of Alector, a member of the Scientific Advisory Board of Moderna, and receives research funding from Shepherd Pharmaceuticals, Novartis, and SecuraBio, all for work unrelated to this manuscript. All other authors report no competing interests.

To accelerate the translation of cancer nanomedicine, we hypothesize that integrated genomic screens will improve understanding of the cellular processes governing nanoparticle trafficking. We developed a massively parallel high-throughput screening method leveraging barcoded, pooled cancer cell lines annotated with multi-omic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. This approach identified both the materials properties and cell-intrinsic features mediating nanoparticle-cell association. Coupling the data with machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers, including gene expression of SLC46A3. We engineered cell lines to validate SLC46A3 as a biomarker whose expression inversely predicts liposomal nanoparticle uptake both in vitro and in vivo. We further demonstrated the predictive capabilities extend beyond liposomal nanoparticles, regulating both uptake and transfection efficacy of solid lipid nanoparticles. Our work establishes the power of massively parallel pooled cell screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations for specific patient populations. Competing Interest Statement The authors have declared no competing interest. Footnotes * We have carried out experiments that expand biomarker findings related to nanoparticle trafficking.