Lipid tethering of breast tumor cells enables real-time imaging of free-floating cell dynamics and drug response

Funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.Free-floating tumor cells located in the blood of cancer patients, known as circulating tumor cells (CTCs), have become key targets for studying metastasis. However, effective strategies to study the free-floating behavior of tumor cells in vitro have been a major barrier limiting the understanding of the functional properties of CTCs. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells while maintaining their free-floating character. We use polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membranes. This coating remains optically clear, allowing capture of high-resolution images and videos of McTNs on viable free-floating cells. In addition, we show that tethering allows for the real-time analysis of McTN dynamics on individual tumor cells and in response to tubulin-targeting drugs. The ability to image detached tumor cells can vastly enhance our understanding of CTCs under conditions that better recapitulate the microenvironments they encounter during metastasis

Reprogramming the Local Lymph Node Microenvironment Promotes Tolerance that Is Systemic and Antigen Specific

Many experimental therapies for autoimmune diseases, such as multiple sclerosis (MS), aim to bias T cells toward tolerogenic phenotypes without broad suppression. However, the link between local signal integration in lymph nodes (LNs) and the specificity of systemic tolerance is not well understood. We used intra-LN injection of polymer particles to study tolerance as a function of signals in the LN microenvironment. In a mouse MS model, intra-LN introduction of encapsulated myelin self-antigen and a regulatory signal (rapamycin) permanently reversed paralysis after one treatment during peak disease. Therapeutic effects were myelin specific, required antigen encapsulation, and were less potent without rapamycin. This efficacy was accompanied by local LN reorganization, reduced inflammation, systemic expansion of regulatory T cells, and reduced T cell infiltration to the CNS. Our findings suggest that local control over signaling in distinct LNs can promote cell types and functions that drive tolerance that is systemic but antigen specific

Modular Vaccine Design Using Carrier-Free Capsules Assembled from Polyionic Immune Signals

New vaccine adjuvants that direct immune cells toward specific fates could support more potent and selective options for diseases spanning infection to cancer. However, the empirical nature of vaccines and the complexity of many formulations has hindered design of well-defined and easily characterized vaccines. We hypothesized that nanostructured capsules assembled entirely from polyionic immune signals might support a platform for simple, modular vaccines. These immune-polyelectrolyte (iPEM) capsules offer a high signal density, selectively expand T cells in mice, and drive functional responses during tumor challenge. iPEMs incorporating clinically relevant antigens could improve vaccine definition and support more programmable control over immunity

Design of Polyelectrolyte Multilayers to Promote Immunological Tolerance

Recent studies demonstrate that excess signaling through inflammatory pathways (<i>e</i>.<i>g</i>., toll-like receptors, TLRs) contributes to the pathogenesis of human autoimmune diseases, including lupus, diabetes, and multiple sclerosis (MS). We hypothesized that codelivery of a regulatory ligand of TLR9, GpG oligonucleotide, along with myelinthe “self” molecule attacked in MSmight restrain the pro-inflammatory signaling typically present during myelin presentation, redirecting T cell differentiation away from inflammatory populations and toward tolerogenic phenotypes such as regulatory T cells. Here we show that myelin peptide and GpG can be used as modular building blocks for co-assembly into immune polyelectrolyte multilayers (iPEMs). These nanostructured capsules mimic attractive features of biomaterials, including tunable cargo loading and codelivery, but eliminate all carriers and synthetic polymers, components that often exhibit intrinsic inflammatory properties that could exacerbate autoimmune disease. <i>In vitro</i>, iPEMs assembled from myelin and GpG oligonucleotide, but not myelin and a control oligonucleotide, restrain TLR9 signaling, reduce dendritic cell activation, and polarize myelin-specific T cells toward tolerogenic phenotype and function. In mice, iPEMs blunt myelin-triggered inflammatory responses, expand regulatory T cells, and eliminate disease in a common model of MS. Finally, in samples from human MS patients, iPEMs bias myelin-triggered immune cell function toward tolerance. This work represents a unique opportunity to use PEMs to regulate immune function and promote tolerance, supporting iPEMs as a carrier-free platform to alter TLR function to reduce inflammation and combat autoimmunity