A 3D in vitro assay to study combined immune cell infiltration and cytotoxicity.

Immune cell-mediated killing of cancer cells in a solid tumor is prefaced by a multi-step infiltration cascade of invasion, directed migration, and cytotoxic activities. In particular, immune cells must invade and migrate through a series of different extracellular matrix (ECM) boundaries and domains before reaching and killing their target tumor cells. These infiltration events are a central challenge to the clinical success of CAR T cells against solid tumors. The current standard in vitro cell killing assays measure cell cytotoxicity in an obstacle-free, two-dimensional (2D) microenvironment, which precludes the study of 3D immune cell-ECM interactions. Here, we present a 3D combined infiltration/cytotoxicity assay based on an oil-in-water microtechnology. This assay measures stromal invasion following extravasation, migration through the stromal matrix, and invasion of the solid tumor in addition to cell killing. We compare this 3D cytotoxicity assay to the benchmark 2D assay through tumor assembloid cocultures with immune cells and engineered immune cells. This assay is amenable to an array of imaging techniques, which allows direct observation and quantification of each stage of infiltration in different immune and oncological contexts. We establish the 3D infiltration/cytotoxicity assay as an important tool for the mechanistic study of immune cell interactions with the tumor microenvironment.

Engineering self-propelled tumor-infiltrating CAR T cells using synthetic velocity receptors.

Chimeric antigen receptor (CAR) T cells express antigen-specific synthetic receptors, which upon binding to cancer cells, elicit T cell anti-tumor responses. CAR T cell therapy has enjoyed success in the clinic for hematological cancer indications, giving rise to decade-long remissions in some cases. However, CAR T therapy for patients with solid tumors has not seen similar success. Solid tumors constitute 90% of adult human cancers, representing an enormous unmet clinical need. Current approaches do not solve the central problem of limited ability of therapeutic cells to migrate through the stromal matrix. We discover that T cells at low and high density display low- and high-migration phenotypes, respectively. The highly migratory phenotype is mediated by a paracrine pathway from a group of self-produced cytokines that include IL5, TNFα, IFNγ, and IL8. We exploit this finding to "lock-in" a highly migratory phenotype by developing and expressing receptors, which we call velocity receptors (VRs). VRs target these cytokines and signal through these cytokines' cognate receptors to increase T cell motility and infiltrate lung, ovarian, and pancreatic tumors in large numbers and at doses for which control CAR T cells remain confined to the tumor periphery. In contrast to CAR therapy alone, VR-CAR T cells significantly attenuate tumor growth and extend overall survival. This work suggests that approaches to the design of immune cell receptors that focus on migration signaling will help current and future CAR cellular therapies to infiltrate deep into solid tumors.

Mechano-induced homotypic patterned domain formation by monocytes.

Matrix stiffness and corresponding mechano-signaling play indispensable roles in cellular phenotypes and functions. How tissue stiffness influences the behavior of monocytes, a major circulating leukocyte of the innate system, and how it may promote the emergence of collective cell behavior is less understood. Here, using tunable collagen-coated hydrogels of physiological stiffness, we show that human primary monocytes undergo a dynamic local phase separation to form highly regular, reversible, multicellular, multi-layered domains on soft matrix. Local activation of the ß2 integrin initiates inter-cellular adhesion, while global soluble inhibitory factors maintain the steady state domain pattern over days. Patterned domain formation generated by monocytes is unique among other key immune cells, including macrophages, B cells, T cells, and NK cells. While inhibiting their phagocytic capability, domain formation promotes monocytes' survival. We develop a computational model based on the Cahn-Hilliard equation of phase separation, combined with a Turing mechanism of local activation and global inhibition suggested by our experiments, and provides experimentally validated predictions of the role of seeding density and both chemotactic and random cell migration on domain pattern formation. This work reveals that, unlike active matters, cells can generate complex cell phases by exploiting their mechanosensing abilities and combined short-range interactions and long-range signals to enhance their survival.

Self-assembling nanofibrous bacteriophage microgels as sprayable antimicrobials targeting multidrug-resistant bacteria.

Nanofilamentous bacteriophages (bacterial viruses) are biofunctional, self-propagating, and monodisperse natural building blocks for virus-built materials. Minifying phage-built materials to microscale offers the promise of expanding the range function for these biomaterials to sprays and colloidal bioassays/biosensors. Here, we crosslink half a million self-organized phages as the sole structural component to construct each soft microgel. Through an in-house developed, biologics-friendly, high-throughput template method, over 35,000 phage-built microgels are produced from every square centimetre of a peelable microporous film template, constituting a 13-billion phage community. The phage-exclusive microgels exhibit a self-organized, highly-aligned nanofibrous texture and tunable auto-fluorescence. Further preservation of antimicrobial activity was achieved by making hybrid protein-phage microgels. When loaded with potent virulent phages, these microgels effectively reduce heavy loads of multidrug-resistant Escherichia coli O157:H7 on food products, leading to up to 6 logs reduction in 9 hours and rendering food contaminant free.