Hydrogel-encapsulation to enhance bacterial diagnosis of colon inflammation.

Bacteria can be genetically programmed to sense and report the presence of disease biomarkers in the gastrointestinal (GI) tract. However, diagnostic bacteria are typically delivered via oral administration of liquid cultures, resulting in poor survival and high dispersal in vivo. These limitations confound recovery and analysis of engineered bacteria from GI or stool samples. Here, we demonstrate that encapsulating bacteria inside of alginate core-shell particles enables robust survival, containment, and diagnostic function in vivo. We demonstrate these benefits by encapsulating a strain engineered to report the presence of the biomarker thiosulfate via fluorescent protein expression in order to diagnose dextran sodium sulfate-induced colitis in rats. Hydrogel-encapsulated bacteria engineered to sense and respond to physiological stimuli should enable minimally invasive monitoring of a wide range of diseases and have applications as next-generation smart therapeutics.

Development of Serum-Free Media for Cryopreservation of Hydrogel Encapsulated Cell-Based Therapeutics.

Introduction: While hydrogel encapsulation of cells has been developed to treat multiple diseases, methods to cryopreserve and maintain the composite function of therapeutic encapsulated cell products are still needed to facilitate their storage and distribution. While methods to preserve encapsulated cells, and post-synthesis have received recent attention, effective preservation mediums have not been fully defined. Methods: We employed a two-tiered screen of an initial library of 32 different cryopreservation agent (CPA) formulations composed of different cell-permeable and impermeable agents. Formulations were assayed using dark field microscopy to evaluate alginate hydrogel matrix integrity, followed by cell viability analyses and measurements of functional secretion activity. Results: The structural integrity of large > 1 mm alginate capsules were highly sensitive to freezing and thawing in media alone but could be recovered by a number of CPA formulations containing different cell-permeable and impermeable agents. Subsequent viability screens identified two top-performing CPA formulations that maximized capsule integrity and cell viability after storage at - 80 °C. The top formulation (10% Dimethyl sulfoxide (DMSO) and 0.3 M glucose) was demonstrated to preserve hydrogel integrity and retain cell viability beyond a critical USA FDA set 70% viability threshold while maintaining protein secretion and resultant cell potency. Conclusions: This prioritized screen identified a cryopreservation solution that maintains the integrity of large alginate capsules and yields high viabilities and potency. Importantly, this formulation is serum-free, non-toxic, and can support the development of clinically translatable encapsulated cell-based therapeutics. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00739-7.

Assessing Gq-GPCR-induced human astrocyte reactivity using bioengineered neural organoids.

Astrocyte reactivity can directly modulate nervous system function and immune responses during disease and injury. However, the consequence of human astrocyte reactivity in response to specific contexts and within neural networks is obscure. Here, we devised a straightforward bioengineered neural organoid culture approach entailing transcription factor-driven direct differentiation of neurons and astrocytes from human pluripotent stem cells combined with genetically encoded tools for dual cell-selective activation. This strategy revealed that Gq-GPCR activation via chemogenetics in astrocytes promotes a rise in intracellular calcium followed by induction of immediate early genes and thrombospondin 1. However, astrocytes also undergo NF-κB nuclear translocation and secretion of inflammatory proteins, correlating with a decreased evoked firing rate of cocultured optogenetic neurons in suboptimal conditions, without overt neurotoxicity. Altogether, this study clarifies the intrinsic reactivity of human astrocytes in response to targeting GPCRs and delivers a bioengineered approach for organoid-based disease modeling and preclinical drug testing.

Activation of Adaptive and Innate Immune Cells via Localized IL2 Cytokine Factories Eradicates Mesothelioma Tumors.

PURPOSE: IL2 immunotherapy has the potential to elicit immune-mediated tumor lysis via activation of effector immune cells, but clinical utility is limited due to pharmacokinetic challenges as well as vascular leak syndrome and other life-threatening toxicities experienced by patients. We developed a safe and clinically translatable localized IL2 delivery system to boost the potency of therapy while minimizing systemic cytokine exposure. EXPERIMENTAL DESIGN: We evaluated the therapeutic efficacy of IL2 cytokine factories in a mouse model of malignant mesothelioma. Changes in immune populations were analyzed using time-of-flight mass cytometry (CyTOF), and the safety and translatability of the platform were evaluated using complete blood counts and serum chemistry analysis. RESULTS: IL2 cytokine factories enabled 150× higher IL2 concentrations in the local compartment with limited leakage into the systemic circulation. AB1 tumor burden was reduced by 80% after 1 week of monotherapy treatment, and 7 of 7 of animals exhibited tumor eradication without recurrence when IL2 cytokine factories were combined with anti-programmed cell death protein 1 (aPD1). Furthermore, CyTOF analysis showed an increase in CD69+CD44+ and CD69-CD44+CD62L- T cells, reduction of CD86-PD-L1- M2-like macrophages, and a corresponding increase in CD86+PD-L1+ M1-like macrophages and MHC-II+ dendritic cells after treatment. Finally, blood chemistry ranges in rodents demonstrated the safety of cytokine factory treatment and reinforced its potential for clinical use. CONCLUSIONS: IL2 cytokine factories led to the eradication of aggressive mouse malignant mesothelioma tumors and protection from tumor recurrence, and increased the therapeutic efficacy of aPD1 checkpoint therapy. This study provides support for the clinical evaluation of this IL2-based delivery system. See related commentary by Palanki et al., p. 5010.

Clinically translatable cytokine delivery platform for eradication of intraperitoneal tumors.

Proinflammatory cytokines have been approved by the Food and Drug Administration for the treatment of metastatic melanoma and renal carcinoma. However, effective cytokine therapy requires high-dose infusions that can result in antidrug antibodies and/or systemic side effects that limit long-term benefits. To overcome these limitations, we developed a clinically translatable cytokine delivery platform composed of polymer-encapsulated human ARPE-19 (RPE) cells that produce natural cytokines. Tumor-adjacent administration of these capsules demonstrated predictable dose modulation with spatial and temporal control and enabled peritoneal cancer immunotherapy without systemic toxicities. Interleukin-2 (IL2)-producing cytokine factory treatment eradicated peritoneal tumors in ovarian and colorectal mouse models. Furthermore, computational pharmacokinetic modeling predicts clinical translatability to humans. Notably, this platform elicited T cell responses in NHPs, consistent with reported biomarkers of treatment efficacy without toxicity. Combined, our findings demonstrate the safety and efficacy of IL2 cytokine factories in preclinical animal models and provide rationale for future clinical testing in humans.

Clinical translation of immunomodulatory therapeutics.

Immunomodulatory therapeutics represent a unique class of drug products that have tremendous potential to rebalance malfunctioning immune systems and are quickly becoming one of the fastest-growing areas in the pharmaceutical industry. For these drugs to become mainstream medicines, they must provide greater therapeutic benefit than the currently used treatments without causing severe toxicities. Immunomodulators, cell-based therapies, antibodies, and viral therapies have all achieved varying amounts of success in the treatment of cancers and/or autoimmune diseases. However, many challenges related to precision dosing, off-target effects, and manufacturing hurdles will need to be addressed before we see widespread adoption of these therapies in the clinic. This review provides a perspective on the progress of immunostimulatory and immunosuppressive therapies to date and discusses the opportunities and challenges for clinical translation of the next generation of immunomodulatory therapeutics.

Targeting the extracellular matrix for immunomodulation: applications in drug delivery and cell therapies.

Host immune cells interact bi-directionally with their extracellular matrix (ECM) to receive and deposit molecular signals, which orchestrate cellular activation, proliferation, differentiation, and function to maintain healthy tissue homeostasis. In response to pathogens or damage, immune cells infiltrate diseased sites and synthesize critical ECM molecules such as glycoproteins, proteoglycans, and glycosaminoglycans to promote healing. When the immune system misidentifies pathogens or fails to survey damaged cells effectively, maladies such as chronic inflammation, autoimmune diseases, and cancer can develop. In these conditions, it is essential to restore balance to the body through modulation of the immune system and the ECM. This review details the components of dysregulated ECM implicated in pathogenic environments and therapeutic approaches to restore tissue homeostasis. We evaluate emerging strategies to overcome inflamed, immune inhibitory, and otherwise diseased microenvironments, including mechanical stimulation, targeted proteases, adoptive cell therapy, mechanomedicine, and biomaterial-based cell therapeutics. We highlight various strategies that have produced efficacious responses in both pre-clinical and human trials and identify additional opportunities to develop next-generation interventions. Significantly, we identify a need for therapies to address dense or fibrotic tissue for the treatment of organ tissue damage and various cancer subtypes. Finally, we conclude that therapeutic techniques that disrupt, evade, or specifically target the pathogenic microenvironment have a high potential for improving therapeutic outcomes and should be considered a priority for immediate exploration. A schematic showing the various methods of extracellular matrix disruption/targeting in both fibrotic and cancerous environments. a Biomaterial-based cell therapy can be used to deliver anti-inflammatory cytokines, chemotherapeutics, or other factors for localized, slow release of therapeutics. b Mechanotherapeutics can be used to inhibit the deposition of molecules such as collagen that affect stiffness. c Ablation of the ECM and target tissue can be accomplished via mechanical degradation such as focused ultrasound. d Proteases can be used to improve the distribution of therapies such as oncolytic virus. e Localization of therapeutics such as checkpoint inhibitors can be improved with the targeting of specific ECM components, reducing off-target effects and toxicity.

The surface topography of silicone breast implants mediates the foreign body response in mice, rabbits and humans.

Silicone is widely used in chronic implants and is generally perceived to be safe. However, textured breast implants have been associated with immune-related complications, including malignancies. Here, by examining for up to one year the foreign body response and capsular fibrosis triggered by miniaturized or full-scale clinically approved breast implants with different surface topography (average roughness, 0-90 µm) placed in the mammary fat pads of mice or rabbits, respectively, we show that surface topography mediates immune responses to the implants. We also show that the surface surrounding human breast implants collected during revision surgeries also differentially alters the individual's immune responses to the implant. Moreover, miniaturized implants with an average roughness of 4 µm can largely suppress the foreign body response and fibrosis (but not in T-cell-deficient mice), and that tissue surrounding these implants displayed higher levels of immunosuppressive FOXP3+ regulatory T cells. Our findings suggest that, amongst the topographies investigated, implants with an average roughness of 4 µm provoke the least amount of inflammation and foreign body response.

Immune-modulatory alginate protects mesenchymal stem cells for sustained delivery of reparative factors to ischemic myocardium.

Paracrine factors secreted by mesenchymal stem cells (MSCs) have been previously shown to improve cardiac function following acute myocardial infarction (MI). However, cell therapy activates the innate immune response, leading to the rapid elimination of transplanted cells and only short-term therapeutic delivery. Herein, we describe a new strategy to deliver sustained paracrine-mediated MSC therapy to ischemic myocardium. Using an immune evasive, small molecule modified alginate, we encapsulated rat MSC cells in a core-shell hydrogel capsule and implanted them in the pericardial sac of post-MI rats. Encapsulated cells allowed diffusion of reparative paracrine factors at levels similar to non-encapsulated cells in vitro. Encapsulation enabled sustained cell survival with localization over the heart for 2 weeks. The effect of the experimental group on ventricular function and fibrosis was compared with blank (cell free) capsules and unencapsulated MSCs injected into infarcted myocardium. MSC capsules improved post-MI ventricular function ∼2.5× greater than MSC injection. After 4 weeks, post-MI fibrosis was reduced ∼2/3 with MSC capsules, but unchanged with MSC injection. MSC encapsulation with alginate core-shell capsules sustains cell survival and potentiates efficacy of therapy.