Metabolic labeling and targeted modulation of dendritic cells.

Targeted immunomodulation of dendritic cells (DCs) in vivo will enable manipulation of T-cell priming and amplification of anticancer immune responses, but a general strategy has been lacking. Here we show that DCs concentrated by a biomaterial can be metabolically labelled with azido groups in situ, which allows for their subsequent tracking and targeted modulation over time. Azido-labelled DCs were detected in lymph nodes for weeks, and could covalently capture dibenzocyclooctyne (DBCO)-bearing antigens and adjuvants via efficient Click chemistry for improved antigen-specific CD8+ T-cell responses and antitumour efficacy. We also show that azido labelling of DCs allowed for in vitro and in vivo conjugation of DBCO-modified cytokines, including DBCO-IL-15/IL-15Rα, to improve priming of antigen-specific CD8+ T cells. This DC labelling and targeted modulation technology provides an unprecedented strategy for manipulating DCs and regulating DC-T-cell interactions in vivo.

Privileged Antigen Presentation in Splenic B Cell Follicles Maximizes T Cell Responses in Prime-Boost Vaccination.

Effector T cells (TEFF) are a barrier to booster vaccination because they can rapidly kill Ag-bearing APCs before memory T cells are engaged. We report in this study that i.v. delivery of rhabdoviral vectors leads to direct infection of follicular B cells in the spleen, where the earliest evidence of secondary T cell responses was observed. This allows booster immunizations to rapidly expand CD8(+) central memory T cells (TCM) during the acute phase of the primary response that is dominated by TEFF Interestingly, although the ablation of B cells before boosting with rhabdoviral vectors diminishes the expansion of memory T cells, B cells do not present Ags directly. Instead, depletion of CD11c(+) dendritic cells abrogates secondary T cell expansion, suggesting that virus-infected follicular B cells may function as an Ag source for local DCs to subsequently capture and present the Ag. Because TCM are located within B cell follicles in the spleen whereas TEFF cannot traffic through follicular regions, Ag production and presentation by follicular APCs represent a unique mechanism to secure engagement of TCM during an ongoing effector response. Our data offer insights into novel strategies for rapid expansion of CD8(+) T cells using prime-boost vaccines by targeting privileged sites for Ag presentation.

Adjuvant-Loaded Subcellular Vesicles Derived From Disrupted Cancer Cells for Cancer Vaccination.

Targeted subunit vaccines for cancer immunotherapy do not capture tumor antigenic complexity, and approaches employing tumor lysate are often limited by inefficient antigen uptake and presentation, and low immunogenicity. Here, whole cancer cells are processed to generate antigen-rich, membrane-enclosed subcellular particles, termed "reduced cancer cells", that reflect the diversity and breadth of the parent cancer cell antigen repertoire, and can be loaded with disparate adjuvant payloads. These vesicular particles enhance the uptake of the adjuvant payload, and potentiate the activation of primary dendritic cells in vitro. Similarly, reduced cancer cell-associated antigens are more efficiently presented by primary dendritic cells in vitro than their soluble counterparts or lysate control. In mice, vaccination using adjuvant-loaded reduced cancer cells facilitates the induction of antigen-specific cellular and humoral immune responses. Taken together, these observations demonstrate that adjuvant-loaded reduced cancer cells could be utilized in cancer vaccines as an alternative to lysate.

Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation.

The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.

Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium.

In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through ß4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6ß4 integrin clustering into hemidesmosomes.

Potentiating cancer immunotherapy using an oncolytic virus.

Oncolytic viruses (OVs) are highly immunogenic and this limits their use in immune-competent hosts. Although immunosuppression may improve viral oncolysis, this gain is likely achieved at the cost of antitumoral immunity. We have developed a strategy wherein the immune response against the OV leads to enhanced therapeutic outcomes. We demonstrate that immunization with an adenoviral (Ad) vaccine before treatment with an oncolytic vesicular stomatitis virus (VSV) expressing the same tumor antigen (Ag) leads to significantly enhanced antitumoral immunity. Intratumoral replication of VSV was minimally attenuated in Ad-immunized hosts but extending the interval between treatments reduced the attenuating effect and further increased antitumoral immunity. More importantly, our combination approach shifted the immune response from viral Ags to tumor Ags and further reduced OV replication in normal tissues, leading to enhancements in both efficacy and safety. These studies also highlight the benefits of using a replicating, OV to boost a pre-existing antitumoral immune response as this approach generated larger responses versus tumor Ag in tumor-bearing hosts than could be achieved in tumor-free hosts. This strategy should be applicable to other vector combinations, tumor Ags, and tumor targets.

Vesicular stomatitis virus as a novel cancer vaccine vector to prime antitumor immunity amenable to rapid boosting with adenovirus.

Vesicular stomatitis virus (VSV) has proven to be an effective vaccine vector for immunization against viral infection, but its potential to induce an immune response to a self-tumor antigen has not been investigated. We constructed a recombinant VSV expressing human dopachrome tautomerase (hDCT) and evaluated its immunogenicity in a murine melanoma model. Intranasal delivery of VSV-hDCT activated both CD4(+) and CD8(+) DCT-specific T-cell responses. The magnitude of these responses could be significantly increased by booster immunization with recombinant adenovirus (Ad)-hDCT, which led to enhanced efficacy against B16-F10 melanoma in both prophylactic and therapeutic settings. Notably, the interval of VSV/Ad heterologous vaccination could be shortened to as few as 4 days, making it a potential regimen to rapidly expand antigen-specific effector cells. Furthermore, VSV-hDCT could increase DCT-specific T-cell responses primed by Ad-hDCT, suggesting VSV is efficient for both priming and boosting of the immune response against a self-tumor antigen.

Anti-tumor immunity induced by ectopic expression of viral antigens is transient and limited by immune escape.

Immunotherapeutic treatments in head and neck cancer clinical trials include cancer vaccines targeting foreign viral antigens or mutational neoantigens derived from cancer-expressed proteins. Anti-tumor immune responses place cancer cells under selective pressure to lose or downregulate target antigens; therefore, vaccination against virus- or host- "driver" oncogenes are proposed as a strategy to overcome immune escape. Herein, we demonstrate the impact of immunogenic viral antigens on anti-tumor response and immune editing in MOC2-E6E7, a syngeneic murine oral cancer cell line expressing HPV-16 E6 and E7 oncoproteins. Using orthotopic syngeneic models, we observed in vivo tumor growth kinetics of MOC2-E6E7 is delayed in immunocompetent mice compared to parental MOC2 tumors. In contrast, tumor growth remained similar in Rag1-/- mice lacking adaptive immunity. MOC2-E6E7 tumors demonstrated an "inflamed" or immune-activated tumor microenvironment and greater infiltration of CD8+ T cells compared to MOC2. By real-time PCR, we detected downregulation of E6 and E7 genes in MOC2-E6E7 tumors only in immunocompetent mice, suggesting the loss of ectopic viral antigen expression due to immune editing. We then assessed the efficacy of a biomaterials-based mesoporous silica rod (MSR) cancer vaccine targeting HPV-16 E7 in our model. Vaccination induced robust infiltration of antigen-specific CD8+ T cells, which led to tumor growth delay and modestly prolonged survival in MOC2-E6E7 tumors. Increased efficacy was seen in a separate head and neck cancer tumor model, mEER, which obligately expresses E7 antigen. Collectively, our data highlight the need for both immunogenicity and 'driver' status of target antigens to be considered in cancer vaccine design.

Scaffolds that mimic antigen-presenting cells enable ex vivo expansion of primary T cells.

Therapeutic ex vivo T-cell expansion is limited by low rates and T-cell products of limited functionality. Here we describe a system that mimics natural antigen-presenting cells (APCs) and consists of a fluid lipid bilayer supported by mesoporous silica micro-rods. The lipid bilayer presents membrane-bound cues for T-cell receptor stimulation and costimulation, while the micro-rods enable sustained release of soluble paracrine cues. Using anti-CD3, anti-CD28, and interleukin-2, we show that the APC-mimetic scaffolds (APC-ms) promote two- to tenfold greater polyclonal expansion of primary mouse and human T cells compared with commercial expansion beads (Dynabeads). The efficiency of expansion depends on the density of stimulatory cues and the amount of material in the starting culture. Following a single stimulation, APC-ms enables antigen-specific expansion of rare cytotoxic T-cell subpopulations at a greater magnitude than autologous monocyte-derived dendritic cells after 2 weeks. APC-ms support over fivefold greater expansion of restimulated CD19 CAR-T cells than Dynabeads, with similar efficacy in a xenograft lymphoma model.

Injectable nanocomposite cryogels for versatile protein drug delivery.

Sustained, localized protein delivery can enhance the safety and activity of protein drugs in diverse disease settings. While hydrogel systems are widely studied as vehicles for protein delivery, they often suffer from rapid release of encapsulated cargo, leading to a narrow duration of therapy, and protein cargo can be denatured by incompatibility with the hydrogel crosslinking chemistry. In this work, we describe injectable nanocomposite hydrogels that are capable of sustained, bioactive, release of a variety of encapsulated proteins. Injectable and porous cryogels were formed by bio-orthogonal crosslinking of alginate using tetrazine-norbornene coupling. To provide sustained release from these hydrogels, protein cargo was pre-adsorbed to charged Laponite nanoparticles that were incorporated within the walls of the cryogels. The presence of Laponite particles substantially hindered the release of a number of proteins that otherwise showed burst release from these hydrogels. By modifying the Laponite content within the hydrogels, the kinetics of protein release could be precisely tuned. This versatile strategy to control protein release simplifies the design of hydrogel drug delivery systems. STATEMENT OF SIGNIFICANCE: Here we present an injectable nanocomposite hydrogel for simple and versatile controlled release of therapeutic proteins. Protein release from hydrogels often requires first entrapping the protein in particles and embedding these particles within the hydrogel to allow controlled protein release. This pre-encapsulation process can be cumbersome, can damage the protein's activity, and must be optimized for each protein of interest. The strategy presented in this work simply premixes the protein with charged nanoparticles that bind strongly with the protein. These protein-laden particles are then placed within a hydrogel and slowly release the protein into the surrounding environment. Using this method, tunable release from an injectable hydrogel can be achieved for a variety of proteins. This strategy greatly simplifies the design of hydrogel systems for therapeutic protein release applications.

Injectable, Tough Alginate Cryogels as Cancer Vaccines.

A covalently crosslinked methacrylated (MA)-alginate cryogel vaccine has been previously shown to generate a potent response against murine melanoma, but is not mechanically robust and requires a large 16G needle for delivery. Here, covalent and ionic crosslinking of cryogels are combined with the hypothesis that this will result in a tough MA-alginate cryogel with improved injectability. All tough cryogels can be injected through a smaller, 18G needle without sustaining any damage, while covalently crosslinked-only cryogels break after injection. Cytosine-phosphodiester-guanine (CpG)-delivering tough cryogels effectively activate dendritic cells (DCs). Granulocyte macrophage colony-stimulating factor releasing tough cryogels recruit four times more DCs than blank gels by day 7 in vivo. The tough cryogel vaccine induces strong antigen-specific cytotoxic T-lymphocyte and humoral responses. These vaccines prevent tumor formation in 80% of mice inoculated with HER2/neu-overexpressing DD breast cancer cells. The MA-alginate tough cryogels provide a promising minimally invasive delivery platform for cancer vaccinations.

Liposomal Delivery Enhances Immune Activation by STING Agonists for Cancer Immunotherapy.

Overcoming the immunosuppressive tumor microenvironment (TME) is critical to realizing the potential of cancer immunotherapy strategies. Agonists of stimulator of interferon genes (STING), a cytosolic immune adaptor protein, have been shown to induce potent anti-tumor activity when delivered into the TME. However, the anionic properties of STING agonists make them poorly membrane permeable, and limit their ability to engage STING in the cytosol of responding cells. In this study, cationic liposomes with varying surface polyethylene glycol (PEG) levels were used to encapsulate cGAMP to facilitate its cytosolic delivery. In vitro studies with antigen-presenting cells (APCs) revealed that liposomal formulations substantially improved the cellular uptake of cGAMP and pro-inflammatory gene induction relative to free drug. Liposomal encapsulation allowed cGAMP delivery to metastatic melanoma tumors in the lung, leading to anti-tumor activity, whereas free drug produced no effect at the same dose. Injection of liposomal cGAMP into orthotopic melanoma tumors showed retention of cGAMP at the tumor site and co-localization with tumor-associated APCs. Liposomal delivery induced regression of injected tumors and produced immunological memory that protected previously treated mice from rechallenge with tumor cells. These results show that liposomal delivery improves STING agonist activity, and could improve their utility in clinical oncology.

In Vivo Enrichment of Diabetogenic T Cells.

Dysfunctional T cells can mediate autoimmunity, but the inaccessibility of autoimmune tissues and the rarity of autoimmune T cells in the blood hinder their study. We describe a method to enrich and harvest autoimmune T cells in vivo by using a biomaterial scaffold loaded with protein antigens. In model antigen systems, we found that antigen-specific T cells become enriched within scaffolds containing their cognate antigens. When scaffolds containing lysates from an insulin-producing ß-cell line were implanted subcutaneously in autoimmune diabetes-prone NOD mice, ß-cell-reactive T cells homed to these scaffolds and became enriched. These T cells induced diabetes after adoptive transfer, indicating their pathogenicity. Furthermore, T-cell receptor (TCR) sequencing identified many expanded TCRs within the ß-cell scaffolds that were also expanded within the pancreata of NOD mice. These data demonstrate the utility of biomaterial scaffolds loaded with disease-specific antigens to identify and study rare, therapeutically important T cells.

Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip.

An in vitro model of the human kidney glomerulus - the major site of blood filtration - could facilitate drug discovery and illuminate kidney-disease mechanisms. Microfluidic organ-on-a-chip technology has been used to model the human proximal tubule, yet a kidney-glomerulus-on-a-chip has not been possible because of the lack of functional human podocytes - the cells that regulate selective permeability in the glomerulus. Here, we demonstrate an efficient (> 90%) and chemically defined method for directing the differentiation of human induced pluripotent stem (hiPS) cells into podocytes that express markers of the mature phenotype (nephrin+, WT1+, podocin+, Pax2-) and that exhibit primary and secondary foot processes. We also show that the hiPS-cell-derived podocytes produce glomerular basement-membrane collagen and recapitulate the natural tissue/tissue interface of the glomerulus, as well as the differential clearance of albumin and inulin, when co-cultured with human glomerular endothelial cells in an organ-on-a-chip microfluidic device. The glomerulus-on-a-chip also mimics adriamycin-induced albuminuria and podocyte injury. This in vitro model of human glomerular function with mature human podocytes may facilitate drug development and personalized-medicine applications.

Biomaterials for enhancing anti-cancer immunity.

Cancer immunotherapy is becoming a standard approach to treat many cancers. However, shortcomings of current methods limit therapeutic benefit in many patients. Rationally designed biomaterial strategies to deliver immune modulatory drugs can potentially show improved safety profiles, while providing multifunctional and spatiotemporally controlled signals to immune cells to improve their anti-cancer activity. This brief review describes biomaterials-based strategies that enhance immune cell function at various tissue sites to improve anti-cancer immunity. Continued collaboration between bioengineers, immunologists, industry, and clinicians is required for biomaterial-based immunotherapy strategies to continue moving to the clinic.

CD44 alternative splicing in gastric cancer cells is regulated by culture dimensionality and matrix stiffness.

Two-dimensional (2D) cultures often fail to mimic key architectural and physical features of the tumor microenvironment. Advances in biomaterial engineering allow the design of three-dimensional (3D) cultures within hydrogels that mimic important tumor-like features, unraveling cancer cell behaviors that would not have been observed in traditional 2D plastic surfaces. This study determined how 3D cultures impact CD44 alternative splicing in gastric cancer (GC) cells. In 3D cultures, GC cells lost expression of the standard CD44 isoform (CD44s), while gaining CD44 variant 6 (CD44v6) expression. This splicing switch was reversible, accelerated by nutrient shortage and delayed at lower initial cell densities, suggesting an environmental stress-induced response. It was further shown to be dependent on the hydrogel matrix mechanical properties and accompanied by the upregulation of genes involved in epithelial-mesenchymal transition (EMT), metabolism and angiogenesis. The 3D cultures reported here revealed the same CD44 alternative splicing pattern previously observed in human premalignant and malignant gastric lesions. These findings indicate that fundamental features of 3D cultures - such as soluble factors diffusion and mechanical cues - influence CD44 expression in GC cells. Moreover, this study provides a new model system to study CD44 dysfunction, whose role in cancer has been in the spotlight for decades.

Click-Crosslinked Injectable Gelatin Hydrogels.

Injectable gelatin hydrogels formed with bioorthogonal click chemistry (ClickGel) are cell-responsive ECM mimics for in vitro and in vivo biomaterials applications. Gelatin polymers with pendant norbornene (GelN) or tetrazine (GelT) groups can quickly and spontaneously crosslink upon mixing, allowing for high viability of encapsulated cells, establishment of 3D elongated cell morphologies, and biodegradation when injected in vivo.

Versatile click alginate hydrogels crosslinked via tetrazine-norbornene chemistry.

Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.

Injectable, porous, and cell-responsive gelatin cryogels.

The performance of biomaterials-based therapies can be hindered by complications associated with surgical implant, motivating the development of materials systems that allow minimally invasive introduction into the host. In this study, we created cell-adhesive and degradable gelatin scaffolds that could be injected through a conventional needle while maintaining a predefined geometry and architecture. These scaffolds supported attachment, proliferation, and survival of cells in vitro and could be degraded by recombinant matrix metalloproteinase-2 and -9. Prefabricated gelatin cryogels rapidly resumed their original shape when injected subcutaneously into mice and elicited only a minor host response following injection. Controlled release of granulocyte-macrophage colony-stimulating factor from gelatin cryogels resulted in complete infiltration of the scaffold by immune cells and promoted matrix metalloproteinase production leading to cell-mediated degradation of the cryogel matrix. These findings suggest that gelatin cryogels could serve as a cell-responsive platform for biomaterial-based therapy.

Influence of the stiffness of three-dimensional alginate/collagen-I interpenetrating networks on fibroblast biology.

Wound dressing biomaterials are increasingly being designed to incorporate bioactive molecules to promote healing, but the impact of matrix mechanical properties on the biology of resident cells orchestrating skin repair and regeneration remains to be fully understood. This study investigated whether tuning the stiffness of a model wound dressing biomaterial could control the behavior of dermal fibroblasts. Fully interpenetrating networks (IPNs) of collagen-I and alginate were fabricated to enable gel stiffness to be tuned independently of gel architecture, polymer concentration or adhesion ligand density. Three-dimensional cultures of dermal fibroblasts encapsulated within matrices of different stiffness were shown to promote dramatically different cell morphologies, and enhanced stiffness resulted in upregulation of key-mediators of inflammation such as IL-10 and COX-2. These findings suggest that simply modulating the matrix mechanical properties of a given wound dressing biomaterial deposited at the wound site could regulate the progression of wound healing.