Durable lymph-node expansion is associated with the efficacy of therapeutic vaccination.

Following immunization, lymph nodes dynamically expand and contract. The mechanical and cellular changes enabling the early-stage expansion of lymph nodes have been characterized, yet the durability of such responses and their implications for adaptive immunity and vaccine efficacy are unknown. Here, by leveraging high-frequency ultrasound imaging of the lymph nodes of mice, we report more potent and persistent lymph-node expansion for animals immunized with a mesoporous silica vaccine incorporating a model antigen than for animals given bolus immunization or standard vaccine formulations such as alum, and that durable and robust lymph-node expansion was associated with vaccine efficacy and adaptive immunity for 100 days post-vaccination in a mouse model of melanoma. Immunization altered the mechanical and extracellular-matrix properties of the lymph nodes, drove antigen-dependent proliferation of immune and stromal cells, and altered the transcriptional features of dendritic cells and inflammatory monocytes. Strategies that robustly maintain lymph-node expansion may result in enhanced vaccination outcomes.

Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors.

Although adoptive T cell therapy provides the T cell pool needed for immediate tumor debulking, the infused T cells generally have a narrow repertoire for antigen recognition and limited ability for long-term protection. Here, we present a hydrogel that locally delivers adoptively transferred T cells to the tumor site while recruiting and activating host antigen-presenting cells with GMCSF or FLT3L and CpG, respectively. T cells alone loaded into these localized cell depots provided significantly better control of subcutaneous B16-F10 tumors than T cells delivered through direct peritumoral injection or intravenous infusion. T cell delivery combined with biomaterial-driven accumulation and activation of host immune cells prolonged the activation of the delivered T cells, minimized host T cell exhaustion, and enabled long-term tumor control. These findings highlight how this integrated approach provide both immediate tumor debulking and long-term protection against solid tumors, including against tumor antigen escape.

Immune-responsive biodegradable scaffolds for enhancing neutrophil regeneration.

Neutrophils are essential effector cells for mediating rapid host defense and their insufficiency arising from therapy-induced side-effects, termed neutropenia, can lead to immunodeficiency-associated complications. In autologous hematopoietic stem cell transplantation (HSCT), neutropenia is a complication that limits therapeutic efficacy. Here, we report the development and in vivo evaluation of an injectable, biodegradable hyaluronic acid (HA)-based scaffold, termed HA cryogel, with myeloid responsive degradation behavior. In mouse models of immune deficiency, we show that the infiltration of functional myeloid-lineage cells, specifically neutrophils, is essential to mediate HA cryogel degradation. Post-HSCT neutropenia in recipient mice delayed degradation of HA cryogels by up to 3 weeks. We harnessed the neutrophil-responsive degradation to sustain the release of granulocyte colony stimulating factor (G-CSF) from HA cryogels. Sustained release of G-CSF from HA cryogels enhanced post-HSCT neutrophil recovery, comparable to pegylated G-CSF, which, in turn, accelerated cryogel degradation. HA cryogels are a potential approach for enhancing neutrophils and concurrently assessing immune recovery in neutropenic hosts.

Matrix viscoelasticity controls spatiotemporal tissue organization.

Biomolecular and physical cues of the extracellular matrix environment regulate collective cell dynamics and tissue patterning. Nonetheless, how the viscoelastic properties of the matrix regulate collective cell spatial and temporal organization is not fully understood. Here we show that the passive viscoelastic properties of the matrix encapsulating a spheroidal tissue of breast epithelial cells guide tissue proliferation in space and in time. Matrix viscoelasticity prompts symmetry breaking of the spheroid, leading to the formation of invading finger-like protrusions, YAP nuclear translocation and epithelial-to-mesenchymal transition both in vitro and in vivo in a Arp2/3-complex-dependent manner. Computational modelling of these observations allows us to establish a phase diagram relating morphological stability with matrix viscoelasticity, tissue viscosity, cell motility and cell division rate, which is experimentally validated by biochemical assays and in vitro experiments with an intestinal organoid. Altogether, this work highlights the role of stress relaxation mechanisms in tissue growth dynamics, a fundamental process in morphogenesis and oncogenesis.

Active tissue adhesive activates mechanosensors and prevents muscle atrophy.

While mechanical stimulation is known to regulate a wide range of biological processes at the cellular and tissue levels, its medical use for tissue regeneration and rehabilitation has been limited by the availability of suitable devices. Here we present a mechanically active gel-elastomer-nitinol tissue adhesive (MAGENTA) that generates and delivers muscle-contraction-mimicking stimulation to a target tissue with programmed strength and frequency. MAGENTA consists of a shape memory alloy spring that enables actuation up to 40% strain, and an adhesive that efficiently transmits the actuation to the underlying tissue. MAGENTA activates mechanosensing pathways involving yes-associated protein and myocardin-related transcription factor A, and increases the rate of muscle protein synthesis. Disuse muscles treated with MAGENTA exhibit greater size and weight, and generate higher forces compared to untreated muscles, demonstrating the prevention of atrophy. MAGENTA thus has promising applications in the treatment of muscle atrophy and regenerative medicine.

Chemotherapy Dose Shapes the Expression of Immune-Interacting Markers on Cancer Cells.

Introduction: Tumor and immune cells interact through a variety of cell-surface proteins that can either restrain or promote tumor progression. The impacts of cytotoxic chemotherapy dose and delivery route on this interaction profile remain incompletely understood, and could support the development of more effective combination therapies for cancer treatment. Methods and Results: Here, we found that exposure to the anthracycline doxorubicin altered the expression of numerous immune-interacting markers (MHC-I, PD-L1, PD-L2, CD47, Fas, and calreticulin) on live melanoma, breast cancer, and leukemia cells in a dose-dependent manner in vitro. Notably, an intermediate dose best induced immunogenic cell death and the expression of immune-activating markers without maximizing expression of markers associated with immune suppression. Bone marrow-derived dendritic cells exposed to ovalbumin-expressing melanoma treated with intermediate doxorubicin dose became activated and best presented tumor antigen. In a murine melanoma model, both the doxorubicin dose and delivery location (systemic infusion versus local administration) affected the expression of these markers on live tumor cells. Particularly, local release of doxorubicin from a hydrogel increased calreticulin expression on tumor cells without inducing immune-suppressive markers, in a manner dependent on the loaded dose. Doxorubicin exposure also altered the expression of immune-interacting markers in patient-derived melanoma cells. Conclusions: Together, these results illustrate how standard-of-care chemotherapy, when administered in various manners, can lead to distinct expression of immunogenic markers on cancer cells. These findings may inform development of chemo-immunotherapy combinations for cancer treatment. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00742-y.

Targeting tumor extracellular matrix activates the tumor-draining lymph nodes.

Disruption of the tumor extracellular matrix (ECM) may alter immune cell infiltration into the tumor and antitumor T cell priming in the tumor-draining lymph nodes (tdLNs). Here, we explore how intratumoral enzyme treatment (ET) of B16 melanoma tumors with ECM-depleting enzyme hyaluronidase alters adaptive and innate immune populations, including T cells, DCs, and macrophages, in the tumors and tdLNs. ET increased CD103+ DC abundance in the tdLNs, as well as antigen presentation of a model tumor antigen ovalbumin (OVA), eliciting local OVA-specific CD8+ T cell responses. Delivered in combination with a distant cryogel-based cancer vaccine, ET increased the systemic antigen-specific CD8+ T cell response. By enhancing activity within the tdLN, ET may broadly support immunotherapies in generating tumor-specific immunity.

Cryogel vaccines effectively induce immune responses independent of proximity to the draining lymph nodes.

The delivery location of traditional vaccines can impact immune responses and resulting efficacy. Cryogel-based cancer vaccines, which are typically injected near the inguinal lymph nodes (iLNs), recruit and activate dendritic cells (DC) in situ, induce DC homing to the iLNs, and have generated potent anti-tumor immunity against several murine cancer models. However, whether cryogel vaccination distance to a draining LN affects the kinetics of DC homing and downstream antigen-specific immunity is unknown, given the heightened importance of the scaffold vaccine site. We hypothesized that vaccination near the iLNs would lead to more rapid DC trafficking to the iLNs, thereby inducing faster and stronger immune responses. Here, mice were injected with cryogel vaccines against ovalbumin either adjacent or distal to the iLNs, and the resultant DC trafficking kinetics, T cell phenotypes, antigen-specific T cell and humoral responses, and prophylactic efficacy in an ovalbumin-expressing tumor model were assessed. Cryogel vaccines induced potent, long-lasting antigen-specific immune responses independent of distance to the iLNs, with no significant differences in DC trafficking kinetics, ovalbumin-specific T cell and antibody responses, or prophylactic efficacy. Moreover, DC trafficking and activation state were not impacted when cryogels were injected near a tumor. These results demonstrate a flexibility in vaccination location for scaffold-based vaccines, independent of draining LN distance.

Ultrasound-triggered release reveals optimal timing of CpG-ODN delivery from a cryogel cancer vaccine.

Recently, several injectable scaffold-based cancer vaccines have been developed that can recruit and activate host dendritic cells (DCs) and generate potent antitumor responses. However, the optimal timing of adjuvant delivery, particularly of the commonly used cytosine-phosphodiester-guanine-oligonucleotide (CpG-ODN), for scaffold-based cancer vaccines remains unknown. We hypothesized that optimally timed CpG-ODN delivery will lead to enhanced immune responses, and designed a cryogel vaccine system where CpG-ODN release can be triggered on-demand by ultrasound. CpG-ODN was first condensed with polyethylenimine and then adsorbed to cryogels. Little adsorbed CpG-ODN was released in vitro. Ultrasound stimulation triggered continuous CpG-ODN release, at an enhanced rate even after ultrasound was turned off, with minimal burst release. In vivo, ultrasound stimulation four days post-vaccination induced a significantly higher antigen-specific cytotoxic T-lymphocyte (CTL) response compared to control mice. Furthermore, ultrasound stimulation at this time point generated a significantly higher IgG2a/c antibody titer than all the groups except ultrasound stimulation eight days post-vaccination. This optimal timing of ultrasound-triggered release coincided with peak DC accumulation in the cryogels. By enabling temporal control of vaccine components through release on-demand, this system is a promising platform to study the optimal timing of delivery of immunomodulatory agents for cancer vaccination.

Biomaterial-based scaffold for in situ chemo-immunotherapy to treat poorly immunogenic tumors.

Poorly immunogenic tumors, including triple negative breast cancers (TNBCs), remain resistant to current immunotherapies, due in part to the difficulty of reprogramming the highly immunosuppressive tumor microenvironment (TME). Here we show that peritumorally injected, macroporous alginate gels loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) for concentrating dendritic cells (DCs), CpG oligonucleotides, and a doxorubicin-iRGD conjugate enhance the immunogenic death of tumor cells, increase systemic tumor-specific CD8 + T cells, repolarize tumor-associated macrophages towards an inflammatory M1-like phenotype, and significantly improve antitumor efficacy against poorly immunogenic TNBCs. This system also prevents tumor recurrence after surgical resection and results in 100% metastasis-free survival upon re-challenge. This chemo-immunotherapy that concentrates DCs to present endogenous tumor antigens generated in situ may broadly serve as a facile platform to modulate the suppressive TME, and enable in situ personalized cancer vaccination.

Cell and tissue engineering in lymph nodes for cancer immunotherapy.

In cancer, lymph nodes (LNs) coordinate tumor antigen presentation necessary for effective antitumor immunity, both at the levels of local cellular interactions and tissue-level organization. In this review, we examine how LNs may be engineered to improve the therapeutic outcomes of cancer immunotherapy. At the cellular scale, targeting the LNs impacts the potency of cancer vaccines, immune checkpoint blockade, and adoptive cell transfer. On a tissue level, macro-scale biomaterials mimicking LN features can function as immune niches for cell reprogramming or delivery in vivo, or be utilized in vitro to enable preclinical testing of drugs and vaccines. We additionally review strategies to induce ectopic lymphoid sites reminiscent of LNs that may improve antitumor T cell priming.

A biomaterial-based vaccine eliciting durable tumour-specific responses against acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a malignancy of haematopoietic origin that has limited therapeutic options. The standard-of-care cytoreductive chemotherapy depletes AML cells to induce remission, but is infrequently curative. An immunosuppressive AML microenvironment in the bone marrow and the paucity of suitable immunotherapy targets limit the induction of effective immune responses. Here, in mouse models of AML, we show that a macroporous-biomaterial vaccine that delivers the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), the Toll-like-receptor-9 agonist cytosine-guanosine oligodeoxynucleotide and one or multiple leukaemia antigens (in the form of a defined peptide antigen, cell lysates or antigens sourced from AML cells recruited in vivo) induces local immune-cell infiltration and activated dendritic cells, evoking a potent anti-AML response. The biomaterial-based vaccine prevented the engraftment of AML cells when administered as a prophylactic and when combined with chemotherapy, and eradicated established AML even in the absence of a defined vaccine antigen. Biomaterial-based AML vaccination can induce potent immune responses, deplete AML cells and prevent disease relapse.

Treating ischemia via recruitment of antigen-specific T cells.

Ischemic diseases are a leading cause of mortality and can result in autoamputation of lower limbs. We explored the hypothesis that implantation of an antigen-releasing scaffold, in animals previously vaccinated with the same antigen, can concentrate TH2 T cells and enhance vascularization of ischemic tissue. This approach may be clinically relevant, as all persons receiving childhood vaccines recommended by the Centers for Disease Control and Prevention have vaccines that contain aluminum, a TH2 adjuvant. To test the hypothesis, mice with hindlimb ischemia, previously vaccinated with ovalbumin (OVA) and aluminum, received OVA-releasing scaffolds. Vaccinated mice receiving OVA-releasing scaffolds locally concentrated antigen-specific TH2 T cells in the surrounding ischemic tissue. This resulted in local angiogenesis, increased perfusion in ischemic limbs, and reduced necrosis and enhanced regenerating myofibers in the muscle. These findings support the premise that antigen depots may provide a treatment for ischemic diseases in patients previously vaccinated with aluminum-containing adjuvants.

CD4 T-cells regulate angiogenesis and myogenesis.

Ischemic diseases, such as peripheral artery disease, affect millions of people worldwide. While CD4+ T-cells regulate angiogenesis and myogenesis, it is not understood how the phenotype of these adaptive immune cells regulate these regenerative processes. The secreted factors from different types of CD4+ T-cells (Th1, Th2, Th17, and Treg) were utilized in a series of in vitro assays and delivered from an injectable alginate biomaterial into a murine model of ischemia to study their effects on vascular and skeletal muscle regeneration. Conditioned medium from Th2 and Th17  T-cells enhanced angiogenesis in vitro and in vivo, in part by directly stimulating endothelial sprouting. Th1 conditioned medium induced vascular regression in vitro and provided no benefit to angiogenesis in vivo. Th1, Th2, and Th17 conditioned medium, to varying extents, enhanced muscle precursor cell proliferation and inhibited their differentiation in vitro, and prolonged early stages of muscle regeneration in vivo. Treg conditioned medium had a moderate or no effect on these processes in vitro and no discernible effect in vivo. These findings suggest that Th2 and Th17 T-cells may enhance angiogenesis and myogenesis in ischemic injuries, which may be useful in the design of immunomodulatory biomaterials to treat these diseases.