Harnessing Degradable Materials to Study and Engineer Lymph Node Function

Vaccines have benefited global health by controlling or eradicating multiple previously fatal diseases. While many early vaccines were efficacious, sophisticated new vaccines and immunotherapies need to address current challenges in the field, including diseases that avoid immune detection or lack strong molecular targets for the immune system. Overcoming these hurdles requires strategies to specifically control the magnitude and type of immune response generated. Biomaterials offer attractive features to achieve this goal, including protection of encapsulated signals, controlled release of cargos, and tunable features for cell targeting. Intriguingly, recent research reveals many common biomaterials activate the immune system, even without other signals. This intrinsic activation results, at least in part, from biomaterial physicochemical features that mimic pathogens and other foreign materials. Surprisingly, although degradable materials are being intensely studied as vaccines carriers, little research has investigated how the intrinsic immunogenicity of these materials changes as polymers degrade. The work in this dissertation reveals parameters impacting material intrinsic immunogenicity and exploits this new understanding to test the influence of biomaterial-based vaccines on the function of lymph nodes (LNs), key tissues that coordinate immunity. In the first aim, the immunostimulatory properties of a library of degradable polymers, poly(beta-amino esters) (PBAEs), were investigated in cell and animal models. PBAEs in soluble forms did not activate innate immune cells (e.g., dendritic cells, DCs). When PBAEs were formulated into particles to mimic a common vaccine strategy, DC activation increased in a molecular weight-specific manner. Using intra-lymph node (i.LN.) injection, a novel technique to control the dose, kinetics, and combination of signals in LNs, PBAE intrinsic immunogenicity was confirmed in mice. In the second aim, microparticles encapsulating immune signals were introduced into mice via i.LN. injection and immune responses were quantified in treated LNs, untreated LNs, and in blood. These results elucidated the interplay between local LN rearrangement and systemic antigen-specific responses which ultimately led to prolonged survival in cancer models. By understanding how the properties and administration of biomaterial-based vaccines impact immunity, this dissertation provides information that can help create new design rules for future vaccines that actively direct the immune system toward a desired response

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

Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells’ (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo–derived senescence signature (SenSig) using a foreign body response–driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and “cartilage-like” fibroblasts as senescent and defined cell type–specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34–CSF1R–TGFβR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.</p

Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells’ (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo–derived senescence signature (SenSig) using a foreign body response–driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and “cartilage-like” fibroblasts as senescent and defined cell type–specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34–CSF1R–TGFβR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.</p

Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells’ (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo–derived senescence signature (SenSig) using a foreign body response–driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and “cartilage-like” fibroblasts as senescent and defined cell type–specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34–CSF1R–TGFβR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.</p

Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells’ (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo–derived senescence signature (SenSig) using a foreign body response–driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and “cartilage-like” fibroblasts as senescent and defined cell type–specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34–CSF1R–TGFβR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.</p

T cell immunoengineering with advanced biomaterials

Recent advances in biomaterials design offer the potential to actively control immune cell activation and behaviour. Many human diseases, such as infections, cancer, and autoimmune disorders, are partly mediated by inappropriate or insufficient activation of the immune system. T cells play a central role in the host immune response to these diseases, and so constitute a promising cell type for manipulation. In vivo, T cells are stimulated by antigen presenting cells (APC), therefore to design immunoengineering biomaterials that control T cell behaviour, artificial interfaces that mimic the natural APC-T cell interaction are required. This review draws together research in the design and fabrication of such biomaterial interfaces, and highlights efforts to elucidate key parameters in T cell activation, such as substrate mechanical properties and spatial organization of receptors, illustrating how they can be manipulated by bioengineering approaches to alter T cell function

Advances in the testing of stereo image acquisition devices

The core problem addressed in the current thesis is to provide a novel test bed for depth map generation and stereo image-based algorithms. The test bed comprises of elements such as (i) the choice and calibration of stereo image acquisition devices; (ii) choice and setup of objects in the test scenes; (iii) light intensity and temperature settings; (iv) camera-object and stereo baseline distance setups and (v) a repeatable test scene. In order to determine each element of the test bed, a number of experiments are presented. During the experiment process, four depth map algorithms are selected which, in conjunction with four different test scenes, are used for the initial measurements where (i) the influence of light intensity on algorithm performance is determined; (ii) the performances of the algorithms at different camera-object distances are determined and (iii) the influence of similar objects in the test scene is described. In order to acquire stereo images, a number of devices are used, from off-the-shelf stereo cameras to custom-built stereo devices. During the stereo image acquisition, a number of possible error sources are noticed, and the problems are mitigated. The experiments show that slight changes in the light intensity, light direction, camera-object distance and stereo base length can have a significant influence on the result of the depth map algorithms. With the help of these results, the details of the test bed are determined, and an on-line database is provided, containing details of the test scene and example stereo test images derived from the scene. This is available online for other researchers to download at http://www.andorko.com/stereo.html. The proposed test scene is provided with a view to having a standardized test scene that can be easily replicated by other researchers for testing of stereo acquisition systems and associated depth map acquisition algorithms