Shyni Varghese

ABSTRACT The use of cellular systems to advance cancer therapeutics has expanded rapidly, spanning cell therapies to patient-specific tumor models. Platforms that recapitulate key features of the tumor microenvironment, including vascular and immune components, hold significant potential to improve the predictive power and translational relevance of preclinical models. Here, we report a vascularized tumor organoid platform that combines self-organizing microvascular networks with patient-derived tumor organoids and tumor-infiltrating lymphocytes. To minimize non-specific endothelial immunogenicity and enable broader compatibility across patient samples, we engineered the vasculature using β2-microglobulin-knockout endothelial cells. Leveraging this system, we established patient-specific, lymphocyte-incorporated tumor models that enabled quantitative assessment of T cell infiltration. In conjunction with immune checkpoint blockade, this platform distinguishes responder and non-responder patient samples, consistent with the clinical observations. Single-cell RNA-sequencing revealed tumor-intrinsic and immune-associated programs underlying this stratification, identifying tumor-driven hyperangiogenic signaling as a barrier to T cell extravasation. Pharmacological co-targeting of PD1 and VEGF restored T cell infiltration in non-responder organoids, shifting them from an immune-excluded to an immune-inflamed state. Together, this vascularized tumor organoid platform provides a predictive and mechanistic framework for modeling patient-specific immunotherapy responses and design of combination therapies.

Osteoporosis arises from an imbalance between bone-resorbing osteoclasts and bone-forming osteoblasts, driving progressive bone loss. Here, we engineer bone-targeting hybrid vesicles by fusing extracellular vesicles (EVs) with lipid nanoparticles (LNPs) to enable coordinated delivery of complementary therapeutics. These vesicles present and carry Osteoprotegerin (OPG) protein and mRNA and are fused with adenosine (Ado)-loaded nanoparticles to form OPG- and Ado-loaded EV-LNP (OPG-EV-LNP-Ado). Acting as a decoy for receptor activator of nuclear factor kappa B ligand (RANKL), OPG directs bone-specific targeting and uptake, enhancing delivery to osteoblasts. Systemic administration results in preferential bone accumulation and significantly attenuates osteoporotic pathology, demonstrating a modular platform for targeted, multi-cargo therapy to restore bone homeostasis.

Abstract In the context of muscle loss, bone repair is impaired, suggesting that muscle derived signals contribute to bone regeneration. However, how muscle surrounding the injury site communicates with the bone repair niche remains unclear. Here we found that CX3CL1 expression was induced in endothelial cells in muscle surrounding a femoral bone injury site. Deletion of Cx3cl1 impaired bone healing, demonstrating a functional role for CX3CL1 in bone repair. A CX3CL1 receptor, CX3CR1, was expressed by PDGFRα⁺ stromal progenitors and lineage tracing showed that CX3CR1 expressing osteoprogenitor lineage cells accumulated at the injury site during repair. PDGFRα⁺ stromal progenitors showed enhanced osteoblastogenesis in response to recombinant CX3CL1. In older mice, local CX3CL1 delivery increased PDGFRα⁺CX3CR1⁺ osteoprogenitor accumulation and improved bone repair. These findings identify a muscle bone signaling pathway in which endothelial CX3CL1 promotes bone repair through CX3CR1 expressing osteoprogenitors.

Exosomes, an acellular regenerative biologic, have demonstrated success in resolving vaginal mesh exposures after pelvic reconstructive surgery; little data exists for their use for prevention of mesh-based complications. This study evaluated the early efficacy of purified exosome product (PEP) for preventing mesh exposures. Ten Yorkshire-crossed pigs underwent mesh sacrocolpopexy with two high-risk-for-exposure configurations: mesh fold ventrally, vaginotomy dorsally. PEP in hyaluronic acid (HA) or HA-only (control) was injected at baseline. Twelve weeks later, animals were euthanized and evaluated for mesh exposure and histologic changes. None of the PEP-treated tissues demonstrated mesh exposure (0/6); all control group animals experienced a mesh exposure (4/4 mesh fold configuration, 2/4 vaginotomy configuration). Control tissues exhibited higher fibrosis (vaginotomy fibrosis score: median(IQR); 3(3,3) control, 2(1,2) PEP; p = 0.03) and greater epithelial apoptosis (mesh fold TUNEL+area fraction: median 18.9 control vs 0.43 PEP; p = 0.02). Our study demonstrated that PEP treatment mitigated the risk of early mesh exposure.

Bone fractures and related complications are a significant concern for older adults, particularly with the growing aging population. Therapeutic interventions that promote bone tissue regeneration are attractive for geriatric fracture repair. Extracellular adenosine plays a key role in bone homeostasis and regeneration. Herein, we examined the changes in extracellular adenosine with aging and the potential of local delivery of adenosine to promote fracture healing using aged mice. Extracellular adenosine level was found to be significantly lower in aged bone tissue compared to young mice. Concomitantly, the ecto-5'-nucleotidase CD73 expression was also lower in aged bone. Local delivery of adenosine using injectable, in situ curing microgel delivery units yielded a pro-regenerative environment and promoted fracture healing in aged mice. This study offers new insights into age-related physiological changes in adenosine levels and demonstrates the therapeutic potential of adenosine supplementation to circumvent the compromised healing of geriatric fractures.

Aging has been identified as a leading risk factor for many diseases, including neurodegenerative disorders. While cellular senescence has been linked to age-related neurodegenerative conditions, its involvement in peripheral stress-associated brain disorders is just beginning to be explored. In this study, we investigated the impact of senescent cells on peripheral stress-induced neuroinflammation using orthopedic surgery as a model. Our results demonstrate an increased accumulation of senescent cells and neuroinflammation in the aged mouse hippocampus following surgery. Intermittent treatment of the mice with the senolytic drugs dasatinib and quercetin (D/Q) showed a significant reduction in surgery-induced senescent cell burden. This reduction in senescent cell accumulation was correlated with reduced surgery-induced neuroinflammation, as evidenced by decreased glial cell activity. Consistent with these observations, we also observed reduced levels of proinflammatory senescence-associated secretory phenotype factors in circulation, following fracture surgery, in mice treated with D/Q. Overall, our findings underscore the pivotal role of cellular senescence in surgery-induced neuroinflammation and highlight the therapeutic potential of eliminating senescent cells as a potential strategy to manage peripheral stress-induced neuroinflammatory conditions.

Bone fractures pose a significant public health challenge, often necessitating surgical interventions to facilitate bone healing and functional recovery. Sensory nerve fibers innervate various compartments of the bone tissue, with the periosteum exhibiting the most extensive innervation that is susceptible to injury during trauma. Despite its importance, the effect of injured periosteum on fracture pain remains unknown. This study examines the impact of extensive periosteal injury on fracture pain by using a mouse model. Periosteal injury is induced by mechanical resection during unilateral transverse fracture and compared to transverse fractures with no periosteal injury. Our results demonstrate that extensive periosteal injury induces severe and long-term pain, as assessed by von Frey and dynamic weight bearing measurements, for up to 12 weeks postfracture. Immunofluorescence staining revealed an increase in local neurofilament heavy polypeptide (NF200 +) nerve innervation and an elevated number of calcitonin gene-related peptide (CGRP +) expressing neurons in the dorsal root ganglion (DRG). Additionally, flow cytometric analyses revealed increased presence of myeloid immune cells in the DRG. Furthermore, bone healing in fractures with extensive periosteal injury exhibited reduced callus size at all time points as assessed by Faxitron X-ray imaging. This study describes a previously unknown effect of extensive periosteal injury in exacerbating fracture pain and establishes a potential model to study long-term orthopedic fracture pain.

Abstract The field of cancer biology and therapeutics has soared in the past several decades with new therapeutic modalities and options for patients, such as chemoradiotherapy, immunotherapy, and combination therapy. This dramatic success in expanding patient options is primarily attributed to the development of various model systems to elucidate drivers of oncogenesis, tumor maturation and evolution, and response to therapeutics. While mouse models have been a workhorse of cancer research, technological progress in ex vivo patient‐derived tumor models has afforded more tunable and scrutable systems for patient‐predictive platforms and mechanistic study. This review explores the technological innovations in 3D solid tumor models and their applicability to various aspects of cancer biology and the identification of therapeutics. Features of the tumor and tumor microenvironment like spatial heterogeneity, multicellular populations, and genomic variations are addressed and elaborated through the establishment of new in vitro models. The integration of perfusable vasculature with 3D tumor models and the potentially wide‐ranging applications of these more complex platforms in precision medicine and cancer immunotherapy are further addressed. Finally, an outlook on the future of experimental cancer models for both biological investigation and bench‐to‐bedside pipeline development is provided.

The role of macrophages (MΦs) remains incompletely understood in kidney injury and repair. The plasticity of MΦs offers an opportunity to polarize them toward mediating injury resolution in both native and transplanted kidneys undergoing ischemia and/or rejection. Here, we show that infiltrating kidney MΦs augmented their own allograft inflammatory factor 1 (AIF-1) expression after injury. Aif1 genetic deletion led to MΦ polarization toward a reparative phenotype while halting the development of kidney fibrosis. The enhanced repair was mediated by higher levels of antiinflammatory and proregenerative markers, leading to a reduction in cell death and an increase in proliferation of kidney tubular epithelial cells after ischemia followed by reperfusion injury (I/RI). Adoptive transfer of Aif1-/- MΦs into Aif1+/+ mice conferred protection against I/RI. Conversely, depletion of MΦs reversed the tissue-reparative effects in Aif1-/- mice. We further demonstrated increased expression of AIF-1 in human kidney biopsies from native kidneys with acute kidney injury or chronic kidney disease, as well as in biopsies from kidney allografts undergoing acute or chronic rejection. We conclude that AIF-1 is a MΦ marker of renal inflammation, and its targeting uncouples MΦ reparative functions from profibrotic functions. Thus, therapies inhibiting AIF-1 when ischemic injury is inevitable have the potential to reduce the global burden of kidney disease.

Alveolar type 2 (AT2) cells play a pivotal role in maintaining lung homeostasis, and the generation of three-dimensional cultures, such as alveolospheres, provides a valuable model for studying lung development, pathologies, and drug responses. Here, we investigate the critical extracellular matrix (ECM) characteristics that influence AT2 alveolosphere formation and growth. By encapsulating AT2 cells in different extracellular matrix-based hydrogels, we identified laminin as a key ECM protein supporting robust alveolosphere formation akin to Matrigel. Our results show that laminin-rich hydrogels support alveolosphere formation across murine and human primary AT2 cells as well as induced human pluripotent stem cell derived AT2 cells (iAT2 cells). Notably, matrix stiffness strongly influenced alveolosphere formation. Hydrogels with a low Young's modulus and high compliance supported a greater number of alveolospheres, exhibiting a broader size distribution and a higher proportion of larger alveolospheres. Moreover, inhibition of matrix degradation and cellular contractility disrupted alveolosphere formation. Leveraging these insights, we developed a multicellular vascularized alveolosphere-on-a-chip model by integrating alveolospheres with endothelial cells and lung fibroblasts within a microfluidic device. Application of this model to assess the inflammatory effects of menthol, a common e-cigarette flavor, demonstrates its utility in evaluating the pulmonary effects of chemical exposures on alveolar cells. Our findings highlight the critical role of matrix physicochemical properties on alveolosphere formation and establish a versatile platform for advancing the study of lung biology, disease mechanisms, and drug discovery.

Asish Pal, Praveen Kumar Vemula and Shyni Varghese introduce the Nanoscale themed collection, Celebrating the 65th birthday of Professor Santanu Bhattacharya.

Abstract Survival for metastatic breast cancer is low and thus, continued efforts to treat and prevent metastatic progression are critical. Estrogen is shown to promote aggressive phenotypes in multiple cancer models irrespective of estrogen receptor (ER) status. Similarly, UDP-Glucose 6-dehydrogenase (UGDH) a ubiquitously expressed enzyme involved in extracellular matrix precursors, as well as hormone processing increases migratory and invasive properties in cancer models. While the role of UGDH in cellular migration is defined, how it intersects with and impacts hormone signaling pathways associated with tumor progression in metastatic breast cancer has not been explored. Here we demonstrate that UGDH knockdown blunts estrogen-induced tumorigenic phenotypes (migration and colony formation) in ER+ and ER- breast cancer in vitro . Knockdown of UGDH also inhibits extravasation of ER- breast cancer ex vivo , primary tumor growth and animal survival in vivo in both ER+ and ER- breast cancer. We also use single cell RNA-sequencing to demonstrate that our findings translate to a human breast cancer clinical specimen. Our findings support the role of estrogen and UGDH in breast cancer progression provide a foundation for future studies to evaluate the role of UGDH in therapeutic resistance to improve outcomes and survival for breast cancer patients.

Perfusable microvascular networks offer promising three-dimensional in vitro models to study normal and compromised vascular tissues as well as phenomena such as cancer cell metastasis. Engineering of these microvascular networks generally involves the use of endothelial cells stabilized by fibroblasts to generate robust and stable vasculature. However, fibroblasts are highly heterogenous and may contribute variably to the microvascular structure. Here, we study the effect of normal and cancer-associated lung fibroblasts on the formation and function of perfusable microvascular networks. We examine the influence of cancer-associated fibroblasts on microvascular networks when cultured in direct (juxtacrine) and indirect (paracrine) contacts with endothelial cells, discovering a generative inhibition of microvasculature in juxtacrine co-cultures and a functional inhibition in paracrine co-cultures. Furthermore, we probed the secreted factors differential between cancer-associated fibroblasts and normal human lung fibroblasts, identifying several cytokines putatively influencing the resulting microvasculature morphology and functionality. These findings suggest the potential contribution of cancer-associated fibroblasts in aberrant microvasculature associated with tumors and the plausible application of such in vitro platforms in identifying new therapeutic targets and/or agents that can prevent formation of aberrant vascular structures.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

electrostatic interactions. We surmised that the electrostatic interactions between the BPL-modified HA molecules (HA-BPL) and the cartilage facilitate localization of the HA molecules to the cartilage surface. The number of BPL molecules on the HA backbone was varied to determine the optimal grafting density for cartilage binding and HA localization. Collectively, our results show that our HA-BPL molecules adhered readily to cartilage and were effective as a lubricant in cartilage-on-cartilage shear measurements where the modified HA molecules significantly reduce the coefficient of friction compared to phosphate-buffered saline or HA alone. This proof-of-concept study shows how the incorporation of cartilage adhering moieties, such as cationic molecules, can be used to enhance cartilage binding and lubrication properties of HA.

Radiotherapy remains a common treatment modality for cancer despite skeletal complications. However, there are currently no effective treatments for radiation-induced bone loss, and the consequences of radiotherapy on skeletal progenitor cell (SPC) survival and function remain unclear. After radiation, leptin receptor-expressing cells, which include a population of SPCs, become localized to hypoxic regions of the bone and stabilize the transcription factor hypoxia-inducible factor-2α (HIF-2α), thus suggesting a role for HIF-2α in the skeletal response to radiation. Here, we conditionally knocked out HIF-2α in leptin receptor-expressing cells and their descendants in mice. Radiation therapy in littermate control mice reduced bone mass; however, HIF-2α conditional knockout mice maintained bone mass comparable to nonirradiated control animals. HIF-2α negatively regulated the number of SPCs, bone formation, and bone mineralization. To test whether blocking HIF-2α pharmacologically could reduce bone loss during radiation, we administered a selective HIF-2α inhibitor called PT2399 (a structural analog of which was recently FDA-approved) to wild-type mice before radiation exposure. Pharmacological inhibition of HIF-2α was sufficient to prevent radiation-induced bone loss in a single-limb irradiation mouse model. Given that ~90% of patients who receive a HIF-2α inhibitor develop anemia because of off-target effects, we developed a bone-targeting nanocarrier formulation to deliver the HIF-2α inhibitor to mouse bone, to increase on-target efficacy and reduce off-target toxicities. Nanocarrier-loaded PT2399 prevented radiation-induced bone loss in mice while reducing drug accumulation in the kidney. Targeted inhibition of HIF-2α may represent a therapeutic approach for protecting bone during radiation therapy.

Bone injuries such as fractures are one major cause of morbidities worldwide. A considerable number of fractures suffer from delayed healing, and the unresolved acute pain may transition to chronic and maladaptive pain. Current management of pain involves treatment with NSAIDs and opioids with substantial adverse effects. Herein, we tested the hypothesis that the purine molecule, adenosine, can simultaneously alleviate pain and promote healing in a mouse model of tibial fracture by targeting distinctive adenosine receptor subtypes in different cell populations. To achieve this, a biomaterial-assisted delivery of adenosine is utilized to localize and prolong its therapeutic effect at the injury site. The results demonstrate that local delivery of adenosine inhibited the nociceptive activity of peripheral neurons through activation of adenosine A1 receptor (ADORA1) and mitigated pain as demonstrated by weight bearing and open field movement tests. Concurrently, local delivery of adenosine at the fracture site promoted osteogenic differentiation of mesenchymal stromal cells through adenosine A2B receptor (ADORA2B) resulting in improved bone healing as shown by histological analyses and microCT imaging. This study demonstrates the dual role of adenosine and its material-assisted local delivery as a feasible therapeutic approach to treat bone trauma and associated pain.

Joint diseases, such as osteoarthritis, often require delivery of drugs to chondrocytes residing within the cartilage. However, intra-articular delivery of drugs to cartilage remains a challenge due to their rapid clearance within the joint. This problem is further exacerbated by the dense and negatively charged cartilage extracellular matrix (ECM). Cationic nanocarriers that form reversible electrostatic interactions with the anionic ECM can be an effective approach to overcome the electrostatic barrier presented by cartilage tissue. For an effective therapeutic outcome, the nanocarriers need to penetrate, accumulate, and be retained within the cartilage tissue. Nanocarriers that adhere quickly to cartilage tissue after intra-articular administration, transport through cartilage, and remain within its full thickness are crucial to the therapeutic outcome. To this end, we used ring-opening polymerization to synthesize branched poly(l-lysine) (BPL) cationic nanocarriers with varying numbers of poly(lysine) branches, surface charge, and functional groups, while maintaining similar hydrodynamic diameters. Our results show that the multivalent BPL molecules, including those that are highly branched (i.e., generation two), can readily adhere and transport through the full thickness of cartilage, healthy and degenerated, with prolonged intra-cartilage retention. Intra-articular injection of the BPL molecules in mouse knee joint explants and rat knee joints showed their localization and retention. In summary, this study describes an approach to design nanocarriers with varying charge and abundant functional groups while maintaining similar hydrodynamic diameters to aid the delivery of macromolecules to negatively charged tissues.

Unlike adult mammals, zebrafish regenerate spinal cord tissue and recover locomotor ability after a paralyzing injury. Here, we find that ependymal cells in zebrafish spinal cords produce the neurogenic factor Hb-egfa upon transection injury. Animals with hb-egfa mutations display defective swim capacity, axon crossing, and tissue bridging after spinal cord transection, associated with disrupted indicators of neuron production. Local recombinant human HB-EGF delivery alters ependymal cell cycling and tissue bridging, enhancing functional regeneration. Epigenetic profiling reveals a tissue regeneration enhancer element (TREE) linked to hb-egfa that directs gene expression in spinal cord injuries. Systemically delivered recombinant AAVs containing this zebrafish TREE target gene expression to crush injuries of neonatal, but not adult, murine spinal cords. Moreover, enhancer-based HB-EGF delivery by AAV administration improves axon densities after crush injury in neonatal cords. Our results identify Hb-egf as a neurogenic factor necessary for innate spinal cord regeneration and suggest strategies to improve spinal cord repair in mammals.

<div>Abstract<p>Recruitment of immune cells to a tumor is determined by the complex interplay between cellular and noncellular components of the tumor microenvironment. <i>Ex vivo</i> platforms that enable identification of key components that promote immune cell recruitment to the tumor could advance the field significantly. Herein, we describe the development of a perfusable multicellular tumor-on-a-chip platform involving different cell populations. Cancer cells, monocytes, and endothelial cells were spatially confined within a gelatin hydrogel in a controlled manner by using 3D photopatterning. The migration of the encapsulated endothelial cells against a chemokine gradient created an endothelial layer around the constructs. Using this platform, we examined the effect of cancer cell–monocyte interaction on T-cell recruitment, where T cells were dispersed within the perfused media and allowed to infiltrate. The hypoxic environment in the spheroid cultures recruited more T cells compared with dispersed cancer cells. Moreover, the addition of monocytes to the cancer cells improved T-cell recruitment. The differences in T-cell recruitment were associated with differences in chemokine secretion including chemokines influencing the permeability of the endothelial barrier. This proof-of-concept study shows how integration of microfabrication, microfluidics, and 3D cell culture systems could be used for the development of tumor-on-a-chip platforms involving heterotypic cells and their application in studying recruitment of cells by the tumor-associated microenvironment.</p>Significance:<p>This study describes how tumor-on-chip platforms could be designed to create a heterogeneous mix of cells and noncellular components to study the effect of the tumor microenvironment on immune cell recruitment.</p></div>

<div>Abstract<p>Recruitment of immune cells to a tumor is determined by the complex interplay between cellular and noncellular components of the tumor microenvironment. <i>Ex vivo</i> platforms that enable identification of key components that promote immune cell recruitment to the tumor could advance the field significantly. Herein, we describe the development of a perfusable multicellular tumor-on-a-chip platform involving different cell populations. Cancer cells, monocytes, and endothelial cells were spatially confined within a gelatin hydrogel in a controlled manner by using 3D photopatterning. The migration of the encapsulated endothelial cells against a chemokine gradient created an endothelial layer around the constructs. Using this platform, we examined the effect of cancer cell–monocyte interaction on T-cell recruitment, where T cells were dispersed within the perfused media and allowed to infiltrate. The hypoxic environment in the spheroid cultures recruited more T cells compared with dispersed cancer cells. Moreover, the addition of monocytes to the cancer cells improved T-cell recruitment. The differences in T-cell recruitment were associated with differences in chemokine secretion including chemokines influencing the permeability of the endothelial barrier. This proof-of-concept study shows how integration of microfabrication, microfluidics, and 3D cell culture systems could be used for the development of tumor-on-a-chip platforms involving heterotypic cells and their application in studying recruitment of cells by the tumor-associated microenvironment.</p>Significance:<p>This study describes how tumor-on-chip platforms could be designed to create a heterogeneous mix of cells and noncellular components to study the effect of the tumor microenvironment on immune cell recruitment.</p></div>

<p>Figure S2 shows the characterization of the endothelial layer.</p>

<p>Supplementary Movie 1</p>

<p>Figures S1 shows the fabrication diagram of the tumor-on-chip device.</p>

<p>Figure S7 shows infiltration of TALL-104 cells into bi-layer GelMA hydrogels containing cancer cells (MDA-MB-231), monocytes (THP-1), and endothelial cells.</p>

<p>Supplementary Movie 2</p>

<p>Figure S5 shows the characterization of MCF7 cultures involving dispersed cells.</p>

<p>Materials and Methods</p>

<p>Figure S4 shows results from the co-culture of cancer cells, monocytes, and endothelial cells within a single hydrogel layer.</p>

<p>Figure S3 shows the mechanical characterization of the GelMA hydrogels.</p>