Biology subjects

Rose, I.

Publications and source records attributed to Rose, I..

5 recordsLinked to original sources

bioRxiv2025

WS6 enables scalable ex vivo expansion and gene editing of epithelial basal stem cells

Modeling human epithelial diseases and developing cell-based therapies require robust methods to expand and manipulate epithelial stem and progenitor cells in vitro. Basal stem/progenitor cells from stratified epithelia can be expanded in 3T3-J2 fibroblast feeder cell co-culture systems, and the addition of the ROCK inhibitor Y-27632 enhances proliferation and culture longevity, a phenomenon described as conditional reprogramming. Here, we present a method incorporating the small molecule WS6 to further improve the proliferation and lifespan of cultured epithelial cells from multiple tissues, including airway, skin, and thymus. Cells maintained in this medium ( EpMED; FAD+Y+WS6) retain basal stem/progenitor cell identity and function, including the capacity to differentiate. We demonstrate their capacity to engraft in vivo in a tracheal transplantation model. In a second application, we generate clonal CRISPR-Cas9 genome edited nasal cultures, introducing targeted knockouts of DNAH5 or DNAI2 to create primary ciliary dyskinesia disease models. We anticipate that our method will have broad applications in epithelial cell biology, disease modeling, and regenerative medicine, while reducing reliance on immortalized or cancer cell lines and animal experimentation.

cell biology↗

bioRxiv2025

Characterization of a Novel Transmembrane Activating STING Agonist using Genetically Humanized Mice

STING is a pattern recognition receptor that activates type I interferon and proinflammatory responses in addition to unrelated molecular processes following exposure of DNA to the cytosol. Its pharmacologic stimulation enhances vaccine potency and generates effective antitumor responses but clinical trials evaluating STING agonists have not led to approval for human use. STING activation can occur through ligand engagement of either cytosolic or transmembrane protein domains, processes to which distinct cellular phenotypes are attributed. However, the only transmembrane STING agonist identified is human selective and in vivo testing in conventional models is not feasible. Here we describe synthesis of novel STING agonists efficacious against allelic variants of the protein. We also describe genetically humanized STING mice and demonstrate their suitability as a model to evaluate in vivo responses following exogenous administration of human-selective agonists. Experiments demonstrate that the lead molecule (termed INI3069) functions through binding to the STING transmembrane region and its comparison with conventional agonists reveals significant differences in molecular and immune effects. INI3069 can also enhance antibody responses to co-administered antigens and antitumor responses. This work both represents the first in vivo examination of the effects of transmembrane STING agonism and demonstrates efficacy of a potential novel vaccine adjuvant and oncological therapeutic.

immunology↗

bioRxiv2025

Comparative Molecular, Innate, and Adaptive Impacts of Chemically Diverse STING Agonists

Pharmacologic activation of the innate immune response is being actively being pursued for numerous clinical purposes including enhancement of vaccine potency and potentiation of anti-cancer immunotherapy. Pattern recognition receptors (PRRs) represent especially useful targets for these efforts as their engagement by agonists can trigger signaling pathways that associate with phenotypes desirable for specific immune outcomes. Stimulator of interferon genes (STING) is an ER-resident PRR reactive to cyclic dinucleotides such as those synthesized endogenously in response to cytosolic dsDNA. STING activation leads to transient generation of type I interferon (IFN-I) and proinflammatory responses that augment immunologically relevant effects including antiviral responses, antigen presentation, immune cell trafficking, and immunogenic cell death. In recent years engineered cyclic dinucleotides and small molecules have been discovered that induce STING and safely confer clinically useful outcomes in animal models such as adjuvanticity of anti-microbial vaccines and tumor clearance. Unfortunately, clinical trials examining the efficacy of STING agonists have thus far failed to satisfactorily recapitulate these positive outcomes and this has prevented their translational advancement. A likely relevant yet perplexingly under investigated aspect of pharmacologic STING activation is the diversity of molecular and immune responses that associate with chemical properties of the agonist. Based on this, a comparative survey of these was undertaken using unrelated STING-activating molecules to characterize the molecular, innate, cellular, and immune outcomes they elicit. This was done to inform and direct future studies aimed at designing and selecting agonists appropriate for desired clinical goals. This revealed demonstrable differences between the agonists in potency, transcriptomes, cytokine secretion profiles, immune cell trafficking, and antigen-directed humoral and cell mediated immune responses. As such, this work illustrates that phenotypes deriving from activation of a protein target can be linked to chemical properties of the engaging agonist and thus heightened scrutiny is necessary when selecting molecules to generate specific in vivo effects.

immunology↗

bioRxiv2020

Monitoring phagocytic uptake of amyloid β into glial cell lysosomes in real time

Phagocytosis by glial cells is essential to regulate brain function during development and disease. Given recent interest in using amyloid {beta} (A{beta})-targeted antibodies as a therapy for patients with Alzheimers disease, removal of A{beta} by phagocytosis is likely protective early in Alzheimers disease, but remains poorly understood. Impaired phagocytic function of glial cells surrounding A{beta} plaques during later stages in Alzheimers disease likely contributes to worsened disease outcomes, but the underlying mechanisms of how this occurs remain unknown. We have developed a human A{beta}1-42 analogue (A{beta}pH) that exhibits green fluorescence upon internalization into the acidic phagosomes of cells but is non-fluorescent at physiological pH. This allowed us to image, for the first time, glial uptake of A{beta}pH in real time in live animals. Microglia phagocytose more A{beta}pH than astrocytes in culture, in brain slices and in vivo. A{beta}pH can be used to investigate the phagocytic mechanisms removing A{beta} from the extracellular space, and thus could become a useful tool to study A{beta} clearance at different stages of Alzheimers disease.

neuroscience↗

bioRxiv2019

Melanoma-secreted Amyloid Beta Supresses Neuroinflammation and Promotes Brain Metastasis

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. We performed unbiased proteomics analysis of melanoma short-term cultures, a novel model for the study of brain metastasis. Intriguingly, we found that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared to those derived from extracranial metastases. This raised the exciting hypothesis that molecular pathways implicated in neurodegenerative disorders are critical for metastatic adaptation to the brain. Here, we show that melanoma cells require amyloid beta (A{beta}), a polypeptide heavily implicated in Alzheimers disease, for growth and survival in the brain parenchyma. Melanoma cells produce and secrete A{beta}, which activates surrounding astrocytes to a pro-metastatic, anti-inflammatory phenotype. Furthermore, we show that pharmacological inhibition of A{beta} decreases brain metastatic burden. Our results reveal a mechanistic connection between brain metastasis and Alzheimers disease - two previously unrelated pathologies, establish A{beta} as a promising therapeutic target for brain metastasis, and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma.

cancer biology↗