Biology subjects

Spatz, J.

Publications and source records attributed to Spatz, J..

7 recordsLinked to original sources

bioRxiv2024

Replicating retroviral delivery of an IL-15 superagonist improves antitumor immunity and long-term survival in poorly immunogenic glioblastoma models

Glioblastoma (GBM) is the most lethal primary brain neoplasm due to its highly immunosuppressive microenvironment and resistance to conventional therapies. To overcome this challenge, we engineered a replicating retrovirus (RRV) to deliver a superagonist interleukin-15 receptor-linked fusion protein (RLI) directly to tumor cells, engineering them into local immunotherapy biofactories. This strategy leverages the tumor-selective replication of RRV to achieve localized and sustained RLI expression within the tumor microenvironment. In two orthotopic poorly immunogenic GBM mouse models, intratumoral administration of RRV RLI significantly reduced tumor growth and prolonged survival compared to controls, with some mice achieving long-term remission and demonstrating immunologic memory upon rechallenge. Transcriptomic and flow cytometric analyses revealed that RRV RLI treatment enhanced infiltration and activation of CD8 T cells, NK cells, and upregulated antigen presentation pathways within the tumor microenvironment. Depletion studies indicated that the therapeutic efficacy of RRV RLI is dependent on both CD4 and CD8 T cells. Notably, combining RRV RLI with the GBM standard of care chemotherapeutic agent temozolomide (TMZ) synergistically improved survival outcomes. Subsequent single-cell RNA and T cell receptor sequencing identified enhanced effector cell activation, antigen presentation, and clonal T cell expansion in the combination therapy group. Further T cell receptor analysis and clustering implied a tumor-specific immune response rather than one targeting the viral delivery vehicle, suggesting that this therapeutic approach could be reapplied without eliciting anti-vector immunity. Our findings suggest that RRV-mediated delivery of RLI effectively transforms GBM tumors into immunostimulatory hubs, eliciting a potent anti-tumor immune response. This novel viral immunotherapy holds significant promise for clinical translation in the treatment of GBM and other difficult-to-treat solid tumors.

cancer biology↗

bioRxiv2022

Binding of His-tagged fluorophores to lipid bilayers and giant vesicles

His-tagged molecules can be attached to lipid bilayers via certain anchor lipids, a method that has been widely used for the bio-functionalization of membranes and vesicles. To observe the membrane-bound molecules, it is useful to consider His-tagged molecules that are fluorescent as well. Here, we study two such molecules, green fluorescence protein (GFP) and green-fluorescent fluorescein isothiocyanate (FITC), both of which are tagged with a chain of six histidines (6H) that bind to the anchor lipids within the bilayers. The His-tag 6H is much smaller than the GFP molecule but somewhat larger than the FITC dye. The lipid bilayers form giant unilamellar vesicles (GUVs), the behavior of which can be directly observed in the optical microscope. We apply and compare three well-established preparation methods for GUVs: electroformation on platinum wire, polyvinyl alcohol (PVA) hydrogel swelling, and electroformation on indium tin oxide (ITO) glass. Microfluidics is used to expose the GUVs to a constant fluorophore concentration in the exterior solution. The brightness of membrane-bound 6H-GFP exceeds the brightness of membrane-bound 6H-FITC, in contrast to the quantum yields of the two fluorophores in solution. In fact, 6H-FITC is observed to be strongly quenched by the anchor lipids which bind the fluorophores via Ni2+ ions. For both 6H-GFP and 6H-FITC, the membrane fluorescence is measured as a function of the fluorophores molar concentration. The theoretical analysis of these data leads to the equilibrium dissociation constants Kd = 37.5 nM for 6H-GFP and Kd = 18.5 nM for 6H-FITC. We also observe a strong pH-dependence of the membrane fluorescence.

biophysics↗

bioRxiv2021

Structure and mechanism of SARS-CoV-2 Spike N679-V687 deletion variant elucidate cell-type specific evolution of viral fitness

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants which is of particular concern due to their potential for increased transmissibility and pathology. In addition to this entrenched variant diversity in circulation, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriS{Delta} variant, originally identified as a viral subpopulation by passaging SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike glycoprotein an eight amino-acid deletion encompassing the furin recognition motif and S1/S2 cleavage site. Here, we elucidate the structure, function and molecular dynamics of this variant spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Moreover, our study reveals long-range allosteric communication between functional regions within the spike that differ in wild-type and deletion variant. Our results support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

biochemistry↗

bioRxiv2021

Identification of Cancer-Associated Fibroblasts in Glioblastoma and Defining Their Pro-tumoral Effects

Despite their identification in some cancers, pro-tumoral cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of primary glioblastoma cultures yielded cells with CAF morphology, CAF transcriptomic profile, and mesenchymal lineage in single-cell RNA-seq. Glioblastoma CAFs were attracted to glioblastoma stem cells (GSCs) and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF-{beta} and TGF-{beta} as mediators of GSC effects on CAFs, and osteopontin and hepatocyte growth factor as mediators of CAF-induced GSC enrichment. Glioblastoma CAFs also induced M2 macrophage polarization by producing the EDA fibronectin variant. Glioblastoma CAFs were enriched in the subventricular zone which houses neural stem cells that produce GSCs. Including CAFs in GSC-derived xenografts induced in vivo growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

cancer biology↗

bioRxiv2020

Unexpected free fatty acid binding pocket in the cryo-EM structure of SARS-CoV-2 spike protein

COVID-19, caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms driving high infectivity, broad tissue tropism and severe pathology. Our cryo-EM structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains (RBDs) tightly and specifically bind the essential free fatty acid (FFA) linoleic acid (LA) in three composite binding pockets. The pocket also appears to be present in the highly pathogenic coronaviruses SARS-CoV and MERS-CoV. Lipid metabolome remodeling is a key feature of coronavirus infection, with LA at its core. LA metabolic pathways are central to inflammation, immune modulation and membrane fluidity. Our structure directly links LA and S, setting the stage for interventions targeting LA binding and metabolic remodeling by SARS-CoV-2. One Sentence SummaryA direct structural link between SARS-CoV-2 spike and linoleic acid, a key molecule in inflammation, immune modulation and membrane fluidity.

biochemistry↗

bioRxiv2020

Substrate Resistance to Traction Forces Controls Fibroblast Polarization

The mechanics of fibronectin-rich extracellular matrix regulate cell physiology in a number of diseases, prompting efforts to elucidate cell mechanosensing mechanisms at the molecular and cellular scale. Here, the use of fibronectin-functionalized silicone elastomers that exhibit considerable frequency-dependence in viscoelastic properties unveiled the presence of two cellular processes that respond discreetly to substrate mechanical properties. Soft elastomers supported efficient focal adhesion maturation and fibroblast spreading due to an apparent stiff surface layer. However, soft elastomers did not enable cytoskeletal and fibroblast polarization; elastomers with high cross-linking and low deformability were required for polarization. The underlying reason for this behavior was the inability of soft elastomeric substrates to resist traction forces, rather than a lack of sufficient traction force generation; accordingly, mild inhibition of actomyosin contractility rescued fibroblast polarization even on the softer elastomers. Our findings help reconcile previously proposed local and global models of cell mechanosensing by demonstrating the differential dependence of substrate mechanics on distinct cellular processes. Statement of SignificanceThe mechanisms cells employ to sense and respond to the mechanical properties of their surroundings remain incompletely understood. In this study we used a commercial silicone elastomer formulation to prepare compliant, fibronectin-coated substrates and investigate the adhesion and polarization of human fibroblasts. Our results suggest the existence of at least two discrete mechanosensing processes regulated at different time and length (force) scales. Focal adhesion assembly and cell spreading were promoted by a stiff surface layer independent from bulk viscoelasticity, whereas effective cell polarization required elevated elastomer stiffness, sufficient to resist applied cell traction. The results presented here have implications on the use of elastomeric substrates as biomaterials for mechanosensing studies or clinical applications.

biophysics↗

bioRxiv2020

Role of c-Met/β1 integrin complex in the metastatic cascade

Metastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. While individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a structural complex between receptor tyrosine kinase c-Met and {beta}1 integrin in metastases. Using novel cell culture and in vivo assays, we found that c-Met/{beta}1 complex induction promotes breast cancer intravasation and adhesion to the vessel wall, but does not increase extravasation. These effects may be driven by the ability of the c-Met/{beta}1 complex to increase mesenchymal and stem cell characteristics. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/{beta}1 complex induction. A {beta}1 integrin point mutation that prevented binding to c-Met reduced intravasation. OS2966, a therapeutic antibody disrupting c-Met/{beta}1 binding, decreased invasion and mesenchymal gene expression and morphology of breast cancer cells. Bone-seeking breast cancer cells exhibited higher c-Met/{beta}1 complex levels than parental controls and preferentially adhere to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/{beta}1 levels than brain metastases. Thus, the c-Met/{beta}1 complex drives breast cancer cell intravasation and preferential affinity for bone tissue-specific matrix. Pharmacological targeting of the complex may prevent metastases, particularly osseous metastases.

cancer biology↗