Biology subjects

Paszek, M. J.

Publications and source records attributed to Paszek, M. J..

13 recordsLinked to original sources

bioRxiv2026

Engineering CAR-T cells to remodel the mucin-rich cancer cell glycocalyx

The dense glycocalyx of cancer cells can restrict immune-cell access to surface antigens and limit CAR-T cell activity. Here, we show that mucin density and epitope position determine how glycocalyx remodeling affects CAR-T cell recognition and killing. We identify KLK5 as a human protease that cleaves tumor-associated mucins, increases access to membrane-proximal antigens, and enhances CAR-T cell function. We then engineer CAR-T cells to display or secrete KLK5, enabling remodeling of the tumor glycocalyx during antigen recognition. KLK5-engineered CAR-T cells improved tumor control across multiple xenograft models, and KLK5-secreting MUC17 CAR-T cells produced the strongest in vivo benefit, prolonging survival compared with conventional MUC17 CAR-T cells. These findings show that CAR-T cells can be engineered to breach the mucin-rich glycocalyx while preserving accessible target epitopes.

bioengineering↗

bioRxiv2026

Functional Differentiation of Type II and Type I Collagen Articular Models in Synovial Fluid Film Formation and Recombinant Lubricin Retention

Collagen type II (Col-II) and collagen type I (Col-I) are major components of articular cartilage present at different ratios at its surface. Understanding how each of these components mediates the assembly of molecular films derived from synovial fluid (SF), the native lubricant of synovial joints, is critical to explain the loss of mechanical performance in pathological conditions, guide the design of biomaterial implants meant to be in contact with SF, and develop molecular therapies to restore SF properties. This work demonstrates that Col-II articular surface model assists in scaffolding of full SF-derived films, while Col-I model lacks SF film scaffolding capabilities. However, when Col-II and Col-I are exposed to recombinant lubricin (rLub) alone, the major boundary lubricant in SF, both adsorbed and retained similar amounts. These insights, deduced from quartz crystal microbalance with dissipation, diffuse reflectance circular dichroism, and atomic force microscopy, reveal possible mechanisms underlying the loss of mechanical performance of synovial joints in pathology, where Col-I becomes the major collagenous component of the articular cartilage surface, as well as considerations for designing functional biomaterial implants. Furthermore, this work reinforces the idea of rLub as an intra-articular osteoarthritis therapy with the ability to bind to Col-II and Col-I, irrespectively. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/726594v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@138a77borg.highwire.dtl.DTLVardef@7b8512org.highwire.dtl.DTLVardef@15d7060org.highwire.dtl.DTLVardef@17ccf20_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Delayed lubricin injection improves cartilage repair tissue quality in an in vivo rabbit osteochondral defect model

Osteochondral lesions (OCL) are common among young patients and often require surgical interventions since cartilage has a poor capacity for self-repair. Bone marrow stimulation (BMS) has been used clinically for decades to treat OCLs, however a persisting challenge with BMS and other cartilage repair strategies is the inferior quality of the resulting fibrocartilaginous repair tissue. Lubrication-based therapies have the potential to improve the quality of cartilage repair tissue as joint lubrication is linked to local cartilage tissue strains and subsequent cellular responses including death and apoptosis. Recently, a full length recombinant human lubricin (rhLubricin) was developed and has been shown to lower friction in cartilage. This study investigated the effect of a single delayed injection of rhLubricin on cartilage repair in an in vivo rabbit OCL model using gross macroscopic evaluation, surface profilometry, histology, and tribology. Moderate improvement in macroscopic scores for cartilage repair were observed. Notably, quantitative analysis of Safranin-O histology showed that rhLubricin treated joints had significantly higher glycosaminoglycan content compared to saline treated joints, and there were no differences in repair integration between groups. Furthermore, rhLubricin treated joints had significantly lower friction coefficients tested across three sliding speeds compared to saline treated joints (rhLubricin: 0.15 {+/-} 0.03 at 0.1 mm/s to 0.12 {+/-} 0.03 at 10 mm/s, Saline: 0.22 {+/-} 0.06 at 0.1 mm/s to 0.19 {+/-} 0.05 at 10 mm/s). Overall, a single delayed injection of rhLubricin improved the quality and lubricating ability of the repair cartilage tissue without inhibiting repair tissue integration.

bioengineering↗

bioRxiv2024

Leucine zipper-based SAIM imaging identifies therapeutic agents to disrupt the cancer cell glycocalyx for enhanced immunotherapy

The abnormally thick glycocalyx of cancer cells can physically limit immune-cell engagement and reduce the efficacy of cellular immunotherapies, but strategies for identifying pharmacological agents that relieve this barrier remain limited. Here, we establish glycocalyx thickness as a pharmacologically actionable physical phenotype. Disruption of mucin-type O-glycosylation reduced glycocalyx thickness and increased NK- and CAR-NK-mediated killing, demonstrating that pharmacological glycocalyx remodeling can enhance innate and CAR-directed cytotoxicity. To screen this phenotype directly, we developed a scanning angle interference microscopy (SAIM) platform using complementary leucine zipper pairs as a stable extracellular plasma-membrane reference. A focused screen of glycosylation and metabolic inhibitors resolved compound- and cell-type-specific nanoscale changes in glycocalyx thickness and identified several thinning agents. Castanospermine, an inhibitor of early N-glycan processing, was among the strongest hits and reduced mucin-probe binding in an independent mucin-rich gastric cancer model. Castanospermine-treated gastric cancer cells retained MUC17 antibody recognition and showed increased physical avidity for MUC17 CAR-T cells, while castanospermine enhanced MUC17 CAR-T-mediated tumor control in real-time co-culture assays. These findings establish direct glycocalyx-thickness measurement as a physical-phenotype-guided screening strategy and identify castanospermine as a candidate modulator for combination with MUC17 CAR-T cells.

bioengineering↗

bioRxiv2024

Dimerization activates the Inversin complex in C. elegans

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through characterization of hyperactive alleles in C. elegans, we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologs of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states--an active dimer and an inactive monomer--gates the output of the Inversin complex.

cell biology↗

bioRxiv2024

Recombinant manufacturing of multispecies biolubricants

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O-glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general.

bioengineering↗

bioRxiv2024

Bio-orthogonal Glycan Imaging of Culture Cells and Whole Animal C. elegans with Expansion Microscopy

Complex carbohydrates called glycans play crucial roles in the regulation of cell and tissue physiology, but how glycans map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O-glycans throughout the entirety of the Caenorhabditis elegans model organism. We construct a library of multifunctional linkers to probe and anchor metabolically labelled glycans in expansion microscopy (ExM), an imaging modality that overcomes the diffraction limit of conventional optical microscopes through the physical expansion of samples embedded in a polyelectrolyte gel matrix. A flexible strategy is demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, we resolve metabolically labelled O-glycans on the gut microvilli and other nanoscale anatomical features using our ExM reagents and optimized protocols. We use transmission electron microscopy images of C. elegans nano-anatomy as ground truth data to validate the fidelity and isotropy of gel expansion. We construct whole organism maps of C. elegans O-glycosylation in the first larval stage and identify O-glycan "hotspots" in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans, we provide validated ExM protocols for nanoscale imaging of metabolically labelled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labelled biomolecules at enhanced resolutions with ExM. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/578333v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@68fb6aorg.highwire.dtl.DTLVardef@52166aorg.highwire.dtl.DTLVardef@13dbfaeorg.highwire.dtl.DTLVardef@f352ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Collagen mineralization decreases NK cell-mediated cytotoxicity of breast cancer cells via increased glycocalyx thickness

Skeletal metastasis is common in patients with advanced breast cancer, and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow, but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, we have utilized a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. Our results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increased their glycocalyx thickness while enhancing resistance to attack by Natural Killer (NK) cells. These changes were functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, our results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

cancer biology↗

bioRxiv2023

Glycocalyx dynamics and membrane curvature under cytosolic pressure

The glycocalyx is a soft material composed of glycosylated proteins and lipids decorating the plasma membrane of cells. Although the glycocalyx is a mechanical conduit between the plasma membrane and the cell surroundings, the coupled dynamics of the glycocalyx and membrane are poorly understood in most cell processes. Here, we construct a dynamic model to predict the coupled mechanical behaviors of the glycocalyx and membrane due to internal cytosolic pressure in a cell interacting with an external substrate. We report how the glycocalyx constituents bear cytosolic loads, physically rearrange, and shape the cell membrane. Simulations of the model predict that highly flexible, polymeric elements in the glycocalyx are uniformly compressed in the cell-substrate contact zone. However, more rigid, fiber-like glycocalyx constituents dynamically rearrange over timescales on the order of 10 milliseconds, concentrating into clusters. This spatial heterogeneity allows close apposition of the plasma membrane with the substrate, a geometry that would be favorable for subsequent adhesive bond formation. Clustered glycocalyx constituents imprint nanotopographical features on the plasma membrane with longitudinal dimensions of 200 - 300 nm. Analysis of the membrane topographies reveals curvatures that could be sufficient to elicit biological responses through curvature sensing proteins, such as BAR-domain proteins. Together, our simulations suggest how instabilities in the compressed glycocalyx could mediate downstream adhesive and signaling processes in the bleb-substrate interface.

biophysics↗

bioRxiv2022

Hyaluronic acid biosynthesis promotes an invasive, stem-like cancer cell phenotype by broadly altering metabolism

Metastasis is the leading cause of breast cancer-related deaths and often driven by invasion and cancer-stem like cells (CSCs). Both the CSC phenotype and invasion have been associated with increased hyaluronic acid (HA) production. How these independent observations are connected, and which role metabolism plays in this process remains unclear due in part to the lack of convergent approaches that integrate engineered model systems, computational tools, and cancer biology. Using microfluidic invasion models, metabolomics, computational flux balance analysis (FBA), and bioinformatic analysis of patient data we investigated the functional links between the stem-like, invasive, and metabolic phenotype of breast cancer cells as a function of HA biosynthesis. Our results suggest that CSCs are more invasive than non-CSCs and that broad metabolic changes caused by overproduction of HA play a role in this process. Accordingly, overexpression of hyaluronic acid synthases (HAS) 2 or 3 induced a metabolic phenotype that promoted breast cancer cell stemness and invasion in vitro and upregulated a transcriptomic signature that was predictive of increased invasion and worse survival in patients. Collectively, this study suggests that HA overproduction leads to metabolic adaptations that help satisfy the energy demands necessary for 3D invasion of breast cancer stem cells further highlighting the importance of engineered model systems and multidisciplinary approaches in cancer research.

cancer biology↗

bioRxiv2022

Mucins form a nanoscale material barrier against immune cell attack

The cancer cell glycocalyx serves as a major line of defense against immune surveillance. However, how specific physical properties of the glycocalyx contribute to immune evasion and how these properties are regulated are not well understood. Here, we uncover how the surface density, glycosylation, and crosslinking of cancer-associated mucins contribute to the nanoscale material thickness of the glycocalyx, and further analyze the effect of the glycocalyx thickness on resistance to effector cell attack. Natural Killer (NK) cell-mediated cytotoxicity exhibits a near perfect inverse correlation with the glycocalyx thickness of target cells regardless of the specific glycan structures present. NK cells expressing a chimeric antigen receptor (CAR) have an enhanced ability to breach the glycocalyx and kill target cells. Equipping the NK cell surface with a mucin-digesting enzyme also improves killing with a performance enhancement that rivals or exceeds CARs in some cases. Together, our results provide new considerations for improving cancer immunotherapies.

biophysics↗

bioRxiv2019

Litmus-Body: a Molecularly Targeted Sensor for Cell-Surface pH Measurements

Precise pH measurements in the immediate environment of receptors is essential for elucidating the mechanisms through which local pH changes associated with diseased phenotypes manifest into aberrant receptor function. However, current pH sensors lack the molecular specificity required to make these measurements. Herein we present the Litmus-body, our recombinant protein-based pH sensor, which through fusion to an anti-mouse IgG nanobody is capable of molecular targeting to specific proteins on the cell surface. By normalizing a pH-dependent green fluorescent protein to a long-Stokes shift red fluorophore or fluorescent protein, we readily report pH independent of sensor concentration using a single 488-nm excitation. Our Litmus-body showed excellent responsiveness in solution, with a greater than 50-fold change across the physiological regime of pH. The sensor was further validated for use on live cells, shown to be specific to the protein of interest, and was able to successfully recapitulate the numerous pH changes along the endocytic pathway.

cell biology↗

bioRxiv2019

High-speed device synchronization in optical microscopy with an open-source hardware control platform

Recent advances in fluorescence microscopy have enabled the visualization of subcellular structures at unprecedented resolution. However, the complexity of state-of-the-art microscopes has increased considerably, often requiring the precise control and synchronization of multiple peripheral devices at high speeds. Drawing inspiration from open-source prototyping systems, like the Arduino, we describe the development of a new control platform that adopts the best features of these systems - affordability, facile programmability, and flexible connectivity - but with the scientific-grade inputs and outputs (I/O) and built-in routines that are necessary to control peripherals in advanced microscopy applications. Notably, our platform includes waveform generators and I/O for point-and azimuthal-scanning of excitation in laser-based applications. As a proof of concept, we show how the integration of waveform generation, multiplexed analog outputs, and native hardware triggers into a single central hub provides a versatile platform for performing fast circle-scanning acquisitions, including ring scanning-angle interference microscopy (SAIM), total internal reflection fluorescence (TIRF and ring TIRF) microscopy, and multiangle TIRF (MA-TIRF). We also demonstrate how the low communication latency of our hardware platform can reduce image intensity and reconstruction artifacts arising from synchronization errors produced by software control. Our complete platform, including hardware design files, firmware, API, software, and all associated source code, will be hosted for community-based development and collaboration.

biophysics↗