Biology subjects

Ponik, S. M.

Publications and source records attributed to Ponik, S. M..

9 recordsLinked to original sources

bioRxiv2026

Integrated Multi-Omics Enabled by Sequential Extraction for Comprehensive Molecular Profiling of Small Extracellular Vesicles

Small extracellular vesicles (sEVs) are membrane-bound particles whose protein, lipid, and metabolite cargo reflects the molecular state of their cells of origin, making them attractive targets for biomarker discovery and therapeutic development. However, comprehensive characterization of sEVs remains challenging due to the extremely limited material available. Here, we present an integrated mass spectrometry-based multi-omics platform for simultaneous characterization of lipids, metabolites, and proteins from a single sEV sample enabled by sequential extraction, maximizing sample utilization. To enhance molecular coverage and analytical depth, the platform combines iterative tandem mass spectrometry for improved small-molecule fragmentation and nano-flow proteomics with data-independent acquisition. We achieved deep and reproducible multi-omic characterization of proteins, lipids, and metabolites using 10 million sEVs. We further demonstrated the compatibility of our multi-omics platform with sEVs isolated from plasma by ultracentrifugation, size-exclusion chromatography with ultrafiltration, and polymer precipitation, revealing purification-dependent differences in molecular profiles associated with tradeoffs in yield and purity of sEVs. By enabling integrated multi-omics from the same sample, this strategy addresses a key challenge in low-input sEV analysis and establishes a robust analytical foundation for synergistic biomarker discovery and therapeutic applications.

biochemistry↗

bioRxiv2025

Exosomes are specialized vehicles to induce fibronectin assembly

Fibronectin is a key stromal matrix molecule whose assembly into fibrils is thought to require cells. In contrast, we find that small exosome-type extracellular vesicles (EVs) are critical initiators of fibronectin assembly. Fibroblasts engineered to be deficient in exosome secretion showed greatly reduced assembly of fibronectin and other stromal matrix molecules in 2D, 3D, and in vivo environments, and led to reduced tumor growth and lung metastasis by breast cancer cells. Furthermore, transgenic mice with defects in exosome secretion had greatly reduced lung fibrosis after treatment with bleomycin. In a direct test of exosome function, we find that the addition of purified small EVs to purified soluble fibronectin in a cell-free system is sufficient to induce fibronectin assembly. The EV-induced fibronectin assembly requires the presence of fibronectin-binding integrins and Syndecan-1 in the EVs. We propose a new model in which secreted exosomes directly drive stromal matrix assembly and tissue fibrosis.

cell biology↗

bioRxiv2025

Inhibiting fibronectin assembly in the breast tumor microenvironment increases cell death and improves response to doxorubicin

PurposeEffective therapies for solid tumors, including breast cancers, are hindered by several roadblocks that can be largely attributed to the fibrotic extracellular matrix (ECM). Fibronectin (FN) is a highly upregulated ECM component in the fibrotic tumor stroma and is associated with poor patient prognosis. This study aimed to investigate the therapeutic potential of an anti-fibrotic peptide that specifically targets FN and blocks the fibrillar assembly of FN. MethodsTo target FN, we used PEGylated Functional Upstream Domain (PEG-FUD), which binds to the 70 kDa N-terminal region of FN with high affinity, localizes to mammary tumors, and potently inhibits FN assembly in vitro and in vivo. Here, we used the 4T1 tumor model to investigate the efficacy and mechanisms of PEG-FUD to inhibit tumor growth. ResultsOur data demonstrates that PEG-FUD monotherapy reduces tumor growth without systemic toxicity. Analysis of the tumor microenvironment revealed that PEG-FUD effectively inhibited FN matrix assembly within tumors and reduced adhesion-mediated signaling through 5 integrin and FAK leading to enhanced tumor cell death. Notably, signaling through FAK has been associated with resistance mechanisms to doxorubicin (DOX). Therefore, we tested the combination of PEG-FUD and Dox, which significantly reduced tumor growth by 60% compared to vehicle control and 30% compared to Dox monotherapy. ConclusionsOur findings demonstrate that PEG-FUD significantly modifies the peritumoral ECM of breast cancer, leading to increased tumor cell death, and potentiates the efficacy of conventional breast cancer therapy.

cancer biology↗

bioRxiv2024

Myeloid-Specific Thrombospondin-1 Deficiency Exacerbates Aortic Rupture via Broad Suppression of Extracellular Matrix Proteins

RationaleRupture of abdominal aortic aneurysms (AAA) is associated with high mortality. However, the precise molecular and cellular drivers of AAA rupture remain elusive. Our prior study showed that global and myeloid-specific deletion of matricellular protein thrombospondin-1 (TSP1) protects mice from aneurysm formation primarily by inhibiting vascular inflammation. ObjectiveTo investigate the cellular and molecular mechanisms that drive AAA rupture by testing how TSP1 deficiency in different cell populations affects the rupture event. Methods and ResultsWe deleted TSP1 in endothelial cells and macrophages --- the major TSP1-expressing cells in aneurysmal tissues ---- by crossbreeding Thbs1 flox/flox mice with VE-cadherin Cre and Lyz2-cre mice, respectively. Aortic aneurysm and rupture were induced by angiotensin II in mice with hypercholesterolemia. Myeloid-specific Thbs1 knockout, but not endothelial-specific knockout, increased the rate of lethal aortic rupture by more than 2 folds. Combined analyses of single-cell RNA sequencing and histology showed a unique cellular and molecular signature of the rupture-prone aorta that was characterized by a broad suppression in inflammation and extracellular matrix production. Visium spatial transcriptomic analysis on human AAA tissues showed a correlation between low TSP1 expression and aortic dissection. ConclusionsTSP1 expression by myeloid cells negatively regulates aneurysm rupture, likely through promoting the matrix repair phenotypes of vascular smooth muscle cells thereby increasing the strength of the vascular wall.

pathology↗

bioRxiv2024

Tumor Stroma Content Regulates Penetration and Efficacy of Tumor-targeting Bacteria

Bacteria-based cancer therapy (BBCT) strains grow selectively in primary tumors and metastases, colonize solid tumors independent of genetics, and kill cells resistant to standard molecular therapy. Clinical trials of BBCT in solid tumors have not reported any survival advantage yet, partly due to the limited bacterial colonization. Collagen, abundant in primary and metastatic solid tumors, has a well-known role in hindering intratumoral penetration of therapeutics. Nevertheless, the effect of collagen content on the intratumoral penetration and antitumor efficacy of BBCT is rarely unexplored. We hypothesized that the presence of collagen limits the penetration and, thereby, the antitumor effects of tumor-selective Salmonella. Typhimurium VNP20009 cheY+. We tested our hypothesis in low and high collagen content tumor spheroid models of triple-negative murine breast cancer. We found that high collagen content significantly hinders bacteria transport in tumors, reducing bacteria penetration and distribution by [~]7-fold. The higher penetration of bacteria in low collagen-content tumors led to an overwhelming antitumor effect ([~]73% increase in cell death), whereas only a 28% increase in cell death was seen in the high collagen-content tumors. Our mathematical modeling of intratumoral bacterial colonization delineates the role of growth and diffusivity, suggesting an order of magnitude lower diffusivity in the high collagen-content tumors dominates the observed outcomes. Finally, our single-cell resolution analysis reveals a strong spatial correlation between bacterial spatial localization and collagen content, further corroborating that collagen acts as a barrier to bacterial penetration despite S. Typhimurium VNP20009 cheY+ motility. Understanding the effect of collagen on BBCT performance could lead to engineering more efficacious BBCT strains capable of overcoming this barrier to colonization of primary tumors and metastases.

biophysics↗

bioRxiv2022

One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant

Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content of exosomes but the current methods require laborious and time-consuming multi-step sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, for rapid and effective exosomal lysis, protein extraction, and digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3,466 proteins and quantified 2,288 proteins using reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. 3,166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.

biochemistry↗

bioRxiv2021

Conversion of cancer-associated fibroblasts from pro- to antitumor improves the sensitivity of pancreatic cancer to chemotherapeutics

Previous therapeutic attempts to deplete cancer-associated fibroblasts (CAFs) or inhibit their proliferation in pancreatic ductal adenocarcinoma (PDAC) were not successful in mice or patients. Thus, CAFs may be tumor suppressive or heterogeneous, with distinct cancer-restraining and -promoting CAFs (rCAFs and pCAFs, respectively). Here, we show that induced expression of the glycosylphosphatidylinositol-anchored protein Meflin, a rCAF-specific marker, in CAFs by genetic and pharmacological approaches improved the chemosensitivity of mouse PDAC. A chemical library screen identified Am80, a synthetic, non-natural retinoid, as a reagent that effectively induced Meflin expression in CAFs. Am80 administration improved the sensitivity of PDAC to chemotherapeutics, accompanied by increases in tumor vessel area and intratumoral drug delivery. Mechanistically, Meflin was involved in the suppression of tissue stiffening by interacting with lysyl oxidase to inhibit its collagen crosslinking activity. These data suggested that modulation of CAF heterogeneity may represent a strategy for PDAC treatment.

cancer biology↗

bioRxiv2020

Cell-Scale Biophysical Cues from Collagen Fiber Architecture Instruct Cell Behavior and the Propagation of Mechanosensory Signals

Mechanosensory cues from the extracellular matrix underpin numerous cellular behaviors including tumor cell migration yet are influenced by the local structure and organization of the matrix in unknown ways. To investigate mechanical cues with respect to local collagen organization, we used a combination of intravital imaging of the mammary tumor microenvironment and 3D collagen gel systems with both migratory MDA-MB-231 cells and acellular pNIPAAm beads. We identified that fiber organization directs a bias in cell response along the axis of alignment. Using innovative methodology, we determined that local collagen alignment resulted in a 30-fold difference in directional cell-scale stiffness and also dramatically altered the rate at which cell-induced fiber displacements decayed over distance. Our results reveal differential mechanical properties across orthogonal directions in aligned matrices that provide sizeable cues to the cell and have important implications for cellular mechanosensing and cell-cell communication within the tissue microenvironment.

biophysics↗

bioRxiv2019

Islet architecture controls synchronous β cell response to glucose in the intact mouse pancreas in vivo

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between {beta} cells, yet testing this in vivo in the intact pancreas is challenging. Robo {beta}KO mice, in which the genes Robo1 and Robo2 are deleted selectively in {beta} cells, provide a unique model of altered islet spatial architecture without loss of {beta} cell differentiation or islet damage from diabetes. Combining Robo {beta}KO mice with intravital microscopy, we show here that Robo {beta}KO islets lose synchronized intra-islet Ca2+ oscillations between {beta} cells in vivo. We provide evidence that this loss is not due to a {beta} cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

developmental biology↗