Structure-functionality relationship of collagen-fibrin interpenetrating hydrogels for engineered tumor-stroma models

Acta Biomaterialia · 2026-03-11

Collagen and fibrin are two major extracellular matrix (ECM) proteins involved in tumorigenesis. During cancer progression, collagen and fibrin undergo sequential and concurrent polymerization and degradation, altering ECM structure and functional properties, which impact cell behavior and drug resistance. While engineered tumor models are recently emerging, including 3D printed tissue models and microphysiological systems (MPS), the interaction of these two proteins and its impact on the structure-functionality are largely overlooked. A critical knowledge gap on the structure-functionality relationship of collagen-fibrin interpenetrating hydrogels limits reliable reconstitution of the ECM of the tumor-stroma microenvironment. To address this, we characterized the fibrillar microstructure and biomechanical properties (e.g., diffusivity, Young's modulus, and cell-derived contraction) of collagen-fibrin interpenetrating hydrogels while varying the protein ratios and sequence of polymerization. The results show that the pore size of the composite matrices decreases as fibrin content increases. This structural change is correlated to the decreased diffusivity, which can be partially recovered after fibrin depletion. In contrast, Young's modulus only increases at lower fibrin content and decreases with increases in fibrin content, even below collagen-only levels. Cell-derived contraction is well correlated with these Young's modulus changes. Confocal microscopy analysis shows that collagen-fibrin gels have distinctly different microstructure depending on polymerization sequence, which highlights how collagen-fibrin interactions during polymerization shape matrix properties, with important implications for the design of engineered tumor models. STATEMENT OF SIGNIFICANCE: Engineered tumor models are emerging to recapitulate the complexity of human tumors with unprecedented cellular and molecular resemblance. Despite recent advancements, efforts to recreate the fibrin deposition caused by the extravascular coagulation due to leaky tumor vasculature is lacking. Furthermore, its impact on the structure-functionality relation of the tumor-stroma tissues poses a significant knowledge gap to reliably reconstitute biomaterials for engineered tumor models. To address this gap, we report how composition and polymerization conditions influence the microstructure and biomechanical properties of collagen-fibrin interpenetrating hydrogels. We show that both the collagen and fibrin concentration and polymerization sequence impact the interpenetrating microstructure and determine the diffusivity, Young's modulus, and contraction index of the hydrogel. These results are important by providing the quantitative knowledge-base for designing and analyzing biomaterials for new and innovative engineered tumor models.

CAR-neutrophils produced in vivo to treat glioma

Nature Biomedical Engineering · 2026-04-24 · 1 citations

Thrombin‐PAR1 signaling in pancreatic cancer promotes an immunosuppressive microenvironment

UNC Libraries · 2026-03-19

Exosome-like biogenesis from the Golgi releases extracellular vesicles lacking conventional tetraspanins that mediate immune evasion in cancer

bioRxiv (Cold Spring Harbor Laboratory) · 2026-05-13

Abstract Immunotherapy has improved survival across multiple malignancies but remains largely ineffective in solid cancers such as lung, breast, and pancreatic cancer. A key driver of resistance is the immunosuppressive tumor microenvironment (TME). Although numerous mediators of TME immunosuppression have been identified, therapeutic targeting has provided limited clinical benefit. Tumor-derived extracellular vesicles (EVs) have recently emerged as contributors to resistance, yet their mechanisms remain unclear. We developed human non-small cell lung cancer models to investigate EV-mediated immunosuppression. We identified a distinct Golgi-derived EV subpopulation that potently suppress T cell function and tumor infiltration. These EVs express the trans -Golgi network marker TGOLN2, and exhibit minimal levels of canonical EV markers. TGOLN2 overexpression drives this suppressive phenotype. Clinically, elevated TGOLN2 associates with poor survival and correlate with an immunosuppressive TME signature across more than 20 cancer types, including NSCLC. Collectively, this work defines a previously unrecognized mechanism of TGOLN2-driven, EV-mediated immunosuppression. Statement of Significance We revealed TGOLN2 overexpression as a new immune evasion mechanism that mediates T cell suppression through increased secretion of a Golgi-derived extracellular vesicle (EV) subpopulation. These findings redefine current paradigms of EV biology and nominate TGOLN2 as a potential biomarker and therapeutic target in immunosuppressive cancers.

Optimizing mRNA delivery with targeted elastin-like polypeptide–based LENN formulations: Insights into the endocytosis mechanism

Proceedings of the National Academy of Sciences · 2026-01-07 · 1 citations

Gene delivery has emerged as a groundbreaking technique for altering gene expression, offering new possibilities in treating a vast array of diseases. We report a layer-by-layer elastin-like polypeptide nucleic acid nanoparticle (LENN) system for mRNA delivery as an attractive alternative to viral vectors and lipid nanoparticle (LNP) systems. This study focuses on determining the physical characteristics of LENN bearing mRNA cargo and assessing their biological performance in T24 bladder tumor cells. Our data show that mRNA encoding luciferase forms stable 30 to 130 nm LENN particles via batch mixing to efficiently encapsulate the mRNA strands, are resistant to heparin challenge, and are capable of storage at -20 °C for 3 d as lyophilized powders while retaining full biological activity after rehydration. We also demonstrate that LENN targeted to the epidermal growth factor receptor (EGFR) can efficiently deliver the mRNA cargo to the cytosol of EGFR+ T24 human bladder cancer cells via clathrin-mediated endocytosis where it is translationally active. Lipid profiling analyses show the significant role that upregulated phospholipid biosynthesis plays in nanoparticle internalization and endosomal escape compared to untargeted LENN, indicating the importance of the clathrin pathway in contributing to the delivery efficiency of LENN. Endocytosis inhibition experiments further support the involvement of the clathrin pathway. These findings highlight the compelling features of LENN with respect to their size, in vitro and in vivo targetability, mRNA encapsulation efficiency, complex stability, gene expression, and "green" manufacturability, offering an attractive alternative to existing methods for gene delivery.

Abstract B037: Validation and translation of therapeutic potential of thrombin-PAR1 signaling in suppressing fibrosis using microphysiological PDAC tumor models

Cancer Research · 2025-09-28

Abstract Pancreatic ductal adenocarcinoma (PDAC) creates complex tumor microenvironment (TME) hallmarked with a desmoplastic stroma that facilitates tumor growth/invasion, chemoresistance, and immunosuppression. It urgently needs the identification and evaluation of stromal components that can be targeted to reprogram the stroma to improve drug delivery and efficacy without making tumors more aggressive. Thus, we hypothesize that the coagulation system in the PDAC TME can be targeted to reprogram PDAC stroma to alleviate chemoresistance and drug delivery barriers. Specifically, the thrombin/protease-activated receptor 1 (PAR1) signaling axis can be targeted to suppress growth/invasion of pancreatic cancer cells (PCCs) and cancer associated fibroblast (CAF)-derived fibrosis. Our underlying rationale is based upon a leaky tumor vasculature in PDAC resulting in the release of circulating coagulation factors and subsequent activation of the coagulation system in the TME. Tissue factor expressed by PCCs initiates the conversion of prothrombin to the active serine protease thrombin, which then activates PAR1, whose signaling is thought to promote PCC growth/invasion and CAF-mediated fibrosis. We developed and employed novel microphysiological systems (MPS) of PDAC tumor-stroma, which were designed to reconstitute extravascular coagulation in the PDAC TME to specifically investigate the role of thrombin-PAR1 signaling events on PCC growth and CAF-mediated fibrosis. Our MPS was a microfluidic platform where PCC and CAF were co-cultured in the 3D extracellular matrix perfused with/without thrombin. In addition, PAR1 expression in murine and human PCCs and CAFs was genetically modified or pharmacologically inhibited. Our MPS enabled systematic and translational analyses on the therapeutic potential of blocking PAR1 signaling in PCCs, CAFs, or both. In murine MPS, genetic deletion of PAR1 drastically decreased thrombin-mediated PCC and CAF growth compared to that of MPS with wildtype cells. Human MPS with varying levels of PAR1 also suggest thrombin stimulates PCC-CAF crosstalk, including CAF growth, elevated expression of a-SMA and secreted collagen levels. Furthermore, pharmacological inhibition of PAR1 by vorapaxar decreases both PCC and CAFs in all human PCC/CAF pairs studied. Finally, we confirm the findings from our MPS using PDAC tumor-stroma xenograft models with both human PCC and CAF. A significant reduction in tumor size is observed with vorapaxar treatment, which attributes primarily to the reduction of CAFs. In summary, we validate and translate the therapeutic potential of thrombin-PAR1 signaling in reprogramming PDAC stroma using novel MPS of PDAC tumor-stroma model. Our study also demonstrates MPS as a promising system for target identification, validation, and streamlining preclinical studies for drug discovery. Citation Format: Sae Rome. Choi, Hye-ran Moon, Natalia Ospina Muñoz, Yun Chang, Xiaoping Bao, Bennett D. Elzey, Meliss L. Fishel, Matthew J. Flick, Bumsoo Han. Validation and translation of therapeutic potential of thrombin-PAR1 signaling in suppressing fibrosis using microphysiological PDAC tumor models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B037.

Identification of alkynyl nicotinamide HSN748 as a RET solvent-front mutant inhibitor with intracranial efficacy

RSC Medicinal Chemistry · 2025-01-01 · 1 citations

This study identified HSN748 as a potent RET kinase solvent-front mutant inhibitor with a high central nervous system (CNS) permeability.

DNA Origami‐Cyanine Nanocomplex for Precision Imaging of KRAS‐Mutant Pancreatic Cancer Cells

Advanced Science · 2025-02-14 · 3 citations

Selective delivery of imaging agents to pancreatic cancer cells (PCCs) within the highly desmoplastic tumors of pancreatic ductal adenocarcinoma (PDAC) represents a significant advancement. This approach allows for precise labeling of PCCs while excluding cancer-associated fibroblasts (CAFs), thereby enhancing both research and diagnostic capabilities. Additionally, it holds the potential to target and eliminate PCCs precisely without harming the surrounding stromal cells in the PDAC tumor microenvironment (TME). In this study, DNA origami-cyanine (Do-Cy) nanocomplexes are synthesized to image KRAS-mutant PCCs selectively in the PDAC TME. These Do-Cy nanocomplexes are hypothesized to be internalized preferentially to KRAS-mutant PCCs over CAFs via elevated macropinocytosis. Several designs of Do-Cy nanocomplexes are synthesized and characterized their cellular uptake using both engineered in vitro and xenograft pancreatic cancer models. The results are further discussed for the implication of precision delivery of therapeutic and imaging agents to KRAS-mutant cancers.

AMPK‐Dependent Epigenetic Regulation of Metabolism Mediates the Anti‐Cancer Action of Pterostilbene in Hepatocellular Carcinoma

Molecular Nutrition & Food Research · 2025-09-01 · 4 citations

Disrupted metabolism, often implicated in hepatocellular carcinoma (HCC), is linked to aberrant epigenetic patterns. Dietary polyphenols, including pterostilbene (PTS), have been demonstrated to remodel epigenetic landscapes and restore metabolic homeostasis by regulating the activity of AMP-activated protein kinase (AMPK), a protein recently shown to orchestrate a diverse set of networks to epigenetically mediate transcription. We therefore explored the mechanistic involvement of AMPK in the epigenetic effects of PTS in HCC. We incorporated PTS into a choline-deficient amino acid defined HCC-inducing diet (CDAA) in male Fisher-344 rats and found significant attenuation in HCC development compared to CDAA alone. Transcriptomics by RNA-sequencing revealed PTS-upregulated targets, that were enriched in key metabolic processes, including the folate (Aldh1l1), methionine (Bhmt), and sarcosine (Dmdgh) cycles. PTS-mediated gene upregulation was linked to lower levels of histone H3-methylation at lysine 27 (H3K27me3) at gene promoters. Mechanistic studies in HCC HepG2 cells revealed that AMPK inhibition abolished epigenetic gene activation in response to PTS, which was accompanied by diminished binding of H3K27me3-demethylase KDM6A at promoters of PTS-target genes. Our findings provide evidence for new disease vulnerabilities that arise from epigenetic/metabolic changes and constitute novel opportunities for preventative and therapeutic success in HCC.

PACT suppresses PKR activation through dsRNA binding and dimerization, and is a therapeutic target for triple-negative breast cancer

RNA · 2025-08-13 · 2 citations

Triple-negative breast cancer (TNBC), the deadliest breast cancer subtype, lacks broadly applicable targeted therapies. Induction of "viral mimicry" by activation of viral double-stranded RNA (dsRNA) sensors has potential therapeutic applications for TNBC and other cancers. Suppressors of dsRNA sensing prevent sensing of endogenous dsRNAs and resulting autoimmunity. Depletion of the suppressor of dsRNA sensing ADAR1 causes activation of dsRNA sensors and cell death in many cancer cell lines. These ADAR1-dependent cells are generally also dependent on the dsRNA-binding protein PACT, which is highly expressed and essential in many TNBC cell lines. While PACT is known as an activator of the dsRNA sensor PKR, overexpression of PACT had no effect on activation of PKR in multiple TNBC cell lines. Conversely, depletion of PACT in PACT-dependent cell lines caused robust activation of PKR and cell death, in addition to induction of integrated stress response genes and NF-κB targets. These phenotypes were entirely dependent on PKR. Rescue experiments revealed that PACT dimerization and dsRNA binding are required to suppress PKR activation. While depletion of PACT alone in ADAR1/PACT-independent cell lines had no effect on PKR activation, combined depletion of both PACT and ADAR1 in those cell lines caused robust PKR activation and cell death, supporting a partially redundant role for ADAR1 and PACT in suppression of dsRNA sensing. Taken together, these findings support a vital role for PACT in suppressing PKR activation and highlight the therapeutic potential of targeting PACT to treat TNBC.