Ovijit Chaudhuri

ABSTRACT Cell division within mechanically confining extracellular matrices (ECMs) is a key regulator of tissue morphogenesis and cancer progression. Although the intracellular force-generation mechanisms that drive volumetric growth and mitotic elongation are well characterized, how ECMs resist these forces remains poorly understood. Unlike linearly elastic materials, fibrillar ECMs exhibit nonlinear and viscoelastic behaviors that fundamentally alter how they oppose cell-generated stresses. Using a fiber-level computational model, we dissected the origins of ECM-mediated mechanical confinement during mitosis. We identified three distinct modes of resistance: compressive resistance at the cell poles, shear resistance from a pericellular shell, and tensile resistance at the cell equator. The relative contributions of these modes depended on fiber architecture and connectivity; however, shear resistance from the pericellular shell—pre-tensed by volumetric growth during G1—consistently dominated as the primary mechanical barrier to mitotic elongation. These findings suggest that the pericellular shell functions as a natural mechanical checkpoint on cell division within collagen-rich microenvironments. Notably, a finite element continuum model, despite being the most widely used framework for tissue mechanics, failed to reproduce these behaviors, underscoring the necessity of fiber-resolution approaches. We propose that overcoming this mechanical checkpoint is a critical step in cancer progression, enabling cells to divide within the dense stromal matrices characteristic of metastatic tumors.

Abstract Purpose: Surgical resection remains the only curative treatment for pancreatic ductal adenocarcinoma (PDAC), yet only 15% of patients are resectable at diagnosis and up to 40% of patients present with locally advanced tumors involving vital vasculature. Even among those who undergo resection, recurrence rates exceed 60%. To address this, we conceived the idea of a locally implantable hydrogel (PancVax) capable of sustained release of an adjuvant therapeutic over a week post-operative to induce a sustained immune response in a murine model pancreatic cancer involving incomplete resection. Materials and Methods: We engineered a mechanically tough interpenetrating network consisting of carboxyethyl chitosan with dynamic covalent crosslinks and ionically crosslinked alginate. Hydrogel biocompatibility was assessed incubating hydrogel components with RAW264.7 macrophage cell line and assessing markers for apoptosis and maturation. Release assays were conducted by assessment of the hydrogel supernatant following incubation. Compression failure testing was performed to assess the compression toughness. Our murine model for incomplete resection of pancreatic cancer involved orthotopically implanting a KPC-embedded collagen hydrogel at D0 and performing an incomplete tumor resection and implantation of PancVax at D12. Treatment response to adjuvant therapeutics embedded in the PancVax was monitored through tumor volume, collagen staining, and immune profiling. Results: Individual hydrogel components were incubated for 24 hours with RAW264.7 macrophages, demonstrating >97% cell viability and <1% MHC-II+ demonstrating cytocompatibility and minimal macrophage activation. The hydrogel released albumin-FITC as a model of cytokines and peptides over a week with a sustained release profile. Hydrogels were microporous, showed a compression toughness of 60.8 kJ/m3, and maintained structural integrity during suture fixation. Implantation of PancVax with encapsulated adjuvants after tumor resection resulted in a significant reduction in tumor volume compared to resection alone (p<0.01). Conclusion: We successfully developed a hydrogel-based vaccine vehicle with properties of sustained release, high mechanical toughness suitable for suture fixation, and sufficient microporous structure permissive for cell trafficking. This work presents a novel perioperative hydrogel-based immunotherapy platform with the potential to shift the treatment paradigm for PDAC by improving post-surgical tumor control and expanding the pool of patients eligible for curative-intent surgery. Citation Format: Peter Yuxin Xie, James P. Agolia, Rosyli F. Reveron-Thornton, Chuner Guo, Maria Moozhiyil Korah, Andrea Delitto, Deshka Foster, Ovijit Chaudhuri, Daniel Delitto. Engineering a hydrogel-based vaccine to prevent recurrence in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6713.

Hydrogels that recapitulate the dynamic mechanical cues of native extracellular matrix are powerful tools that can be leveraged for tissue engineering. Despite growing recognition that cues such as stress relaxation and plasticity modulate cell-matrix interactions, the influence of these properties on mesenchymal stromal cell (MSC) chondrogenesis has yet to be elucidated across a broad range of relaxation timescales and in the absence of confounding biochemical cues. Here, we report the adaptable sliding hydrogel (ASG) with tunable stress relaxation and plasticity as a novel MSC cell niche. By incorporating reversible hydrazone crosslinks into polyethylene glycol (PEG)-based sliding hydrogels (SG), ASG achieves a wide range of tunable stress relaxation and plasticity that are distinct from other dynamic hydrogels used for MSC chondrogenesis. Notably, increasing stress relaxation and plasticity in ASG promotes rapid and robust cartilage formation by human MSCs and supports long-term cell viability. Mechanistically, ASG facilitates local matrix remodeling and enables MSCs to form "pericellular pockets" in 3D that correlate with enhanced nascent extracellular matrix deposition and reorganization, integrin signaling, and nuclear dynamics. Overall, the ASG platform provides a tunable, synthetic microenvironment that helps probe the relationship between dynamic mechanical cues and stem cell fate and informs next-generation material design within the field of tissue engineering.

Complex and robust tissue self-organization requires defined initial conditions and dynamic boundaries-neighbouring tissues and extracellular matrix that actively evolve to guide morphogenesis. A major challenge in tissue engineering is identifying material properties that are compatible with controlling initial culture conditions while mimicking dynamic tissue boundaries. Here we describe a highly tunable granular biomaterial, MAGIC matrix, that supports both long-term bioprinting and gold-standard tissue self-organization. We identify that significant stress relaxation at the long timescales and large deformation magnitudes relevant to self-organization is required for optimal morphogenesis. We apply optimized MAGIC matrices toward precise extrusion bioprinting of saturated cell suspensions directly into three-dimensional culture. Carefully controlling initial conditions for tissue growth yields dramatic increases in organoid reproducibility and complexity across multiple tissue types, enabling high-throughput generation of organoid arrays and perfusable three-dimensional microphysiological systems. Our results identify key biomaterial parameters for optimal organoid morphogenesis and lay the foundation for fabricating more complex and reproducible self-organized tissues.

Cell migration is pivotal in cancer metastasis, where cells navigate the extracellular matrix (ECM) and invade distant tissues. While the ECM is viscoelastic, exhibiting time-dependent stress relaxation, its influence on cell migration remains poorly understood. Here, we employ an integrated experimental and modeling approach to investigate filopodial cancer cell migration on viscoelastic substrates and uncover a striking transition from sub-diffusive to super-diffusive behavior driven by the substrate's viscous relaxation timescale. Conventional motor-clutch based migration models fail to capture these anomalous migration modes, as they overlook the complex adhesion dynamics shaped by broad distribution of adhesion lifetimes. To address this, we develop a glassy motor-clutch model that incorporates the rugged energy landscape of adhesion clusters, where multiple metastable states yield long-tailed adhesion timescales. Our model reveals that migration dynamics are governed by the interplay between cellular and substrate timescales: slow-relaxing substrates prolong trapping, leading to sub-diffusion, while fast-relaxing substrates promote larger steps limiting trapping, leading to super-diffusion. Additionally, we uncover the role of actin polymerization and contractility in modulating adhesion dynamics and driving anomalous migration. These findings establish a mechanistic framework linking substrate viscoelasticity to cell motility, with implications for metastasis and cancer progression.

Available mouse models for pancreatic ductal adenocarcinoma (PDAC) are limited by slow tumor development and failure to recapitulate key stromal and immune characteristics. Here, we present a protocol for generating a collagen hydrogel mouse model for orthotopic PDAC. We describe steps for embedding mouse pancreatic cancer cells in a dense collagen hydrogel and surgically implanting it into the mouse pancreas. Mouse PDAC tumors typically reach 1 cm in diameter by 10 days after implantation and show immune and stromal cell recruitment. For complete details on the use and execution of this protocol, please refer to Korah et al. 1 • Protocol for embedding and culturing KPC cells in a collagen gel matrix • Surgical procedure for implanting tumor hydrogels into the mouse pancreas • Guidance on characterization of the hydrogel and measurement of mouse tumors • Instructions for limiting variability and ensuring consistent tumor growth Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Available mouse models for pancreatic ductal adenocarcinoma (PDAC) are limited by slow tumor development and failure to recapitulate key stromal and immune characteristics. Here, we present a protocol for generating a collagen hydrogel mouse model for orthotopic PDAC. We describe steps for embedding mouse pancreatic cancer cells in a dense collagen hydrogel and surgically implanting it into the mouse pancreas. Mouse PDAC tumors typically reach 1 cm in diameter by 10 days after implantation and show immune and stromal cell recruitment.

In the version of this article initially published, due to mistakes when outputting final source data, the plot shown in Fig. 1n and Fig. 1n source data were incorrect. The conclusions are unaffected by the change. The source data and figure panel are updated in the HTML and PDF versions of the article.

T cells migrate through soft tissues to target infected and abnormal cells and regulate immunity. T cell migration is typically studied in microfluidic devices or other contexts where there is a pre-existing migration path; how they create paths in confining nanoporous extracellular matrices (ECM), such as can occur during fibrosis and around tumors, remains unclear. Here, we studied T cell migration in confining collagen-rich matrices with a range of stiffness, viscoelasticity, mechanical plasticity, and shear strength, or the stress at which the material fails. Strikingly, only shear strength, the stress at which a material fails, not stiffness or viscoelasticity, correlates with migration. During migration, T-cells extend thin actin-rich, finger-like protrusions into the ECM, which then undergo a divergent breaststroke-like motion. Thus, T cells tear apart confining matrices using a breaststroke-like motion to generate migration paths.

Pancreatic ductal adenocarcinoma (PDAC) continues to carry a dismal prognosis. The disease is characterized by a uniquely dense fibrotic matrix generated by cancer-associated fibroblasts (CAFs). We have previously demonstrated that fibroblast-driven chronic inflammation suppresses T cell function through a myeloid differentiation primary response protein 88 (MYD88)-dependent mechanism. While extensively studied in myeloid cells, the role of MYD88 signaling in CAFs and its effects on PDAC remain poorly understood. In this study, we identify a MYD88-driven inflammatory CAF population in PDAC using a combination of bulk, single-cell, and spatial transcriptomic studies. Using an innovative collagen gel implantation model, we demonstrate that loss of MYD88 in CAFs enhances T cell infiltration and suppresses tumor growth. Combining MYD88 inhibition with immune checkpoint blockade significantly reduces tumor size and enhances antitumor immune responses, underscoring its potential as a therapeutic target in PDAC.

ABSTRACT The extracellular matrix component hyaluronan (HA) plays important roles in inflammation and immune regulation. However, its involvement in lymphoid tissue during autoreactive processes remains poorly understood. Here, using the DORmO mouse model of autoimmune diabetes, we demonstrate that HA progressively accumulates in lymph nodes and splenic germinal centers during disease development. This accumulation is accompanied by a shift from high to low molecular mass HA fragments. Through immunofluorescence microscopy, we observed extensive colocalization between HA deposits and B cells in germinal centers. Inhibition of HA synthesis using 4‐methylumbelliferone (4‐MU) increased lymph node stiffness. Treatment with 4‐MU decreased immunoglobulin production and reduced B cell activation and differentiation into plasmablasts following immunization. These findings reveal a previously unrecognized role for HA in regulating B cell responses within lymphoid tissues during autoimmune reactions and suggest that targeting HA synthesis may represent a novel therapeutic strategy for conditions involving dysregulated B cell responses.

Circulating monocytes are recruited to the tumor microenvironment, where they can differentiate into macrophages that mediate tumor progression. To reach the tumor microenvironment, monocytes must first extravasate and migrate through the type-1 collagen rich stromal matrix. The viscoelastic stromal matrix around tumors not only stiffens relative to normal stromal matrix, but often exhibits enhanced viscous characteristics, as indicated by a higher loss tangent or faster stress relaxation rate. Here, we studied how changes in matrix stiffness and viscoelasticity impact the three-dimensional (3D) migration of monocytes through stromal-like matrices. Interpenetrating networks of type-1 collagen and alginate, which enable independent tunability of stiffness and stress relaxation over physiologically relevant ranges, were used as confining matrices for 3D culture of monocytes. Increased stiffness and faster stress relaxation independently enhanced the 3D migration of monocytes. Migrating monocytes have an ellipsoidal or rounded wedge-like morphology, reminiscent of amoeboid migration, with accumulation of actin at the trailing edge. Matrix adhesions were dispensable for monocyte migration in 3D, but migration did require actin polymerization and myosin contractility. Mechanistic studies indicate that actin polymerization at the leading edge generates protrusive forces that open a path for the monocytes to migrate through in the confining viscoelastic matrices. Taken together, our findings implicate matrix stiffness and stress relaxation as key mediators of monocyte migration and reveal how monocytes use pushing forces at the leading edge mediated by actin polymerization to generate migration paths in confining viscoelastic matrices.

<p>Patient demographics.</p>

<p>Epithelial cell substates</p>

<p>Expression of basement membrane genes</p>

<p>Cell state analysis</p>

<p>Suppl Figure Legend</p>

<div>Abstract<p>Ductal carcinoma <i>in situ</i> (DCIS) is a risk factor for subsequent invasive breast cancer (IBC). To identify events in DCIS that lead to invasive cancer, we performed single-cell RNA sequencing on DCIS lesions and matched normal breast tissue. Inferred copy-number variation was used to identify neoplastic epithelial cells from clinical specimens, which contained a mixture of DCIS and normal ducts. Phylogenetic analysis demonstrated intratumoral clonal heterogeneity that was associated with significant gene expression differences. Classification of epithelial cells into mammary cell states revealed that subclones contained a mixture of cell states, suggesting an ongoing pattern of differentiation after neoplastic transformation. Cell state proportions were significantly different based on estrogen receptor expression, with estrogen receptor–negative DCIS more closely resembling the distribution in the normal breast, particularly with respect to cells with basal characteristics. Specific alterations in cell state proportions were associated with progression to invasive cancer in a cohort of DCIS with longitudinal outcome. Ongoing transcription of key basement membrane (BM) genes occurred in specific subsets of epithelial cell states, including basal/myoepithelial, which are diminished in DCIS. In the transition to IBC, the BM protein laminin, but not COL4, was altered in DCIS adjacent to invasion. Loss of COL4, but not laminin, in an <i>in vitro</i> DCIS model led to an invasive phenotype. These findings suggest that the process of invasion is a loss-of-function event due to an imbalance in critical cell populations essential for BM integrity rather than a gain of an invasive phenotype by neoplastic cells.</p>Significance:<p>Single-cell analyses reveal ductal carcinoma <i>in situ</i> comprises multiple genetic clones with significant phenotypic diversity and link alterations in epithelial cell states and basement membrane integrity with invasive breast cancer progression.</p></div>

<p>Decision rules for CNV categorization.</p>

<p>Bulk sample analysis</p>

<p>CNV phylogenetic trees</p>

<p>QC plots</p>

<p>Complete list of differentially expressed genes from DCIS (DD) to normal cells (NN) comparison.</p>

<p>Sample sets.</p>

<p>Differentially enriched genes within the cell states between normal cells (DN/NN) and ER+ and ER- DCIS cells (DD) cell populations.</p>

<p>Differentially enriched genes within the cell states between ER+ and ER- DCIS to normal.</p>

The biological function and clinical translation of bioprinted cell-laden constructs largely depend on the bioink printability and biomechanical cues presented to embedded cells. Despite multiple biomaterials and crosslinking reactions have been explored for bioink design, how the type and density of crosslinks used in bioink development determine the relationship between bioink printability and viscoelasticity , and how in turn the resulting alterations in viscoelasticity regulate cell behavior within 3D bioprinted constructs remain largely unknown. Here, we developed double crosslinked bioinks with controllable printability and time-dependent mechanical properties by varying the density of reversible (ionic) to static (thioether) crosslinks in the gel network. We utilized these bioinks to investigate how the altered density of physical and chemical crosslinks affects the viscoelasticity of bioprinted cell constructs and how they regulate fundamental cellular responses in 3D. From our results, it was evident that increased density of reversible bonds in bioprinted constructs significantly promotes not only rapid cell spreading, but also the formation of interconnected cellular networks by enhancing matrix viscoelasticity and stress relaxation. By co-printing bioinks whose viscoelasticity can be adjusted independently from its cell-adhesiveness by varying the degree of covalent crosslinking via photoclick thiol-ene reaction, we showed that cell spreading and morphology are spatially regulated in step-gradient hydrogels by the viscoelasticity of their surrounding environment. Our findings reveal that bioinks with similar printability elicit distinct cell responses in bioprinted 3D constructs via altered matrix viscoelasticity, which is determined by the type and density of crosslinks employed for bioink crosslinking. Taken together, these results underscore matrix viscoelasticity as a key parameter in the rational design of mechano-instructive bioinks for bioprinting applications in tissue repair and in vitro tissue modelling.

Collective migration of epithelial tissues is a critical feature of developmental morphogenesis and tissue homeostasis. Coherent motion of cell collectives requires large-scale coordination of motion and force generation and is influenced by mechanical properties of the underlying substrate. While tissue viscoelasticity is a ubiquitous feature of biological tissues, its role in mediating collective cell migration is unclear. Here, we have investigated the impact of substrate stress relaxation on the migration of micropatterned epithelial monolayers. Epithelial monolayers exhibit faster collective migration on viscoelastic alginate substrates with slower relaxation timescales, which are more elastic, relative to substrates with faster stress relaxation, which exhibit more viscous loss. Faster migration on slow-relaxing substrates is associated with reduced substrate deformation, greater monolayer fluidity, and enhanced leader cell formation. In contrast, monolayers on fast-relaxing substrates generate substantial substrate deformations and are more jammed within the bulk, with reduced formation of transient lamellipodial protrusions past the monolayer edge leading to slower overall expansion. This work reveals features of collective epithelial dynamics on soft, viscoelastic materials and adds to our understanding of cell-substrate interactions at the tissue scale.

Cell migration occurs throughout development, tissue homeostasis and regeneration, as well as in diseases such as cancer. Cells migrate along two-dimensional (2D) surfaces or interfaces, within microtracks, or in confining three-dimensional (3D) extracellular matrices. Although the basic mechanisms of 2D migration are known, recent studies have elucidated unexpected migration behaviors associated with more complex substrates and have provided insights into their underlying molecular mechanisms. Studies using engineered biomaterials for 3D culture and microfabricated channels to replicate cell confinement observed in vivo have revealed distinct modes of migration. Across these contexts, the mechanical features of the surrounding microenvironment have emerged as major regulators of migration. In this Cell Science at a Glance article and the accompanying poster, we describe physiological contexts wherein 2D and 3D cell migration are essential, report how mechanical properties of the microenvironment regulate individual and collective cell migration, and review the mechanisms mediating these diverse modes of cell migration.

Ductal carcinoma in situ (DCIS) is a risk factor for subsequent invasive breast cancer (IBC). To identify events in DCIS that lead to invasive cancer, we performed single-cell RNA sequencing on DCIS lesions and matched normal breast tissue. Inferred copy-number variation was used to identify neoplastic epithelial cells from clinical specimens, which contained a mixture of DCIS and normal ducts. Phylogenetic analysis demonstrated intratumoral clonal heterogeneity that was associated with significant gene expression differences. Classification of epithelial cells into mammary cell states revealed that subclones contained a mixture of cell states, suggesting an ongoing pattern of differentiation after neoplastic transformation. Cell state proportions were significantly different based on estrogen receptor expression, with estrogen receptor-negative DCIS more closely resembling the distribution in the normal breast, particularly with respect to cells with basal characteristics. Specific alterations in cell state proportions were associated with progression to invasive cancer in a cohort of DCIS with longitudinal outcome. Ongoing transcription of key basement membrane (BM) genes occurred in specific subsets of epithelial cell states, including basal/myoepithelial, which are diminished in DCIS. In the transition to IBC, the BM protein laminin, but not COL4, was altered in DCIS adjacent to invasion. Loss of COL4, but not laminin, in an in vitro DCIS model led to an invasive phenotype. These findings suggest that the process of invasion is a loss-of-function event due to an imbalance in critical cell populations essential for BM integrity rather than a gain of an invasive phenotype by neoplastic cells. SIGNIFICANCE: Single-cell analyses reveal ductal carcinoma in situ comprises multiple genetic clones with significant phenotypic diversity and link alterations in epithelial cell states and basement membrane integrity with invasive breast cancer progression.

Cell migration is pivotal in cancer metastasis, where cells navigate the extracellular matrix (ECM) and invade distant tissues. While the ECM is viscoelastic-exhibiting time-dependent stress relaxation-its influence on cell migration remains poorly understood. Here, we employ an integrated experimental and modeling approach to investigate filopodial cancer cell migration on viscoelastic substrates and uncover a striking transition from sub-diffusive to super-diffusive behavior driven by the substrate's viscous relaxation timescale. Conventional motor-clutch based migration models fail to capture these anomalous migration modes, as they overlook the complex adhesion dynamics shaped by broad distribution of adhesion lifetimes. To address this, we develop a glassy motor-clutch model that incorporates the rugged energy landscape of adhesion clusters, where multiple metastable states yield long-tailed adhesion timescales. Our model reveals that migration dynamics are governed by the interplay between cellular and substrate timescales: slow-relaxing substrates prolong trapping, leading to sub-diffusion, while fast-relaxing substrates promote larger steps limiting trapping, leading to super-diffusion. Additionally, we uncover the role of actin polymerization and contractility in modulating adhesion dynamics and driving anomalous migration. These findings establish a mechanistic framework linking substrate viscoelasticity to cell motility, with implications for metastasis and cancer progression.

In breast cancer (BC), radial alignment of collagen fibers at the tumor-matrix interface facilitates collective invasion of cancer cells into the surrounding stromal matrix, a critical step toward metastasis. Collagen remodeling is driven by proteases and cellular forces, mediated by matrix mechanical plasticity, or irreversible matrix deformation in response to force. However, the specific mechanisms causing collagen radial alignment remain unclear. Here, we study collective invasion of BC tumor spheroids in collagen-rich matrices. Increasing plasticity to BC-relevant ranges facilitates invasion, with increasing stiffness potentiating a transition from single cell to collective invasion. At enhanced plasticity, cells radially align collagen at the tumor-matrix interface prior to invasion. Surprisingly, cells migrate tangentially to the tumor-matrix interface in a swirling-like motion, perpendicular to the direction of alignment. Mechanistically, swirling generates local shear stresses, leading to distally propagating contractile radial stresses due to negative normal stress, an underappreciated property of collagen-rich matrices. These contractile stresses align collagen fibers radially, facilitating collective invasion. The basement membrane (BM), which separates epithelia from stroma in healthy tissues, acts as a mechanical insulator by preventing swirling cells from aligning collagen. Thus, after breaching the BM, swirling of BC cells at the tumor-stroma interface radially aligns collagen to facilitate invasion.

Dynamic covalent cross-linked (DCC) hydrogels represent a significant advance in biomaterials for regenerative medicine and mechanobiology, offering viscoelasticity, and self-healing properties that more closely mimic in vivo tissue mechanics than traditional, predominantly elastic, covalent hydrogels. However, the effects of varying cross-linker architecture on DCC hydrogel viscoelasticity have not been thoroughly investigated. This study introduces hydrazone-based alginate hydrogels to explore how cross-linker architectures impact stiffness and viscoelasticity. In hydrogels with side-chain cross-linker (SCX), higher cross-linker concentrations enhance stiffness and decelerate stress relaxation, while an off-stoichiometric hydrazine-to-aldehyde ratio reduces stiffness and shortens relaxation time. In hydrogels with telechelic cross-linking, maximal stiffness and relaxation time occurs at intermediate cross-linker mixing ratio for both linear cross-linker (LX) and star cross-linker (SX), with higher cross-linker valency further enhancing these properties. Further, the ranges of stiffness and viscoelasticity accessible with the different cross-linker architectures are found to be distinct, with SCX hydrogels leading to slower stress relaxation relative to the other architectures, and SX hydrogels providing increased stiffness and slower stress relaxation versus LX hydrogels. This research underscores the pivotal role of cross-linker architecture in defining hydrogel stiffness and viscoelasticity, providing insights for designing DCC hydrogels with tailored mechanical properties for specific biomedical applications.