Cellular and molecular bioengineering最新文献

Purpose: Adipose tissues have long been recognized for their diverse endocrine functions that serve to regulate tissue homeostasis. Although adipose depot-specific secretory profiles can differentially regulate fibroblast fate and fibrotic tissue remodeling, systematic investigation of how adipose depots of varying phenotypes influence fibroblast mechanobiology remains lacking.

Methods: By integrating secretome profiling with functional and mechanobiological assays in a controlled adipose-fibroblast coculture platform, we mechanistically investigate how canonical brown (BAT), white (WAT), and beige adipose tissues (thoracic and abdominal perivascular adipose tissue; T-PVAT and A-PVAT, respectively) govern fibroblast-to-myofibroblast transition (FMT) through depot-specific paracrine signaling.

Results: Thermogenic depots (BAT and T-PVAT) enhance fibroblast proliferation, collagen deposition, and stiffness (1.9-fold increase in single cell modulus; p < 0.0001) alongside specific adipokines that regulate metabolism (ANGPTL3, FGF1) and inhibit calcification (Fetuin A). In contrast, lipid-storing depots (WAT and A-PVAT) promote fibroblast migration (79% scratch wound closure at 12 h; p < 0.01) and matrix degradation through upregulated pro-inflammatory cytokines (CCL2, IL-6) and fibrogenic mediators (PAI-1, TIMP-1). Mechanistically, BAT and T-PVAT secretomes induce pro-contractile and matrix-producing fibroblast phenotypes, while A-PVAT and WAT promote migratory and matrix-remodeling behaviors. Further, T-PVAT uniquely combines BAT-like genetic identity with elevated RAGE-DPP4 signaling, resulting in excessive fibroblast activation, stiffening, and collagen production that can be attenuated via pharmacological inhibition of DPP4.

Conclusions: Our findings establish adipose depots as distinct modulators of fibroblast phenotype, wherein depot-specific mediators promote mechanobiological alterations that balance reparative and pathological remodeling processes related to fibrosis. Differential biological responses by fibroblasts exposed to diverse adipose phenotypes underscores the potential for adipose-driven stromal crosstalk to mitigate fibrotic remodeling alongside metabolic and cardiovascular disease comorbidities.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00864-z.

Motivation: Directed cell migration is essential in many biological processes and is driven by a variety of directional cues, including aligned fibrils in the extracellular matrix (ECM), a phenomenon known as contact guidance. How different cells respond to aligned fibrils and how internal regulators like formins and Arp2/3 control contact guidance is unknown.

Methods: In this study, a unique system to assemble aligned collagen fibrils on mica and to transfer them onto controllable substrates is used to probe contact guidance.

Results: This fibril alignment system reveals that cytoskeletal regulation through myosin contractility and not receptor expression drives contact guidance ability. Highly contractile cells exhibit high-fidelity contact guidance, weakly contractile cells ignore cues and moderately contractile cells use a mixture of both parallel and perpendicular migration strategies on aligned collagen fibrils. In addition to myosin contractility, formins and Arp2/3 control contact guidance in a reciprocal manner across a variety of cell types. Formins, mediators of linear F-actin structures, enhance contact guidance and Arp2/3, a mediator of branched F-actin structures, diminishes contact guidance.

Conclusion: This controlled materials system reveals the importance of both myosin-mediated contractility as well as the antagonistic action of formins and Arp2/3 on contact guidance, providing potential targets to tune contact guidance.

Graphical abstract:

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00868-9.

[This corrects the article DOI: 10.1007/s12195-025-00860-3.].

[This corrects the article DOI: 10.1007/s12195-025-00853-2.].

Introduction: Triple negative breast cancer (TNBC) has significantly worse outcomes compared to other subtypes. Strains in the tumor microenvironment (TME) generated by cancer-associated fibroblasts (CAFs) can regulate TNBC progression. Recent studies suggest that expression of VEGFR-2 on TNBC is linked to decreased survival, while our prior studies show strains activate VEGFR-2 to drive angiogenesis. We hypothesized that VEGFR-2 on TNBC can be mechanically activated to alter migration and proliferation.

Methods: We utilized MDA-MB-231 TNBC cells loaded into the center chamber of a multi-microtissue TME model; opposing side chambers were loaded with CAFs or normal breast fibroblasts (NBFs). A second series of studies utilized magnetic beads to generate strains in the model without secretion of growth factors. Microtissues were analyzed for TNBC migration and proliferation via Ki67 staining.

Results: TNBC cells migrated significantly more towards CAFs compared to NBFs (5×); TME models with magnetic beads showed a 2× increase in migration compared to no strain controls. TNBC cells treated with shRNA against VEGFR-2 demonstrated decreased overall migration but still significantly more towards CAFs vs. NBFs (2×). Proliferation analyses showed strain significantly increased Ki67 in control cells (10%+ vs. 28%+) but not in shVEGFR-2 TNBC (~ 10% all conditions).

Discussion: These studies demonstrate that strain in the TME drives increased migration and proliferation of TNBC. Loss of VEGFR-2 suppresses migration and growth, even with mechanical stimulation. Therefore, our results suggest that mechanosignaling via VEGFR-2 on TNBC may regulate disease progression and potentially explain failure of anti-VEGFR-2 drugs in breast cancer patients.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00866-x.

Purpose: Despite the success of immune checkpoint inhibitors (ICIs) that target immunosuppressive interactions, treatment resistance remains a major clinical challenge. The tumor microenvironment is comprised of tumor, immune, and stromal cell types that communicate through secreted and cell surface proteins. This can be represented by a weighted, directed network where pairs of cell types communicate via multiple ligand-receptor interactions with varying strengths. Identifying interaction network motifs that are linked with outcome or evolve pre- to post-ICI presents a rational framework to identify combination therapeutic targets.

Methods: Interaction inference was performed on publicly available single-cell RNA-sequencing data from melanoma patients. The constructed patient-specific networks were input to multivariate statistical learning approaches to identify network motifs that predicted response pre-treatment and that shifted pre- to post-treatment. Relevance of interactions was validated by (1) differential expression of related pathways in single cell RNA sequencing (scRNA-seq) data, (2) survival associations in an independent bulk RNA-seq dataset, and (3) repeated analyses of scRNA-seq data in a second cohort.

Results: Immune-immune interactions with roles in T cell activation, chemotaxis, and adhesion were upregulated in patients who respond to therapy pre-treatment. Related pathways were perturbed in involved immune cells and expression of these genes was associated with improved survival. The interactome also distinguished pre- and post-treatment biopsies with high accuracy despite no significant differences in individual interactions. Analysis in the validation dataset with mixed responses pre-treatment recapitulated results from the discovery analyses.

Conclusion: Unbiased analysis of interaction networks and their evolution is a powerful framework to guide prognostic indicators and novel combination targets to improve patient outcomes.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00857-y.

Purpose: Sex differences in cellular biology significantly influence cell responses in culture. Yet, the sex-specific effects of culture reagents such as fetal bovine serum (FBS) remain understudied. Increased adoption of cell-based models such as microphysiological systems (MPS) as replacements for animal models demands a greater understanding sex-specific responses to common media formulations. This study examined the effects of FBS and hormone-free charcoal-stripped serum (CSS) on male (XY) and female (XX) cells in 2D and 3D MPS culture models to demonstrate profound sex-specificity in bioassays and inform the development of future sex-specific cell culture protocols and methods.

Methods: Primary human endothelial cells and fibroblasts from multiple organ sources were cultured in 2D and in 3D MPS models. Cells were cultured with either FBS or CSS. Endothelial specific gene expression, cytoskeletal spreading, and cell cycle status were analyzed in 2D culture. Vascular network formation, macromolecular leakage, and directional angiogenic sprouting were assessed in 3D MPS models.

Results: FBS promoted significant upregulation of genes associated with endothelial function in XX endothelial cells, but the same gene clusters were downregulated in XY cells. FBS increased cytoskeletal spreading and cell cycle participation of XX endothelial cells and fibroblasts relative to culture with CSS. Conversely, culture with CSS increased these 2D metrics in XY cells. Measurement of 40 kDa FITC-dextran leakage in a single vessel MPS model revealed that culture with FBS significantly decreased XX endothelial barrier permeability relative to culture with CSS. In line with 2D assays, CSS conversely enhanced XY endothelial barrier permeability relative to culture with FBS. Culture with FBS increased metrics of vasculogenesis in XX tissues relative to CSS cultures, whereas prolonged cultured in CSS supported vasculogenesis in XY models. MPS angiogenesis assays revealed increased sprouting in XX tissues cultured with FBS, while only minimal sprouting was observed in all other conditions.

Conclusions: FBS imparted significant sex-specific effects on the gene expression patterns, morphology, and cell cycle status of human endothelial cells and fibroblasts in 2D culture. Sex-specific effects measured in 2D culture assays carried over to 3D MPS assays of endothelial barrier function, vasculogenesis, and angiogenesis. Notably, FBS significantly enhanced XX cell functions relative to XY cells in all 2D and 3D MPS assays. Thus, accounting for the sex-specific effects of culture media components will be imperative to improve reproducibility and translational relevance of MPS in preclinical research.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00860-3.

Purpose: In muscle tissues, anisotropic cell alignment is essential for optimal muscle fiber development and function. Biomaterials for muscle tissue engineering must guide cellular alignment while supporting cell proliferation and myogenic differentiation.

Methods: Here, we describe the fabrication of a tissue-engineered construct consisting of a scaffold of aligned poly(ε-caprolactone) (PCL) microfibers coated in a dynamic covalent hydrazone crosslinked hyaluronic acid (HA) hydrogel to support myoblast attachment, myoblast alignment, and myotube formation. Norbornene modification of HA further enabled functionalization with fibronectin-derived arginine-glycine-aspartic acid (RGD) peptide. Scaffolds were fabricated using melt electrowriting (MEW), a three-dimensional (3D)-printing technique that uses stabilization of fluid columns to produce precisely aligned polymeric microfibers. We evaluated C2C12 mouse skeletal myoblasts cultured on non-coated, HA-coated, and HA-RGD-coated MEW scaffolds with fiber diameters of 10, 20, and 30 µm using immunocytochemistry and creatine kinase activity assays. We further evaluated the mechanical properties of 20 µm fiber scaffolds and their effect on myogenic gene expression and alpha-actinin protein expression of C2C12 myoblasts undergoing differentiation.

Results: HA-coated and HA-RGD-coated scaffolds increased attachment of C2C12 myoblasts on all fiber diameters compared to non-coated scaffolds, with HA-RGD-coated scaffolds demonstrating the highest cell attachment. All scaffolds supported cellular alignment along the fibers. Cells differentiated on scaffolds showed anisotropic alignment with increased myotube formation on HA-coated and HA-RGD-coated scaffolds as demonstrated by myosin heavy chain (MHC) staining and by the presence of striations on HA-coated scaffolds visualized with alpha-actinin staining. Increased creatine kinase activity and myogenic gene expression on day 5 further indicated myotube formation on all scaffolds, with HA-coated scaffolds significantly increasing the expression of several key myogenic markers.

Conclusion: This unique combination of tunable biophysical and biochemical cues enables the creation of a biomimetic tissue engineered scaffold, providing a platform for new therapeutic approaches for muscle regeneration.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00865-y.

Purpose: In this study, we aimed to develop a dynamic on-chip platform to study macrophage polarization in a more physiologically relevant way by incorporating mechanical forces which have been recently shown to play important roles in macrophage biology.

Methods: We developed polymethyl methacrylate (PMMA) based platform. We examined the effects of the dynamic microenvironment on polarization states of human monocyte derived macrophages (HMDMs) towards the M1 and M2a phenotypes using lipopolysaccharide (LPS)/interferon-γ (IFN-γ) and interleukin-4 (IL-4) respectively for both static and dynamic conditions. M1 and M2 polarization levels were assessed by qPCR and flow cytometry analyses.

Results: M1 and M2 polarization was achieved successfully under dynamic and static conditions. Our platform establishes that the mechanotransductive stimulation through shear stress during polarization has direct synergistic effects with stimulants on TNF-α secretion within HMDMs. Exposure to media flow rates of 0.5, 2.5, and 5 µl/min without stimulants is insufficient to induce macrophage polarization.

Conclusion: The dynamic environment present inside our dynamic on-chip culture platform influences the human monocyte-derived macrophages (HMDMs) to become polarized into M1 phenotype at a greater level.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00863-0.

Scope: Extracellular vesicles (EVs) have emerged as promising cell-free delivery vehicles for cancer therapy due to their inherent biocompatibility, low immunogenicity, and natural targeting capabilities. EVs derived from various cellular sources offer distinct advantages in drug-loading capacity and therapeutic effectiveness. However, their clinical application is limited by challenges such as poor cargo stability, potential immunogenicity, and off-target effects. These limitations necessitate further surface functionalization of EVs to optimize vesicle stability, targeting precision, and safety of pharmacological cargos. Paclitaxel (PTX), a first-line chemotherapeutic agent effective against multiple cancers, is limited by poor solubility and significant systemic toxicity, highlighting the need for targeted delivery systems.

Methods: A literature search was conducted to identify relevant articles published between 1993 and 2025. This review provides a comprehensive overview of EV biogenesis and cellular origins, highlighting recent advances in engineering strategies for PTX delivery. Current progress in employing engineered EVs for PTX delivery in both in vitro and in vivo cancer models, along with practical challenges and future directions in the clinical translation of EV-based PTX delivery, are discussed.

Results: Preclinical studies demonstrate that engineered EVs can effectively encapsulate and deliver PTX to tumor sites, improving therapeutic outcomes while minimizing systemic side effects. Despite these advances, challenges remain in optimizing EV isolation, surface modification, PTX loading efficiency, and precise recognition of tumor cells.

Conclusion: Engineered EVs represent a promising platform for PTX delivery, combining targeted therapeutic potential with reduced systemic toxicity. Continued research to address technical and translational barriers will be critical for advancing EV-based PTX therapies toward clinical application.

Graphical abstract: