Journal: bioRxiv

Article Title: Matrix stiffness induces endothelial network senescence

doi: 10.1101/2025.10.05.680536

Figure Lengend Snippet: Endothelial networks cultured in hydrogels of increasing stiffness show elevated expression of senescence markers: (A) CDKN1A and CDKN2A mRNA levels measured by qRT-PCR, (B-C) p21 protein levels assessed by Western blot, and (D-E) p16 protein expression visualized by immunofluorescence staining. Representative confocal images show microvascular networks stained for VE-Cadherin in green, p16 in while, nuclei in blue. Scale bar is 100 µm. N=3. (F-G) SA-β-Gal activity increased with matrix stiffening. Representative confocal images show networks stained for F-actin (red), SA-β-Gal (green), and nuclei (blue). Scale bar: 100 µm. N=3. (H-J) Senescence-associated secretory phenotype (SASP), including cytokines, chemokines, and MMPs analyzed using a Luminex assay. Values were normalized to the amount of dsDNA isolated from each hydrogel construct. N=3. Significance levels were set at ns= not significant (p > 0.05), **p ≤ 0.01, and ****p ≤ 0.0001.

Article Snippet: The constructs were incubated with primary antibodies, including anti rabbit-p16 (1:400, Proteintech) and anti mouse-VE-CAD (1:400, Santa Cruz) overnight at 4 °C.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Activity Assay, Luminex, Isolation, Construct

Journal: bioRxiv

Article Title: Matrix stiffness induces endothelial network senescence

doi: 10.1101/2025.10.05.680536

Figure Lengend Snippet: (A) qRT-PCR analysis revealed significant upregulation of Notch1 receptor, ligands JAG1 and JAG2 , and downstream target HEY1 under stiff conditions. Dll4 ligand gene expression remained unchanged upon matrix stiffening. N=3. (B) Matrix stiffening also induced MAPK8 (encoding JNK1), FOS , and JUN gene expression, key components of the JNK-AP-1 signaling axis, as measured by qRT-PCR. N=3. (C-F) Western blot analysis confirmed increased protein levels of cleaved-Notch1 (NICD), JAG1, and JAG2 in stiffened matrices. GAPDH was used as a protein loading control. N=3. (G-K) Matrix stiffening increased phosphorylation of JNK (p-JNK) in both cytoplasmic and nuclear fractions (N=4), while phosphorylation of cJUN (p-c-JUN) was elevated only in the cytoplasm (N=3). This suggest that nuclear p-c-JUN activation is independent of stiffness-mediated Notch signaling. GAPDH and PCNA were used as protein loading controls for cytoplasm and nucleus, respectively. (L) Treatment with nirogacestat (Niro), a γ-secretase inhibitor, reduced stiffness-induced expression of Notch1 , MAPK8 , FOS , CDKN1A , and CDKN2A (N=3), indicating that Notch signaling regulates JNK-FOS signaling and senescence-associated cell cycle arrest. (M-N) Immunofluorescence staining exhibited reduced p16 expression in nirogacestat-treated microvascular networks compared to untreated stiff controls. Representative confocal images show endothelial networks stained for VE-Cadherin in green, p16 in white, nuclei in blue. Scale bar is 100 µm. N=3. (O) Schematic illustration of the proposed mechanotransductive pathway: matrix stiffening enhances Notch activation, NICD release, which in turn activates the JNK-FOS axis, promoting endothelial senescence and an immunomodulatory phenotype. Significance levels indicated as ns= not significant (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Article Snippet: The constructs were incubated with primary antibodies, including anti rabbit-p16 (1:400, Proteintech) and anti mouse-VE-CAD (1:400, Santa Cruz) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Gene Expression, Western Blot, Control, Phospho-proteomics, Activation Assay, Expressing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Matrix stiffness induces endothelial network senescence

doi: 10.1101/2025.10.05.680536

Figure Lengend Snippet: (A) Immunofluorescence staining of fibrotic capsules surrounding surgically explanted synthetic breast implants and adjacent fat tissue showed ECs (CD31) in green, senescence marker (p16 INK4a ) in orange, Notch1 receptor in magenta, nuclei in blue. Representative images of both soft and fibrotic tissues were obtained from the same patient. Scale bar = 100 μm (left), 20 μm (right). (B) Increased p16 expression was observed in ECs within fibrotic capsule regions compared to soft control tissues, including fat and muscle. (C) The proportion of non-senescent ECs expressing Notch1 (CD31 + Notch1 + ) was comparable between soft control and fibrotic tissues, suggesting Notch1 expression in non-senescent ECs is not altered by stiffness. (D) A significantly higher frequency of triple-positive (CD31 + p16 + Notch1 + ) cells in fibrotic regions indicates that Notch 1 expression is enriched specifically within senescent ECs in stiffened environments. (E) Violin plots confirm EC identity through expression of standard EC markers VE-Cadherin (CDH5), PECAM1, and von Willebrand factor (vWF). (F) UMAP projection identifies 132 p16⁺ ECs out of 1,836 total ECs. (G) Volcano plot showing differentially expressed genes between p16⁺ and p16⁻ ECs. Genes meeting adjusted p < 0.05 and log₂ fold change > 0.5 are highlighted. (H) Dot plot displays enrichment of Notch- and JNK-associated genes in senescent ECs. (I) p16⁺ ECs show increased expression of genes related to cell cycle arrest (e.g., CDKN2A/B/C, CCNG1), SASP and inflammatory signaling (e.g., BCL2, BAX, CXCL12/14/16, IL11RA), and ECM remodeling (e.g., MMP3/11/14, PLAUR, SERPINE2, CCN1), supporting their active role in fibrosis-associated tissue remodeling.

Article Snippet: The constructs were incubated with primary antibodies, including anti rabbit-p16 (1:400, Proteintech) and anti mouse-VE-CAD (1:400, Santa Cruz) overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Capsules, Marker, Expressing, Control

Journal: Nature Biomedical Engineering

Article Title: Bioengineered immunocompetent preclinical trial-on-chip tool enables screening of CAR T cell therapy for leukaemia

doi: 10.1038/s41551-025-01428-2

Figure Lengend Snippet: 2D time-lapse imaging showed CAR T cell extravasation, related to Extended Data Fig. 3a. T cells are in red, Reh B-ALL leukaemia blasts are in green, and vessels (HUVECs) are in blue.

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs; catalogue number C2519A, Lonza), VE-CAD-GFP-expressing HUVECs (catalogue number cAP-0001VECAD-GFP, Angio-Proteomie) and RFP-expressing HUVECs (catalogue number cAP-0001RFP, Angio-Proteomie) were cultured in Endothelial Cell Growth Medium-2 BulletKit (EGM-2; catalogue number CC-3162, Lonza) and used within passage 5.

Techniques:

Journal: Nature Biomedical Engineering

Article Title: Bioengineered immunocompetent preclinical trial-on-chip tool enables screening of CAR T cell therapy for leukaemia

doi: 10.1038/s41551-025-01428-2

Figure Lengend Snippet: 2D time-lapse imaging showed CAR T cell infiltration, related to Extended Data Fig. 3b. T cells are in red, Reh B-ALL leukaemia blasts are in green, and vessels (HUVECs) are in blue.

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs; catalogue number C2519A, Lonza), VE-CAD-GFP-expressing HUVECs (catalogue number cAP-0001VECAD-GFP, Angio-Proteomie) and RFP-expressing HUVECs (catalogue number cAP-0001RFP, Angio-Proteomie) were cultured in Endothelial Cell Growth Medium-2 BulletKit (EGM-2; catalogue number CC-3162, Lonza) and used within passage 5.

Techniques:

Journal: Nature Biomedical Engineering

Article Title: Bioengineered immunocompetent preclinical trial-on-chip tool enables screening of CAR T cell therapy for leukaemia

doi: 10.1038/s41551-025-01428-2

Figure Lengend Snippet: Confocal time-lapse imaging showed the process of extravasation, infiltration and killing of healthy donor-derived CAR T cell in the leukaemia bone marrow chip. Vessel was formed with VE-CAD-GFP-expressing HUVECs (in green). Leukaemia blast was K562-meso-19-mCherry (in yellow). CAR T cells were stained with DiD dye (in red). The video was captured at 5 min per frame within 14 h using Nikon C2i confocal microscopy and a 20× objective.

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs; catalogue number C2519A, Lonza), VE-CAD-GFP-expressing HUVECs (catalogue number cAP-0001VECAD-GFP, Angio-Proteomie) and RFP-expressing HUVECs (catalogue number cAP-0001RFP, Angio-Proteomie) were cultured in Endothelial Cell Growth Medium-2 BulletKit (EGM-2; catalogue number CC-3162, Lonza) and used within passage 5.

Techniques:

Journal: Nature Biomedical Engineering

Article Title: Bioengineered immunocompetent preclinical trial-on-chip tool enables screening of CAR T cell therapy for leukaemia

doi: 10.1038/s41551-025-01428-2

Figure Lengend Snippet: Confocal time-lapse imaging showed the process of extravasation, infiltration and killing of patient-derived CAR T cell in the leukaemia bone marrow chip. Vessel was formed with VE-CAD-GFP-expressing HUVECs (in green). Leukaemia blast was K562-meso-19-mCherry (in yellow). CAR T cells were stained with DiD dye (in red). The video was captured at 5 min per frame within 14 h using Nikon C2i confocal microscopy and a 20× objective.

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs; catalogue number C2519A, Lonza), VE-CAD-GFP-expressing HUVECs (catalogue number cAP-0001VECAD-GFP, Angio-Proteomie) and RFP-expressing HUVECs (catalogue number cAP-0001RFP, Angio-Proteomie) were cultured in Endothelial Cell Growth Medium-2 BulletKit (EGM-2; catalogue number CC-3162, Lonza) and used within passage 5.

Techniques:

Journal: Nature Biomedical Engineering

Article Title: Bioengineered immunocompetent preclinical trial-on-chip tool enables screening of CAR T cell therapy for leukaemia

doi: 10.1038/s41551-025-01428-2

Figure Lengend Snippet: a , The compartmentalized bone marrow chip (middle) was populated with human bone marrow cells (right) to replicate the in vivo counterpart (left). The whole scanning of the leukaemia bone marrow chip (bottom right) with central sinus (RFP-HUVECs in red), medullary cavity and endosteum (DiD-labelled osteoblasts in yellow). The 3D view of perivascular niche (top right) in bone marrow with hematopoietic cells (CD45 + in red), vascular cells (CD31 + in orange) and nuclei in blue (DAPI). Representative images were from one of the three technical replicates with similar results ( n = 3). Schematic (left part) was adapted from smart.servier.com . Schematic (right part) was created in BioRender (C.M. (2025), https://BioRender.com/wlbhoxu ), b , scRNA-seq profiling of the bone marrow cellularity on-chip, highlighting the presence of most hematopoietic (immune) and non-hematopoietic (bone marrow stroma) cells. c , scRNA-seq profiling of primary bone marrow mononuclear cells, which was comparatively mapped to that of on-chip bone marrow niche. d , The cellularity where B cell populations were excluded to reveal bone marrow niche immune populations. In addition to the bone marrow stromal cell populations seeded to build the stromal environment, macrophage, basophil/mast cells and megakaryocyte/platelets were generated on-chip during culture. e , The presence of stromal compartment. HUVECs in red (RFP), Reh B-ALL cells in green (GFP) and mesenchymal cells in purple (DiD labelling). The white arrowheads indicate Reh B-ALL cells. f , The presence of hematopoietic cells. Lymphoid (left): CD8 + T cells in cyan and CD4 + T cells in red. The top white arrowheads indicate CD4 + T cells, and the bottom arrowheads indicates CD8 + T cells. Myeloid (right): monocyte (CD14 + ) in cyan, HUVECs (CD31 + ) in green and Reh B-ALL cells (CD19 + ) in red. The white arrowheads indicate monocytes. g , The deposition of ECMs such as laminin (green) and collagen IV (purple). Reh B-ALL cells in red. The white arrowheads indicate Reh B-ALL cells. Representative images were from one of the three technical replicates with similar results ( n = 3).

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs; catalogue number C2519A, Lonza), VE-CAD-GFP-expressing HUVECs (catalogue number cAP-0001VECAD-GFP, Angio-Proteomie) and RFP-expressing HUVECs (catalogue number cAP-0001RFP, Angio-Proteomie) were cultured in Endothelial Cell Growth Medium-2 BulletKit (EGM-2; catalogue number CC-3162, Lonza) and used within passage 5.

Techniques: In Vivo, Generated