vegf164  (Novoprotein)

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Other drugs and compounds used in this study included: GW9662 (MCE, HY-16578; intraperitoneal injection, 1 mg/kg body weight/day, administered continuously for 4 weeks), T0070907 (Selleck, S2871; intraperitoneal injection, 2 mg/kg body weight/day, administered continuously for 4 weeks), rapamycin (MCE, HY-10219; subcutaneous injection, 3 mg/kg body weight/day, administered continuously for 4 weeks), Rosiglitazone (MCE, HY-17386; oral gavage, 3 mg/kg body weight/day, administered continuously for 2 weeks), LY294002 (Selleck, S1105; intraosseous injection, 10 μM, 5 μL per dose per week, administered for 1 or 4 weeks), DMH1 (Selleck, S7146; intraperitoneal injection, 5 mg/kg body weight/day, administered continuously for 4 weeks), Noggin (PeproTech, 250-38; intraosseous injection, 50 ng per dose, twice per week, administered for 2 or 4 weeks), LDN-193189 (Selleck, S2618; intraperitoneal injection, 3 mg/kg body weight/day, administered for 1 or 4 weeks), IGF-1 (PeproTech, 250-19; intraosseous injection, 4 μg per dose per week, administered for 2 weeks), IGF-1 neutralizing antibody (R&D Systems, AF-791; intraosseous injection, 2 μg per dose, twice per week, administered for 2 or 4 weeks), VEGF neutralizing antibody (R&D Systems, AF-493-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-AA neutralizing antibody (R&D Systems, AF-221-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-BB neutralizing antibody (R&D Systems, AF-220-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β1 neutralizing antibody (R&D Systems, MAB2401; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β2 neutralizing antibody (R&D Systems, AB-112-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks).

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Other drugs and compounds used in this study included: GW9662 (MCE, HY-16578; intraperitoneal injection, 1 mg/kg body weight/day, administered continuously for 4 weeks), T0070907 (Selleck, S2871; intraperitoneal injection, 2 mg/kg body weight/day, administered continuously for 4 weeks), rapamycin (MCE, HY-10219; subcutaneous injection, 3 mg/kg body weight/day, administered continuously for 4 weeks), Rosiglitazone (MCE, HY-17386; oral gavage, 3 mg/kg body weight/day, administered continuously for 2 weeks), LY294002 (Selleck, S1105; intraosseous injection, 10 μM, 5 μL per dose per week, administered for 1 or 4 weeks), DMH1 (Selleck, S7146; intraperitoneal injection, 5 mg/kg body weight/day, administered continuously for 4 weeks), Noggin (PeproTech, 250-38; intraosseous injection, 50 ng per dose, twice per week, administered for 2 or 4 weeks), LDN-193189 (Selleck, S2618; intraperitoneal injection, 3 mg/kg body weight/day, administered for 1 or 4 weeks), IGF-1 (PeproTech, 250-19; intraosseous injection, 4 μg per dose per week, administered for 2 weeks), IGF-1 neutralizing antibody (R&D Systems, AF-791; intraosseous injection, 2 μg per dose, twice per week, administered for 2 or 4 weeks), VEGF neutralizing antibody (R&D Systems, AF-493-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-AA neutralizing antibody (R&D Systems, AF-221-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-BB neutralizing antibody (R&D Systems, AF-220-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β1 neutralizing antibody (R&D Systems, MAB2401; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β2 neutralizing antibody (R&D Systems, AB-112-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks).

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Digital spatial profiling reveals additive effects of triple therapy on tumor microenvironment: anti-PD-L1, anti-VEGF, and PARP inhibition in mouse models

doi: 10.1007/s00262-026-04312-3

Figure Lengend Snippet: Experimental design and analysis protocol. a Dual combination therapy protocol for MC38 and HM-1 tumor models. Anti-PD-L1 and anti-VEGF treatments initiated on day 6 post-implantation in established tumors. b Triple combination therapy protocol for HM-1 model. Anti-PD-L1, anti-VEGF, and PARPi with eight treatment groups evaluated. Spatial analysis of cellular compositions performed on days 8 and 10, RNA-seq analysis on day 13, and treatment response evaluation on days 10 and 20. c Spatial distribution and quantitative analysis of immunohistochemically positive cells using HALO software. Quantification of stained positive cell density (cells/mm 2 ) stratified by distance from tumor tissue edge

Article Snippet: Sections were stained with the following primary antibodies: anti-Granzyme B (rabbit anti-mouse; Abcam, #ab4059), anti-CD8 (rabbit anti-mouse; Cell Signaling, #98,941), anti-PD-L1 (rabbit monoclonal clone D5V3B; Cell Signaling, #64,988), anti-VEGF (goat polyclonal; R&D SYSTEMS, #AF-493-NA), anti-PARP1 (rabbit polyclonal; Abcam, #ab227244), anti-CD31 (rabbit monoclonal clone EPR17259 ; Abcam, #ab182981), and anti-MECA79 (rat monoclonal clone MECA79; BioLegend, #120,801).

Techniques: RNA Sequencing, Software, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Digital spatial profiling reveals additive effects of triple therapy on tumor microenvironment: anti-PD-L1, anti-VEGF, and PARP inhibition in mouse models

doi: 10.1007/s00262-026-04312-3

Figure Lengend Snippet: Therapeutic effects and CD8 + T cell distribution in MC38 and HM-1 tumor models. a Tumor volumes were measured at day 10 following anti-VEGF and anti-PD-L1 treatment. MC38 tumors showed significant reduction with anti-PD-L1 (2.0-fold reduction, 30.4 ± 4.79 mm 3 , p = 0.02), anti-VEGF (1.8-fold reduction, 33.0 ± 2.46 mm 3 , p = 0.02), and anti-PD-L1 + anti-VEGF (2.8-fold reduction, 21.6 ± 5.15 mm 3 , p = 0.01) vs. Control (60.7 ± 7.29 mm 3 ). No differences were observed in HM-1 tumors; anti-PD-L1 (46.3 ± 2.80 mm 3 ), anti-VEGF (56.1 ± 3.10 mm 3 ), and anti-PD-L1 + anti-VEGF (43.3 ± 5.16 mm 3 ) vs. Control (57.0 ± 3.34 mm 3 ). b CD8 + T cell distribution was analyzed on days 8 and 10. Images display one representative tumor from each treatment group. MC38 tumors showed enhanced CD8 + infiltration compared to HM-1 across all treatments including Control. * p < 0.05

Article Snippet: Sections were stained with the following primary antibodies: anti-Granzyme B (rabbit anti-mouse; Abcam, #ab4059), anti-CD8 (rabbit anti-mouse; Cell Signaling, #98,941), anti-PD-L1 (rabbit monoclonal clone D5V3B; Cell Signaling, #64,988), anti-VEGF (goat polyclonal; R&D SYSTEMS, #AF-493-NA), anti-PARP1 (rabbit polyclonal; Abcam, #ab227244), anti-CD31 (rabbit monoclonal clone EPR17259 ; Abcam, #ab182981), and anti-MECA79 (rat monoclonal clone MECA79; BioLegend, #120,801).

Techniques: Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Digital spatial profiling reveals additive effects of triple therapy on tumor microenvironment: anti-PD-L1, anti-VEGF, and PARP inhibition in mouse models

doi: 10.1007/s00262-026-04312-3

Figure Lengend Snippet: Quantitative analysis of CD8 + T cell density by distance from tumor margin. CD8 + cell density was compared between HM-1 and MC38 models by distance from tumor margin (0 to – 450 μm in 150 μm intervals) on days 8 and 10. a In the HM-1 treatment groups, CD8 + density was low across all distances and time points. b Representative CD8 + T cell immunohistochemical staining of HM-1 tumor tissue on day 10. c In the MC38 model, all treatment groups showed significant increases in CD8 + cell density on day 10 compared to day 6. The anti-PD-L1 + anti-VEGF combination showed the largest fold increases among all groups at each depth region: 4.9-fold ( p = 0.004) at 0 to – 150 μm, 11.9-fold ( p = 0.004) at − 150 to − 300 μm, and 28.2-fold ( p = 0.004) at − 300 to − 450 μm regions. d Representative CD8 + T cell immunohistochemical staining of MC38 tumor tissues on day 10. * p < 0.05, ** p < 0.01

Article Snippet: Sections were stained with the following primary antibodies: anti-Granzyme B (rabbit anti-mouse; Abcam, #ab4059), anti-CD8 (rabbit anti-mouse; Cell Signaling, #98,941), anti-PD-L1 (rabbit monoclonal clone D5V3B; Cell Signaling, #64,988), anti-VEGF (goat polyclonal; R&D SYSTEMS, #AF-493-NA), anti-PARP1 (rabbit polyclonal; Abcam, #ab227244), anti-CD31 (rabbit monoclonal clone EPR17259 ; Abcam, #ab182981), and anti-MECA79 (rat monoclonal clone MECA79; BioLegend, #120,801).

Techniques: Immunohistochemical staining, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Digital spatial profiling reveals additive effects of triple therapy on tumor microenvironment: anti-PD-L1, anti-VEGF, and PARP inhibition in mouse models

doi: 10.1007/s00262-026-04312-3

Figure Lengend Snippet: Triple combination therapy effects in HM-1 tumor model. a Tumor volumes were measured at day 20 in HM-1 model. anti-PD-L1 + anti-VEGF (1.5-fold reduction, 112.1 ± 13.93 mm 3 , p = 0.04) and anti-PD-L1 + anti-VEGF + PARPi (1.7-fold reduction, 99.2 ± 12.17 mm 3 , p = 0.03) showed significant reduction vs. Control (170.5 ± 8.58 mm 3 ). b CD8 + infiltration was analyzed on day 10. The anti-PD-L1 + anti-VEGF + PARPi group showed significant increases compared to all monotherapies in the − 150 to – 300 μm region (Control: 3.1-fold, p = 0.03; anti-PD-L1: 3.3-fold, p = 0.03; anti-VEGF: 2.7-fold, p = 0.04; PARPi: 5.8-fold, p = 0.02). In the 0 to − 150 μm region, anti-PD-L1 + anti-VEGF + PARPi showed significant increase compared to PARPi (9.9-fold, p = 0.03). c Representative CD8 + T cell immunohistochemical staining of HM-1 tumor tissues on days 10. * p < 0.05

Article Snippet: Sections were stained with the following primary antibodies: anti-Granzyme B (rabbit anti-mouse; Abcam, #ab4059), anti-CD8 (rabbit anti-mouse; Cell Signaling, #98,941), anti-PD-L1 (rabbit monoclonal clone D5V3B; Cell Signaling, #64,988), anti-VEGF (goat polyclonal; R&D SYSTEMS, #AF-493-NA), anti-PARP1 (rabbit polyclonal; Abcam, #ab227244), anti-CD31 (rabbit monoclonal clone EPR17259 ; Abcam, #ab182981), and anti-MECA79 (rat monoclonal clone MECA79; BioLegend, #120,801).

Techniques: Control, Immunohistochemical staining, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Digital spatial profiling reveals additive effects of triple therapy on tumor microenvironment: anti-PD-L1, anti-VEGF, and PARP inhibition in mouse models

doi: 10.1007/s00262-026-04312-3

Figure Lengend Snippet: Dual Immunofluorescence Staining for CD8 and Granzyme B across MC38 and HM-1 Tumor Models. a CD8 + Granzyme B + cell density at day 10 in MC38 tumors. In the − 150 to − 300 μm region, anti-PD-L1 + anti-VEGF significantly increased cell density compared to Control (1.9-fold, p = 0.01), anti-VEGF alone (2.7-fold, p = 0.02), and anti-PD-L1 alone (1.8-fold, p = 0.01). b CD8 + Granzyme B + cell density at day 10 in HM-1 tumors. In the 0 to − 150 μm region, anti-PD-L1 + anti-VEGF + PARPi significantly increased density compared to Control (2.8-fold, p = 0.02), anti-VEGF alone (1.9-fold, p = 0.03), and PARPi alone (3.3-fold, p = 0.02). Anti-PD-L1 + anti-VEGF also significantly increased density compared to Control (2.5-fold, p = 0.03) and PARPi alone (3.0-fold, p = 0.03) c Representative dual immunostaining of CD8 + Granzyme B + cells in HM-1 tumors at day 10. CD8 + cells appear green, Granzyme B + cells appear red, and dual-positive CD8 + Granzyme B + cells appear yellow/orange in merged images. Tumor boundaries are indicated by dashed lines. Scale bars: 100 μm. * p < 0.05

Article Snippet: Sections were stained with the following primary antibodies: anti-Granzyme B (rabbit anti-mouse; Abcam, #ab4059), anti-CD8 (rabbit anti-mouse; Cell Signaling, #98,941), anti-PD-L1 (rabbit monoclonal clone D5V3B; Cell Signaling, #64,988), anti-VEGF (goat polyclonal; R&D SYSTEMS, #AF-493-NA), anti-PARP1 (rabbit polyclonal; Abcam, #ab227244), anti-CD31 (rabbit monoclonal clone EPR17259 ; Abcam, #ab182981), and anti-MECA79 (rat monoclonal clone MECA79; BioLegend, #120,801).

Techniques: Immunofluorescence, Staining, Control, Immunostaining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Digital spatial profiling reveals additive effects of triple therapy on tumor microenvironment: anti-PD-L1, anti-VEGF, and PARP inhibition in mouse models

doi: 10.1007/s00262-026-04312-3

Figure Lengend Snippet: Evaluation of CD31 + MECA79 + HEV formation in HM-1 tumors. a Quantitative analysis of CD31 + MECA79 + high endothelial venules (HEV) counts per high-power field (HPF) at × 200 magnification in HM-1 tumors at day 10. Both anti-PD-L1 + anti-VEGF and the triple combination (anti-PD-L1 + anti-VEGF + PARPi) groups exhibited a significantly higher number of HEVs compared to Control, all monotherapy groups, and other doublet combinations. b Representative fluorescent immunohistochemical images of HM-1 tumors double-stained for CD31 (red) and MECA79 (green). CD31 + MECA79 + cells (arrowhead) are prominently detected in the anti-PD-L1 + anti-VEGF and triple combination settings, whereas they are nearly absent or only sporadically observed in all other experimental groups. Tumor boundaries are indicated by dashed lines. Scale bars: 100 μm. ** p < 0.01

Article Snippet: Sections were stained with the following primary antibodies: anti-Granzyme B (rabbit anti-mouse; Abcam, #ab4059), anti-CD8 (rabbit anti-mouse; Cell Signaling, #98,941), anti-PD-L1 (rabbit monoclonal clone D5V3B; Cell Signaling, #64,988), anti-VEGF (goat polyclonal; R&D SYSTEMS, #AF-493-NA), anti-PARP1 (rabbit polyclonal; Abcam, #ab227244), anti-CD31 (rabbit monoclonal clone EPR17259 ; Abcam, #ab182981), and anti-MECA79 (rat monoclonal clone MECA79; BioLegend, #120,801).

Techniques: Control, Immunohistochemical staining, Staining