anti igfbp5  (R&D Systems)

Journal: Cancer Cell International

Article Title: Insulin-like growth factor-binding protein 5 promotes prostate cancer metastasis and osteoblastic activity by inducing chemokines and activating NF-κB signaling

doi: 10.1186/s12935-026-04317-z

Figure Lengend Snippet: IGFBP5 is upregulated in advanced prostate cancer, associated with metastasis, M2 macrophage infiltration, higher Gleason score, and stemness features. A Representative IHC staining of IGFBP5 in tumor versus adjacent tissue. Tissue microarray (TMA) images from six paired prostate cancer cases illustrating stronger IGFBP5 staining in tumor tissues compared with adjacent controls. B . Quantification of IGFBP5 protein expression across the TMA cohort. Bar chart comparing IGFBP5 IHC scores between all prostate cancer tissues ( n = 72) and adjacent controls ( n = 36), showing significantly higher expression in tumors (* p < 0.05). C . Paired analysis of IGFBP5 expression. Paired dot plots of IGFBP5 IHC scores for 34 matched tumor–adjacent pairs, demonstrating consistently elevated IGFBP5 in tumors (* p < 0.05). D . IGFBP5 expression in stemness-enriched prostate cancer cells ( GSE228203 ). Differential expression analysis comparing parental DU145 cells with spheroid-derived, stem-like cells, IGFBP5 is significantly increased in spheroids (* p < 0.05), linking IGFBP5 to stemness features associated with metastasis. E . Correlation between IGFBP5 and M2 macrophage infiltration in TCGA-PRAD. Scatter plot depicting the positive correlation between IGFBP5 expression and the abundance of M2 macrophages (Spearman’s rho = 0.514, p < 0.01). Given that M2 macrophages promote angiogenesis and metastasis, this association supports a pro-metastatic role for IGFBP5. Purity indicates the proportion of tumor cells in the analyzed samples. F . IGFBP5 expression by metastatic status in TCGA-PRAD. Bar chart showing elevated IGFBP5 mRNA in cases with extraprostatic metastasis compared with non-metastatic cases, suggesting a link to metastatic potential (* p < 0.05)

Article Snippet: After blocking with 5% nonfat milk in TBST (0.1% Tween-20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against IGFBP5 (Abclonal, 1:1000), p50 (CST, 1:1000), phospho–NF-κB p65 (CST, 1:1000), total NF-κB p65 (CST, 1:1000), and GAPDH (Santa Cruz, 1:5000).

Techniques: Immunohistochemistry, Microarray, Staining, Expressing, Quantitative Proteomics, Derivative Assay

Journal: Cancer Cell International

Article Title: Insulin-like growth factor-binding protein 5 promotes prostate cancer metastasis and osteoblastic activity by inducing chemokines and activating NF-κB signaling

doi: 10.1186/s12935-026-04317-z

Figure Lengend Snippet: IGFBP5 promotes EMT, migration and invasion in prostate cancer cells. ( A ) Western blot analysis confirming stable overexpression of IGFBP5 in PC3 and DU145 cells. GAPDH was used as a loading control. * P < 0.05. ( B ) Quantitative PCR analysis showing increased mRNA levels of EMT markers CDH2 (N-cadherin), Vimentin, and Snail1 in IGFBP5-overexpressing PC3 cells compared to vector controls. Data are presented as mean ± SD; p < 0.05. ( C ) Transwell migration assays demonstrating enhanced migratory capacity in IGFBP5-overexpressing PC3 and DU145 cells compared to vector controls. Representative images and quantification are shown. Data are presented as mean ± SD; p < 0.05. ( D ) Matrigel-coated Transwell invasion assays showing increased invasive potential in IGFBP5-overexpressing PC3 and DU145 cells. Representative images and quantification are shown. Scale bar, 100 μm. Data are presented as mean ± SD; p < 0.05

Article Snippet: After blocking with 5% nonfat milk in TBST (0.1% Tween-20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against IGFBP5 (Abclonal, 1:1000), p50 (CST, 1:1000), phospho–NF-κB p65 (CST, 1:1000), total NF-κB p65 (CST, 1:1000), and GAPDH (Santa Cruz, 1:5000).

Techniques: Migration, Western Blot, Over Expression, Control, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Cancer Cell International

Article Title: Insulin-like growth factor-binding protein 5 promotes prostate cancer metastasis and osteoblastic activity by inducing chemokines and activating NF-κB signaling

doi: 10.1186/s12935-026-04317-z

Figure Lengend Snippet: The overexpression of IGFBP5 enhances pulmonary metastasis, EMT-like features, and angiogenesis in a tail-vein model. A , Schematic of the experimental design. PC3 cells stably overexpressing IGFBP5 (PC3-IGFBP5) or vector control PC3 cells were injected via the tail vein into BALB/c nude mice; lungs were harvested 4 weeks post-injection. B , Gross lung images showing metastatic nodules. Mice receiving PC3-IGFBP5 cells developed more visible pulmonary metastatic foci than controls (red arrows and Figure S3). C , H&E staining of lung sections demonstrating tumor clusters within the lung parenchyma in the IGFBP5-OE group (red boxes). D – F , Representative IHC images of lung lesions stained for IGFBP5 ( D ), Vimentin ( E ), and VEGF ( F ), showing stronger immunoreactivity in the IGFBP5-OE group compared with controls. G – I , Quantification of IHC staining intensity/positive area for IGFBP5 ( G ), Vimentin ( H ), and VEGF ( I ), indicating significant upregulation in the IGFBP5-OE group ( p < 0.05)

Article Snippet: After blocking with 5% nonfat milk in TBST (0.1% Tween-20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against IGFBP5 (Abclonal, 1:1000), p50 (CST, 1:1000), phospho–NF-κB p65 (CST, 1:1000), total NF-κB p65 (CST, 1:1000), and GAPDH (Santa Cruz, 1:5000).

Techniques: Over Expression, Stable Transfection, Plasmid Preparation, Control, Injection, Staining, Immunohistochemistry

Journal: Cancer Cell International

Article Title: Insulin-like growth factor-binding protein 5 promotes prostate cancer metastasis and osteoblastic activity by inducing chemokines and activating NF-κB signaling

doi: 10.1186/s12935-026-04317-z

Figure Lengend Snippet: IGFBP5 promotes bone metastasis–associated osteogenic and angiogenic phenotypes. A . Emission computed tomography (ECT) imaging of a prostate cancer patient reveals abnormal bone formation indicative of bone metastasis. Metastatic lesions are detected in the scapula ( a ) and lumbar vertebrae ( b ), with increased radiotracer (99mTc) uptake and abnormal bone formation highlighted by red frames. B . IGFBP5 enhances osteogenic mineralization under hypoxia. MC3T3-E1 pre-osteoblasts were cultured with conditioned media (CM) collected from PC3 cells overexpressing IGFBP5 or vector controls under normoxic or hypoxic conditions. CM was refreshed every three days; Alizarin Red S staining was performed on day 14. Representative images and quantification show a pronounced increase in mineralized matrix deposition in the IGFBP5–hypoxia CM group compared with all other groups, indicating that IGFBP5, particularly under hypoxia, augments osteogenic activity at the mineralization stage (* p < 0.05). C . IGFBP5-conditioned media promotes angiogenesis. HUVEC Matrigel tube-formation assays were performed using CM from IGFBP5-overexpressing PC3 and DU145 cells or their respective vector controls. Representative images and quantitative metrics demonstrate that IGFBP5-CM drives more extensive and interconnected capillary-like networks than control CM, consistent with a pro-angiogenic effect (* p < 0.05)

Article Snippet: After blocking with 5% nonfat milk in TBST (0.1% Tween-20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against IGFBP5 (Abclonal, 1:1000), p50 (CST, 1:1000), phospho–NF-κB p65 (CST, 1:1000), total NF-κB p65 (CST, 1:1000), and GAPDH (Santa Cruz, 1:5000).

Techniques: Computed Tomography, Imaging, Cell Culture, Plasmid Preparation, Staining, Activity Assay, Control

Journal: Cancer Cell International

Article Title: Insulin-like growth factor-binding protein 5 promotes prostate cancer metastasis and osteoblastic activity by inducing chemokines and activating NF-κB signaling

doi: 10.1186/s12935-026-04317-z

Figure Lengend Snippet: Transcriptomic profiling identifies NF-κB signaling as a key pathway downstream of IGFBP5. A . Differential gene expression overview. Volcano plot summarizing RNA-seq results from PC3 cells overexpressing IGFBP5 (PC3-IGFBP5) versus vector controls. Thresholds for significance (e.g., |log2FC| > 1, adjusted p < 0.05) are indicated. In total, 1,506 genes were significantly upregulated and 2,509 were downregulated in IGFBP5-overexpressing cells. B . Unsupervised clustering and sample separation. Heatmap of differentially expressed genes (DEGs) showing clear segregation between IGFBP5-OE and control samples with minimal intermixing of biological replicates, indicating a coherent IGFBP5-driven transcriptional program. C . GO enrichment analysis. Top enriched Gene Ontology biological processes among DEGs, highlighting functions related to DNA replication, cell-cycle progression, and cell adhesion relevant to prostate cancer biology. Bar or dot plots display enrichment significance (adjusted p-values) and gene ratios. D . Pathway–gene connectivity map. Category–gene network (cnetplot) of KEGG terms illustrating shared gene membership across key pathways and emphasizing the centrality of NF-κB-related genes within the IGFBP5-regulated network. E . Gene set enrichment analysis (GSEA). Enrichment plot demonstrates significant enrichment of the NF-κB signaling pathway in IGFBP5-overexpressing cells (control vs. overexpression; NES = − 1.92, Padj < 0.01), supporting transcriptional activation of NF-κB target programs. F. NF-κB activation by IGFBP5. Western blots showing increased NF-κB subunits p50 and p65, and enhanced phosphorylation of p65 (RELA) in IGFBP5-overexpressing cells compared with controls, consistent with pathway activation

Article Snippet: After blocking with 5% nonfat milk in TBST (0.1% Tween-20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against IGFBP5 (Abclonal, 1:1000), p50 (CST, 1:1000), phospho–NF-κB p65 (CST, 1:1000), total NF-κB p65 (CST, 1:1000), and GAPDH (Santa Cruz, 1:5000).

Techniques: Gene Expression, RNA Sequencing, Plasmid Preparation, Control, Over Expression, Activation Assay, Western Blot, Phospho-proteomics

Journal: Cancer Cell International

Article Title: Insulin-like growth factor-binding protein 5 promotes prostate cancer metastasis and osteoblastic activity by inducing chemokines and activating NF-κB signaling

doi: 10.1186/s12935-026-04317-z

Figure Lengend Snippet: IGFBP5 promotes EMT and motility via NF-κB signaling. A . RNA-seq showing decreased E-cadherin (CDH1) and increased N-cadherin (CDH2) in IGFBP5-overexpressing prostate cancer cells, consistent with EMT induction. B . qRT-PCR analysis of EMT markers following NF-κB inhibition. Treatment of PC3-IGFBP5 and DU145-IGFBP5 cells with BAY 11-7082 (2.5µM) significantly reduced IGFBP5-induced mRNA levels of CDH2, Vimentin, and Snail. * P < 0.05. C . Transwell migration assays demonstrating that BAY 11-7082 (2.5 and 5µM) attenuates IGFBP5-driven cell migration. * P < 0.05. D . Matrigel-coated Transwell invasion assays demonstrating that BAY 11-7082 (2.5 and 5µM) attenuates IGFBP5-driven cell invasion. * P < 0.05

Article Snippet: After blocking with 5% nonfat milk in TBST (0.1% Tween-20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against IGFBP5 (Abclonal, 1:1000), p50 (CST, 1:1000), phospho–NF-κB p65 (CST, 1:1000), total NF-κB p65 (CST, 1:1000), and GAPDH (Santa Cruz, 1:5000).

Techniques: RNA Sequencing, Quantitative RT-PCR, Inhibition, Migration

Journal: Cancer Cell International

Article Title: Insulin-like growth factor-binding protein 5 promotes prostate cancer metastasis and osteoblastic activity by inducing chemokines and activating NF-κB signaling

doi: 10.1186/s12935-026-04317-z

Figure Lengend Snippet: IGFBP5 upregulates CXCL8 and CCL2 to promote osteogenesis in prostate cancer. A . Correlation analyses between IGFBP5 and chemokines in TCGA-PRAD database. Scatter plots showing positive correlations of IGFBP5 with CXCL8 ( R = 0.32, P < 0.01) and CCL2 ( R = 0.30, P < 0.01). B . ELISA quantification of secreted chemokines in prostate cancer cells. CXCL8 and CCL2 levels are significantly elevated in IGFBP5-overexpressing (IGFBP5-OE) cells compared with vector controls.* P < 0.05. C .ELISA validation of CCL2 and CXCL8 depletion from conditioned medium. Conditioned medium from IGFBP5-overexpressing (IGFBP5-OE) cells was treated with neutralizing antibodies followed by immunoprecipitation to deplete CCL2 and CXCL8. ELISA assays confirmed a significant reduction in the levels of both chemokines compared to IgG controls. * P < 0.05. D Osteogenic potential is reduced after depletion of CCL2 or CXCL8 from conditioned medium.The conditioned medium, after immunoprecipitation-based removal of CCL2 or CXCL8, was applied to osteogenic assays. Osteogenic differentiation was assessed by mineralization. Removal of either chemokine resulted in a significant decrease in osteogenic activity compared to the IGFBP5-OE medium without depletion. * P < 0.05

Article Snippet: After blocking with 5% nonfat milk in TBST (0.1% Tween-20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against IGFBP5 (Abclonal, 1:1000), p50 (CST, 1:1000), phospho–NF-κB p65 (CST, 1:1000), total NF-κB p65 (CST, 1:1000), and GAPDH (Santa Cruz, 1:5000).

Techniques: Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Biomarker Discovery, Immunoprecipitation, Activity Assay

Journal: Cancer Cell International

Article Title: Insulin-like growth factor-binding protein 5 promotes prostate cancer metastasis and osteoblastic activity by inducing chemokines and activating NF-κB signaling

doi: 10.1186/s12935-026-04317-z

Figure Lengend Snippet: Proposed model of IGFBP5-mediated promotion of prostate cancer metastasis

Article Snippet: After blocking with 5% nonfat milk in TBST (0.1% Tween-20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against IGFBP5 (Abclonal, 1:1000), p50 (CST, 1:1000), phospho–NF-κB p65 (CST, 1:1000), total NF-κB p65 (CST, 1:1000), and GAPDH (Santa Cruz, 1:5000).

Techniques:

Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Volcano plot of DEGs in ligament cells following ADAM12 knockdown. B Representative enriched pathways from GO, KEGG, and REACTOME analysis of genes altered by ADAM12 knockdown. C Correlation analysis between ADAM12 expression and glycolysis pathway activity from transcriptomic data. D , E Western blot analysis of IGFBP5 and cleaved IGFBP5 protein levels after ADAM12 knockdown and overexpression. n = 3. F , G Western blot analysis of total and phosphorylated levels of IGF1R, PI3K, AKT, and mTOR proteins after ADAM12 knockdown and overexpression. n = 3. H , I IGF1 concentration in cell culture supernatant measured by ELISA after ADAM12 knockdown and overexpression. n = 5. J Western blot analysis of glycolytic enzymes (HK2, LDHA) and osteogenic markers (RUNX2, ALP) in ADAM12-overexpressing cells treated with Linsitinib or Rapamycin. n = 3. K , L Glucose uptake and lactate production in ADAM12-overexpressing cells treated with Linsitinib or Rapamycin. n = 3. M ECAR measurements in ADAM12-overexpressing cells treated with Linsitinib or Rapamycin. n = 3. Data are presented as mean ± standard deviation.

Article Snippet: Primary antibodies used were as follows: ADAM12 (Proteintech, 14139-1-AP), RUNX2 (Abclonal, A11753), OSX (Abclonal, A18699), ALP (Zenbio, 381009), HK2 (Zenbio, R24552 ), PKM2 (Zenbio, R381318), GLUT1 (Zenbio, R380464), PDK1 (Zenbio, R381931), LDHA (Zenbio, R22873 ), cytochrome c (Zenbio, R22867 ), ATP5A (Zenbio, R381760), PGC1α (Zenbio, R381615), mtTFA (Zenbio, R22594 ), IGFBP5 (Proteintech, 55205-1-AP), IGFBP3 (Proteintech, 10189-2-AP), IGF1R (Zenbio, R26882 ), phosphorylated IGF1R (Zenbio, R24720 ), PI3K (Zenbio, R25368 ), phosphorylated PI3K (Zenbio, 341468), AKT (Zenbio, 342529), phosphorylated AKT (Zenbio, 310021), mTOR (Zenbio, R380411), phosphorylated mTOR (Zenbio, R25033 ), and β-actin (Zenbio, R380624).

Techniques: Knockdown, Expressing, Activity Assay, Western Blot, Over Expression, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: bioRxiv

Article Title: IGFBPs define distinct pro-tumorigenic CAF subtypes in lung cancer tumour-microenvironment

doi: 10.64898/2025.12.03.692056

Figure Lengend Snippet: a) Dot plot representing IGFBP5 (left), IGFBP6 (centre) and IGFBP7 (right) expressions in different cell types in publicly available lung single-cell RNA sequencing datasets. Data was generated from the Curated Cancer Cell Atlas. The size of the circle dot represents the percentage of expressing cells, while the colour represents the mean log2 expression level, as indicated in the scale bar (below), b) mRNA expression (fold change) of IGFBP5, 6 and 7 in patient-derived lung normal fibroblasts (NF) and cancer-associated fibroblasts (CAF). Data are presented as mean ± SEM (n=3 biological triplicates). Statistical analysis was conducted using an unpaired t-test with Welch’s correction; exact p-values are represented in the graph, c) Immunoblots (IBs) showing IGFBP5, 6 and 7 expressions in whole cell lysates of NF and CAF. β-Actin served as the loading control. Densitometric analysis was done using Image Lab (v6.1.0), d) IBs showing secreted and cellular (corresponding serum-starved cell lysates) expression of IGFBP5, 6 and 7. β-Actin served as the loading control for serum-starved cellular lysates, while Ponceau blot served as the loading control for secretory lysates. All IBs are representative of three biological triplicates.

Article Snippet: Cells were lysed directly with RIPA buffer or Laemmli buffer and subjected to SDS PAGE and primary antibodies of α-Smooth Muscle Actin (#19245, CST, 1:1000), FAP (#66562, CST, 1:1000), S100A4 (#13018, CST, 1:1000), IGFBP5 (sc-515184, Santa Cruz, 1:1000), IGFBP6 (AF876, R&D Systems, 1:1000), IGFBP7(sc-365293, Santa Cruz 1:750), Phospho-p44/42 MAPK (Thr202/Tyr204) (#9101, CST, 1:2000), p44/42 MAPK (Thr202/Tyr204) (137F5)(#4695, CST, 1:2000), Phospho-Akt (Ser473) (#4060, CST, 1:2000), Akt (pan) (#2920, CST, 1:2000), Phospho-Insulin R(Y1162/Y1163)/IGF-1R (Y1132/Y1136) (AF2507, R&D Systems, 1:1000), IGF-I R/IGF1R (MAB391, R&D Systems, 1:1000), Phospho-Stat3 (Tyr705) (#9145, CST, 1:2000), Stat3 (#9139, CST, 1:2000), IL6 (#12153, CST, 1:1000), β-Actin (8H10D10) (#3700, CST, 1:2000), GAPDH (#2118, CST, 1:10,000).

Techniques: RNA Sequencing, Generated, Expressing, Derivative Assay, Western Blot, Control

Journal: bioRxiv

Article Title: IGFBPs define distinct pro-tumorigenic CAF subtypes in lung cancer tumour-microenvironment

doi: 10.64898/2025.12.03.692056

Figure Lengend Snippet: a) Schematics showing investigation of IGFBP5, 6 and 7 roles in CAF biology through transient and stable knockdown approaches. Illustration was prepared using Biorender.com, b) Immunoblots showing knockdown validation of IGFBP5, 6 and 7 in CAF upon transient transfection. GAPDH and β-Actin served as the loading control. The IBs represent three biological replicates, c) Representative immunofluorescence images showing F-actin staining (red) and DAPI (blue) in CAF upon individual and pooled knockdown of IGFBPs, scale bar 45μm, d) Quantification of cell area for (c). Quantification was performed for a minimum of thirty cells, which were included from three biological replicates. Data are presented as mean ± SEM, with open circles indicating individual cells. Statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s multiple comparison; exact p-values are indicated in the graph, e) Schematics showing collagen contraction experiment. Illustration was prepared using Biorender.com, f) Image representing technical duplicates of collagen contraction upon individual and pooled knockdown of IGFBP5, 6 and 7. Yellow dotted lines mark the boundary of the gel, g) Quantification of the area of the gel disk is represented as a bar plot with mean ± SEM, n=4 biological replicates, h) Representative brightfield images of migrated cells (crystal violet stained) after IGFBP5, 6 and 7 knockdowns, scale bar 200μm. Images were captured 16 hours after wiping off the non-migrated cells in the upper chamber, i) Cell count of the migrated cells is taken as an average of four different fields of one experiment. Data represent mean (relative cell count with respect to control (%) ± SEM, n= 3 biological replicates, (g, i) To determine statistical significance, One-way ANOVA with Dunnett’s multiple comparison was used; exact p-values are indicated in the graph.

Article Snippet: Cells were lysed directly with RIPA buffer or Laemmli buffer and subjected to SDS PAGE and primary antibodies of α-Smooth Muscle Actin (#19245, CST, 1:1000), FAP (#66562, CST, 1:1000), S100A4 (#13018, CST, 1:1000), IGFBP5 (sc-515184, Santa Cruz, 1:1000), IGFBP6 (AF876, R&D Systems, 1:1000), IGFBP7(sc-365293, Santa Cruz 1:750), Phospho-p44/42 MAPK (Thr202/Tyr204) (#9101, CST, 1:2000), p44/42 MAPK (Thr202/Tyr204) (137F5)(#4695, CST, 1:2000), Phospho-Akt (Ser473) (#4060, CST, 1:2000), Akt (pan) (#2920, CST, 1:2000), Phospho-Insulin R(Y1162/Y1163)/IGF-1R (Y1132/Y1136) (AF2507, R&D Systems, 1:1000), IGF-I R/IGF1R (MAB391, R&D Systems, 1:1000), Phospho-Stat3 (Tyr705) (#9145, CST, 1:2000), Stat3 (#9139, CST, 1:2000), IL6 (#12153, CST, 1:1000), β-Actin (8H10D10) (#3700, CST, 1:2000), GAPDH (#2118, CST, 1:10,000).

Techniques: Knockdown, Western Blot, Biomarker Discovery, Transfection, Control, Immunofluorescence, Staining, Comparison, Cell Counting

Journal: bioRxiv

Article Title: IGFBPs define distinct pro-tumorigenic CAF subtypes in lung cancer tumour-microenvironment

doi: 10.64898/2025.12.03.692056

Figure Lengend Snippet: a) Volcano plot representing differentially expressed genes between CAF siControl vs siIGFBP5 b) CAF siControl vs siIGFBP6 c) CAF siControl vs siIGFBP7 (n=3 biological replicates). A log2 fold change of 0.5 was set as the threshold. Selected genes for validation are marked in blue. d) Enriched pathways based on the differentially expressed genes upon IGFBP5 knockdown e) IGFBP6 knockdown f) IGFBP7 knockdown g) Heatmap comparing the published CAF subtype gene sets with CAF siControl versus siIGFBP5 h) CAF siControl versus siIGFBP6 i) CAF siControl versus siIGFBP7 (n=2 biological replicates)

Article Snippet: Cells were lysed directly with RIPA buffer or Laemmli buffer and subjected to SDS PAGE and primary antibodies of α-Smooth Muscle Actin (#19245, CST, 1:1000), FAP (#66562, CST, 1:1000), S100A4 (#13018, CST, 1:1000), IGFBP5 (sc-515184, Santa Cruz, 1:1000), IGFBP6 (AF876, R&D Systems, 1:1000), IGFBP7(sc-365293, Santa Cruz 1:750), Phospho-p44/42 MAPK (Thr202/Tyr204) (#9101, CST, 1:2000), p44/42 MAPK (Thr202/Tyr204) (137F5)(#4695, CST, 1:2000), Phospho-Akt (Ser473) (#4060, CST, 1:2000), Akt (pan) (#2920, CST, 1:2000), Phospho-Insulin R(Y1162/Y1163)/IGF-1R (Y1132/Y1136) (AF2507, R&D Systems, 1:1000), IGF-I R/IGF1R (MAB391, R&D Systems, 1:1000), Phospho-Stat3 (Tyr705) (#9145, CST, 1:2000), Stat3 (#9139, CST, 1:2000), IL6 (#12153, CST, 1:1000), β-Actin (8H10D10) (#3700, CST, 1:2000), GAPDH (#2118, CST, 1:10,000).

Techniques: Biomarker Discovery, Knockdown

Journal: bioRxiv

Article Title: IGFBPs define distinct pro-tumorigenic CAF subtypes in lung cancer tumour-microenvironment

doi: 10.64898/2025.12.03.692056

Figure Lengend Snippet: a) Ligand activity analysis showing the list of potential ligands that can regulate differentially expressed genes upon IGFBP5 knockdown in CAF, d) IGFBP6 knockdown, g) IGFBP7 knockdown, b) Ligand-target matrix showing the regulatory potential of potential ligands with CAF siControl vs siIGFBP5, e) CAF siControl vs siIGFBP6, h) CAF siControl vs siIGFBP7, c) Western blot analysis of CAF siControl and siIGFBP5, f) CAF siControl and siIGFBP6, i) CAF siControl and siIGFBP7, after treatment with TGFβ1[10 ng/mL] and IL6 [10 ng/mL] for 48 hours. Densitometric analysis was done using Image Lab (v6.1.0). All IBs are representative of three biological triplicates.

Article Snippet: Cells were lysed directly with RIPA buffer or Laemmli buffer and subjected to SDS PAGE and primary antibodies of α-Smooth Muscle Actin (#19245, CST, 1:1000), FAP (#66562, CST, 1:1000), S100A4 (#13018, CST, 1:1000), IGFBP5 (sc-515184, Santa Cruz, 1:1000), IGFBP6 (AF876, R&D Systems, 1:1000), IGFBP7(sc-365293, Santa Cruz 1:750), Phospho-p44/42 MAPK (Thr202/Tyr204) (#9101, CST, 1:2000), p44/42 MAPK (Thr202/Tyr204) (137F5)(#4695, CST, 1:2000), Phospho-Akt (Ser473) (#4060, CST, 1:2000), Akt (pan) (#2920, CST, 1:2000), Phospho-Insulin R(Y1162/Y1163)/IGF-1R (Y1132/Y1136) (AF2507, R&D Systems, 1:1000), IGF-I R/IGF1R (MAB391, R&D Systems, 1:1000), Phospho-Stat3 (Tyr705) (#9145, CST, 1:2000), Stat3 (#9139, CST, 1:2000), IL6 (#12153, CST, 1:1000), β-Actin (8H10D10) (#3700, CST, 1:2000), GAPDH (#2118, CST, 1:10,000).

Techniques: Activity Assay, Knockdown, Western Blot

Journal: bioRxiv

Article Title: IGFBPs define distinct pro-tumorigenic CAF subtypes in lung cancer tumour-microenvironment

doi: 10.64898/2025.12.03.692056

Figure Lengend Snippet: a) H460 cells (masked-yellow) treated with conditioned media (CM) derived from CAF individual and pooled knockdowns of IGFBP5, 6 and 7, image shown here is a representative for the 72-hour timepoint, b) Graph represents mean ± SEM showing the proliferation of tumour cells represented as confluence percentage over a 72-hour time interval, the line colour represents different conditions, n=3, * indicates p-value less than 0.05, c) Representative brightfield images of invaded cells (crystal violet stained) of tumour cells towards conditioned media derived from individual and pooled IGFBP5, 6 and 7 knockdowns in CAF, scale bar 200μm. Images were captured 16 hours after wiping off the non-invaded cells in the upper chamber. d) Cell count of the invaded cells is taken as an average of four different fields of one experiment. Data represent mean ± SEM, n=3 biological replicates. To determine statistical significance, Oneway ANOVA with Dunnett’s multiple comparison was used; exact p-values are indicated in the graph, e) H460 cells (masked- yellow) treated with the IC 50 values of cisplatin [4.8 μM] with CM derived from CAF individual and pooled knockdowns of IGFBP5, 6 and 7, image shown here is a representative for the 72-hour time-point, f) Graph represents mean ± SEM showing the tumour cells represented as confluence percentage over a 72-hour time interval, the line colour represents different conditions, n=3, * indicates p-value less than 0.05, **** indicates pvalue less than 0.0001. g) Schematics showing in-vivo experiment design, illustration was prepared using Biorender.com. h) m-Cherry expressing H460 tumour cells were injected subcutaneously alone (0.5 * 10 6 cells) or co-injected with GFP-expressing CAF individual IGFBP5, 6 or 7 knockdown or shControl (1.5 * 10 6 cells) in male NOD-SCID mice. Tumours were excised on the 16 th day. i) Tumour volume and j) tumour weight were measured after the tumours were excised; data are represented for 6 mice from three independent experiments. Dunnet’s multiple comparison test was used to determine statistical significance.

Article Snippet: Cells were lysed directly with RIPA buffer or Laemmli buffer and subjected to SDS PAGE and primary antibodies of α-Smooth Muscle Actin (#19245, CST, 1:1000), FAP (#66562, CST, 1:1000), S100A4 (#13018, CST, 1:1000), IGFBP5 (sc-515184, Santa Cruz, 1:1000), IGFBP6 (AF876, R&D Systems, 1:1000), IGFBP7(sc-365293, Santa Cruz 1:750), Phospho-p44/42 MAPK (Thr202/Tyr204) (#9101, CST, 1:2000), p44/42 MAPK (Thr202/Tyr204) (137F5)(#4695, CST, 1:2000), Phospho-Akt (Ser473) (#4060, CST, 1:2000), Akt (pan) (#2920, CST, 1:2000), Phospho-Insulin R(Y1162/Y1163)/IGF-1R (Y1132/Y1136) (AF2507, R&D Systems, 1:1000), IGF-I R/IGF1R (MAB391, R&D Systems, 1:1000), Phospho-Stat3 (Tyr705) (#9145, CST, 1:2000), Stat3 (#9139, CST, 1:2000), IL6 (#12153, CST, 1:1000), β-Actin (8H10D10) (#3700, CST, 1:2000), GAPDH (#2118, CST, 1:10,000).

Techniques: Derivative Assay, Staining, Cell Counting, Comparison, In Vivo, Expressing, Injection, Knockdown

Journal: bioRxiv

Article Title: IGFBPs define distinct pro-tumorigenic CAF subtypes in lung cancer tumour-microenvironment

doi: 10.64898/2025.12.03.692056

Figure Lengend Snippet: a) Formalin-fixed paraffin-embedded NSCLC tumour sections were stained with hematoxylin and eosin, Sirius Red, antibodies against α-SMA, IGFBP5, 6 and 7. Cases 1 and 2 represent two different patients. ‘T’ indicates tumour and ‘S’ indicates stroma. The arrow indicates the expression of the respective proteins in fibroblasts; scale bar, 50 μm. b) Representative multiplex immunofluorescence staining images of IGFBP5+, IGFBP6+ and IGFBP7+ CAFs. DAPI (blue), αSMA (green), FAP (pink), PanCK (red), IGFBP5 (grey), IGFBP6 (yellow), IGFBP7 (orange) in human non-small-cell lung cancer tissue sections. Scale bars 50 μm. c) survival between high and low expression of CAF_IGFBP5 gene signatures d) CAF_IGFBP6 gene signatures e) CAF_IGFBP7 gene signatures in TCGA-LUAD datasets, pvalues were obtained from two-sided log-Rank tests.

Article Snippet: Cells were lysed directly with RIPA buffer or Laemmli buffer and subjected to SDS PAGE and primary antibodies of α-Smooth Muscle Actin (#19245, CST, 1:1000), FAP (#66562, CST, 1:1000), S100A4 (#13018, CST, 1:1000), IGFBP5 (sc-515184, Santa Cruz, 1:1000), IGFBP6 (AF876, R&D Systems, 1:1000), IGFBP7(sc-365293, Santa Cruz 1:750), Phospho-p44/42 MAPK (Thr202/Tyr204) (#9101, CST, 1:2000), p44/42 MAPK (Thr202/Tyr204) (137F5)(#4695, CST, 1:2000), Phospho-Akt (Ser473) (#4060, CST, 1:2000), Akt (pan) (#2920, CST, 1:2000), Phospho-Insulin R(Y1162/Y1163)/IGF-1R (Y1132/Y1136) (AF2507, R&D Systems, 1:1000), IGF-I R/IGF1R (MAB391, R&D Systems, 1:1000), Phospho-Stat3 (Tyr705) (#9145, CST, 1:2000), Stat3 (#9139, CST, 1:2000), IL6 (#12153, CST, 1:1000), β-Actin (8H10D10) (#3700, CST, 1:2000), GAPDH (#2118, CST, 1:10,000).

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Expressing, Multiplex Assay, Immunofluorescence

Journal: bioRxiv

Article Title: IGFBPs define distinct pro-tumorigenic CAF subtypes in lung cancer tumour-microenvironment

doi: 10.64898/2025.12.03.692056

Figure Lengend Snippet: Tumour cells secrete TGFβ1 and IL6, which regulate IGFBP5, 6 and 7 expressions in cancer-associated fibroblasts. These high IGFBP-expressing CAFs show pro-tumorigenic effects and are associated with poor patient outcomes. On the other hand, knocking down IGFBP5, 6, and 7 selectively pushes CAF towards distinct subtypes, which suppresses tumour properties and correlates with better survival.

Article Snippet: Cells were lysed directly with RIPA buffer or Laemmli buffer and subjected to SDS PAGE and primary antibodies of α-Smooth Muscle Actin (#19245, CST, 1:1000), FAP (#66562, CST, 1:1000), S100A4 (#13018, CST, 1:1000), IGFBP5 (sc-515184, Santa Cruz, 1:1000), IGFBP6 (AF876, R&D Systems, 1:1000), IGFBP7(sc-365293, Santa Cruz 1:750), Phospho-p44/42 MAPK (Thr202/Tyr204) (#9101, CST, 1:2000), p44/42 MAPK (Thr202/Tyr204) (137F5)(#4695, CST, 1:2000), Phospho-Akt (Ser473) (#4060, CST, 1:2000), Akt (pan) (#2920, CST, 1:2000), Phospho-Insulin R(Y1162/Y1163)/IGF-1R (Y1132/Y1136) (AF2507, R&D Systems, 1:1000), IGF-I R/IGF1R (MAB391, R&D Systems, 1:1000), Phospho-Stat3 (Tyr705) (#9145, CST, 1:2000), Stat3 (#9139, CST, 1:2000), IL6 (#12153, CST, 1:1000), β-Actin (8H10D10) (#3700, CST, 1:2000), GAPDH (#2118, CST, 1:10,000).

Techniques: Expressing