Journal: Advanced Science

Article Title: Steroid Receptor Coactivator‐1 Drives Tumor‐Associated Macrophage Reprogramming by Mediating MMP12 Transcription in Pancreatic Cancer Perineural Invasion

doi: 10.1002/advs.202416575

Figure Lengend Snippet: TAMs reprogrammed by SRC‐1 secretion MMP12 induce PNI. A) BMDMs were induced into TAMs from WT and SRC‐1 ‐/‐ mice, and CM was collected for differential protein analysis by mass spectrometry (n = 3). B) Volcano plot of differentially expressed proteins between WT and SRC‐1 ‐/‐ groups. C) qPCR analysis revealed decreased MMP12 mRNA levels upon SRC‐1 downregulation.n = 3. Significance was calculated with Student's t ‐test. **** P< 0.0001. D, E) Western blotting detected the reduced MMP12 protein expression following SRC‐1 down‐regulation. n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05. F) Immunofluorescence staining to detect the expression of MMP12 in mouse tissues from the sciatic nerve injection models. G,H) Western blot confirmation demonstrated successful SRC‐1 knockdown and MMP12 over‐expression. n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05, **** P< 0.0001. I) CCK8 assays were utilized to indicate the enhanced proliferation of AsPC‐1 cells upon SRC‐1 down‐regulation and MMP12 over‐expression in TAMs. n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. *** P< 0.001, **** P< 0.0001. J) Rescue experiments assessing changes in in vitro PNI following SRC‐1 down‐regulation and MMP12 over‐expression. The red arrow indicates the leading edge of pancreatic cancer cell invasion. K) The neural invasion index of the rescue experiments. n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05, ** P< 0.01. L–O) Wound healing and Trans‐well assays demonstrated the increased migration and invasion ability of AsPC‐1 cells upon SRC‐1 down‐regulation and MMP12 over‐expression in TAMs.n = 3. For wound healing assays: the scale bar is 500 µm. For the Transwell assay, the scale bar is 400 µm.n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Wet transfer was performed at 350 mA for 1.5 h. The membrane was blocked with 5% non‐fat dry milk at room temperature(25 °C) for 1.5 h. Primary antibodies were diluted according to recommended ratios: SRC‐1 (CST, 2191, 1:1000), CD206 (Proteintech, 60 143, 1:5000), CD163 (WANLEI, WL03026, 1:1000), iNOS (Proteintech, 22 226, 1:1000), MMP12 (Proteintech, 22 989, 1:500), STAT1 (CST, 9172, 1:1000), p‐STAT1 (CST, 7649, 1:1000), and GAPDH (Proteintech, 10 494, 1:5000).

Techniques: Mass Spectrometry, Western Blot, Expressing, Immunofluorescence, Staining, Injection, Knockdown, Over Expression, In Vitro, Migration, Transwell Assay

Journal: Advanced Science

Article Title: Steroid Receptor Coactivator‐1 Drives Tumor‐Associated Macrophage Reprogramming by Mediating MMP12 Transcription in Pancreatic Cancer Perineural Invasion

doi: 10.1002/advs.202416575

Figure Lengend Snippet: SRC‐1 as a coactivator of STAT1, promotes MMP12 promoter activity. A, B) Western blot analysis of p‐STAT1 and STAT1 expression in TAMs under different treatments. n = 3. Significance was calculated with Student's t ‐test. * P< 0.05. C, D) Western blot analysis of p‐NF‐κB, NF‐κB, IRF4, and PPAR‐γ expression in TAMs under different treatments.n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05, ** P< 0.01, ns, no significant difference. E, F) Co‐immunoprecipitation experiments detecting the interaction between SRC‐1 and STAT1. n = 3. Significance was calculated with Student's t ‐test. * P< 0.05, ** P< 0.01, *** P< 0.001. G) Immunofluorescence validation of co‐localization of STAT1 (green) and SRC‐1 (red) in TAMs. H) Prediction by Jaspar of STAT1 binding sites on the MMP12 promoter region. I, J) ChIP assay confirming recruitment of STAT1 to the MMP12 promoter region. K, L) Luciferase assays demonstrating changes in MMP12 promoter luciferase activity upon knockdown or over‐expression of SRC‐1. n = 3. Significance was calculated with Student's t ‐test. * P < 0.05, ** P < 0.01.

Article Snippet: Wet transfer was performed at 350 mA for 1.5 h. The membrane was blocked with 5% non‐fat dry milk at room temperature(25 °C) for 1.5 h. Primary antibodies were diluted according to recommended ratios: SRC‐1 (CST, 2191, 1:1000), CD206 (Proteintech, 60 143, 1:5000), CD163 (WANLEI, WL03026, 1:1000), iNOS (Proteintech, 22 226, 1:1000), MMP12 (Proteintech, 22 989, 1:500), STAT1 (CST, 9172, 1:1000), p‐STAT1 (CST, 7649, 1:1000), and GAPDH (Proteintech, 10 494, 1:5000).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Immunofluorescence, Biomarker Discovery, Binding Assay, Luciferase, Knockdown, Over Expression

Journal: Advanced Science

Article Title: Steroid Receptor Coactivator‐1 Drives Tumor‐Associated Macrophage Reprogramming by Mediating MMP12 Transcription in Pancreatic Cancer Perineural Invasion

doi: 10.1002/advs.202416575

Figure Lengend Snippet: Inhibition of MMP12 can effectively alleviate PNI both in vivo and in vitro. A, B) Paralysis severity in the hind limbs of mice from various groups following sciatic nerve co‐injection of tumor and macrophage mixtures. Groups: (1) WT BMDM, (2) SRC‐1‐KO BMDM, (3) WT BMDM + vehicle, and (4) WT BMDM + MMP408 (5 mg/kg). n = 19 C) Tumor growth and invasion in the sciatic nerve of mice were recorded. D) Body weight changes in mice over a 21‐day period following sciatic nerve co‐injection of tumor and macrophage mixtures were recorded. E) Sciatic functional index in mice over 21 days following co‐injection of tumor and macrophage mixtures was recorded. WT‐BMDM+KRAS/p53 /m/+ group: n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test at the final time point. ** P< 0.01. F) Sciatic functional scores in mice over 21 days following co‐injection of tumor and macrophage mixtures were recorded. WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test at the final time point. **** P< 0.0001. G) Tumor weight in mice harvested at 21 days post‐injection was recorded. WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05, ** P< 0.01, ns, no significant difference. H) The length of invasion in the mouse groups was recorded. WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test. ** P< 0.01. I) Quantitative analysis of CD206 + SRC‐1 + dual‐positive cells was performed in murine tissue using dual‐immunofluorescence staining.WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with Student's t ‐test. * P< 0.05. J) Neural invasion index was recorded in the DRG‐tumor cells co‐culture assay. WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test. ** P< 0.01, *** P< 0.001. K) Multiplex immunofluorescence co‐staining demonstrating expression of SRC‐1 (red), CD206 (yellow), and S100β (green) in murine tumor tissues. L) A DRG and tumor cell (red) co‐culture model was utilized to quantitatively assess tumor cell PNI capacity ex vivo.

Article Snippet: Wet transfer was performed at 350 mA for 1.5 h. The membrane was blocked with 5% non‐fat dry milk at room temperature(25 °C) for 1.5 h. Primary antibodies were diluted according to recommended ratios: SRC‐1 (CST, 2191, 1:1000), CD206 (Proteintech, 60 143, 1:5000), CD163 (WANLEI, WL03026, 1:1000), iNOS (Proteintech, 22 226, 1:1000), MMP12 (Proteintech, 22 989, 1:500), STAT1 (CST, 9172, 1:1000), p‐STAT1 (CST, 7649, 1:1000), and GAPDH (Proteintech, 10 494, 1:5000).

Techniques: Inhibition, In Vivo, In Vitro, Injection, Functional Assay, Immunofluorescence, Staining, Co-culture Assay, Multiplex Assay, Expressing, Co-Culture Assay, Ex Vivo

Journal: Advanced Science

Article Title: Steroid Receptor Coactivator‐1 Drives Tumor‐Associated Macrophage Reprogramming by Mediating MMP12 Transcription in Pancreatic Cancer Perineural Invasion

doi: 10.1002/advs.202416575

Figure Lengend Snippet: Schematic illustrating working model for the mechanisms by which SRC‐1 drives TAMs reprogramming by mediating MMP12 transcription in pancreatic cancer PNI. SRC‐1 binds to STAT1 to enhance the transcriptional activity of the MMP12 promoter, thereby increasing MMP12 secretion from TAMs.

Article Snippet: Wet transfer was performed at 350 mA for 1.5 h. The membrane was blocked with 5% non‐fat dry milk at room temperature(25 °C) for 1.5 h. Primary antibodies were diluted according to recommended ratios: SRC‐1 (CST, 2191, 1:1000), CD206 (Proteintech, 60 143, 1:5000), CD163 (WANLEI, WL03026, 1:1000), iNOS (Proteintech, 22 226, 1:1000), MMP12 (Proteintech, 22 989, 1:500), STAT1 (CST, 9172, 1:1000), p‐STAT1 (CST, 7649, 1:1000), and GAPDH (Proteintech, 10 494, 1:5000).

Techniques: Activity Assay

Journal: International Journal of Cancer

Article Title: Integrin β6 expression in colorectal cancer cells promotes liver metastasis through enhanced adhesion to endothelial fibronectin

doi: 10.1002/ijc.35504

Figure Lengend Snippet: Integrin β6 is a proteolysis‐resistant mediator of CRC tumour cell adhesion to endothelial cells. (A) Lysates of HT‐29 and DLD‐1 cells harvested in the presence of 0.05% trypsin or 5 mM EDTA‐PBS were collected, and the expression of integrins αv, β6, β1 and β5 was analysed using western blotting. Blots show the full‐length proteins as well as the trypsin‐induced protein fragments. GAPDH was used as a loading control. (B) Adhesion of cytopainter‐stained ITGB6 KO or NT cells harvested by trypsinization or 5 mM PBS‐EDTA to an IL‐1β‐stimulated HUVEC monolayer under static conditions. Arrow points towards an adherent cell. Scale bar: 250 μm. The quantification is shown below. Mann–Whitney U test; ns, not significant; ** p < 0.01. (C) The cancer‐related survival (60‐month cut‐off) of CRC patients who did not receive neoadjuvant therapy and had no residual tumour (R0) was explored. Based on MMP11 and MMP12 RNA expression levels, patients were divided into high‐ and low‐MMP groups. ITGB6 ‐dependent survival was investigated in these groups. The number of patients per group is indicated by ‘ n ’. Cox proportional hazards survival regression. (D) Recombinant integrins αvβ6, αvβ1, and αvβ5 were incubated with MMP‐12 at an enzyme‐to‐substrate ratio of 1:10 for 1 h. Proteolytic cleavage of integrins was evaluated via western blotting.

Article Snippet: MMP‐12 (R&D Systems, Catalogue #: 917‐MPB) was activated in a concentration of 50 ng/μL by incubation in TCN buffer (10 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5) at 37°C for 2 h. Following enzyme activation, the recombinant human integrins (integrin αvβ6; Catalogue #: T6‐H52E1, integrin αvβ1; Catalogue #: IT1‐H52E1, integrin αvβ5; Catalogue #: IT5‐H52W5, AcroBiosystems, Newark, DE, USA) were added in an enzyme‐to‐substrate ratio of 1:10 in TCN buffer for 1 h (MMP‐12) at 37°C.

Techniques: Expressing, Western Blot, Control, Staining, MANN-WHITNEY, RNA Expression, Recombinant, Incubation

Journal: Advanced Science

Article Title: Resident Macrophage‐Orchestrated Immune and Fibroblast Interactions in Immune Checkpoint Inhibitor‐Associated Nephrotoxicity

doi: 10.1002/advs.202505445

Figure Lengend Snippet: Transcriptomic and protein‐level evidence of MMP12 and CXCL9 upregulation in ICI‐associated nephrotoxicity (ICI‐AN). A) Volcano plot of differentially expressed genes in mouse kidney tissue after ICI treatment, highlighting significantly upregulated genes (e.g., MMP12, CXCL9, CXCL10, and CD74) in red. Differentially expressed genes were identified using a threshold of |log 2 (Fold Change)| > 1.5 and FDR < 0.05. Data were obtained from the GEO database. B) Biological process (BP) enrichment analysis for the upregulated genes in ICI‐AN. C) KEGG Pathway enrichment analysis. D) Immunohistochemical staining of MMP12 in human kidney tissue from control and ICI‐AN group. Scale bars: 50 µm; magnification: 400×. E) CXCL9 levels in urine ( n = 10 control, n = 9 AIN, n = 6 ICI‐AN) and serum ( n = 10 control, n = 10 AIN, n = 7 ICI‐AN). F) Correlation analysis between urinary or serum CXCL9 levels and resident macrophage frequency. Spearman's rank correlation coefficients (R) and P ‐values are shown. Data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA with Tukey's post hoc test. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Membranes were blocked with 5% non‐fat milk and incubated overnight at 4 °C with primary antibodies targeting MMP12 (Novus Biologicals, Centennial, CO, USA), α‐SMA (Proteintech, Wuhan, China), NGAL (R&D systems, USA), and β‐actin (Proteintech, Wuhan, China), followed by incubation with HRP‐conjugated secondary antibodies.

Techniques: Immunohistochemical staining, Staining, Control

Journal: Advanced Science

Article Title: Resident Macrophage‐Orchestrated Immune and Fibroblast Interactions in Immune Checkpoint Inhibitor‐Associated Nephrotoxicity

doi: 10.1002/advs.202505445

Figure Lengend Snippet: Resident macrophages mediate regional enrichment of MMP12 and induce CXCL9 production. A) Western blot analysis of fibrosis and inflammatory markers. B) Quantitative polymerase chain reaction (qPCR) analysis of pro‐inflammatory and fibrosis‐related genes. C) Immunohistochemical staining of MMP12 in kidney tissue from mice treated with either anti‐IgG or anti‐PD‐1 antibody. Red arrowheads indicate MMP12‐positive cells. Scale bars: 50 µm; magnification: 400×. D) Kidney sections stained with DAPI (blue, nuclei), tdTomato (red, resident macrophages), and MMP12 (green). The merged image shows the colocalization of MMP12 with resident macrophages, highlighted in the magnified region (dashed box). TdTomato signal intensity may vary across figures due to differences in imaging settings and sample preparation. Scale bars: 100 µm; magnification: 630×. E) CXCL9 levels in mouse serum. F) qPCR results of CD8 + T cells co‐cultured with resident macrophages ( n = 3 per group). G) qPCR results of resident macrophages isolated from mouse kidneys treated with anti‐PD‐1 antibody or control IgG ( n = 3 per group). Control, n = 3; LLC, n = 3; LLC + anti‐IgG, n = 3; LLC + anti‐PD‐1, n = 5; Resident macrophage depleted + LLC + anti‐IgG, n = 3; Resident macrophage depleted + LLC + anti‐PD‐1, n = 5. Data are presented as mean ± SD. Statistical comparisons were made using an unpaired t ‐test or one‐way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Membranes were blocked with 5% non‐fat milk and incubated overnight at 4 °C with primary antibodies targeting MMP12 (Novus Biologicals, Centennial, CO, USA), α‐SMA (Proteintech, Wuhan, China), NGAL (R&D systems, USA), and β‐actin (Proteintech, Wuhan, China), followed by incubation with HRP‐conjugated secondary antibodies.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Imaging, Sample Prep, Cell Culture, Isolation, Control

Journal: Advanced Science

Article Title: Resident Macrophage‐Orchestrated Immune and Fibroblast Interactions in Immune Checkpoint Inhibitor‐Associated Nephrotoxicity

doi: 10.1002/advs.202505445

Figure Lengend Snippet: Pharmacological inhibition of MMP12 and neutralization of CXCL9 attenuate renal injury and immune activation in ICI‐associated nephrotoxicity (ICI‐AN). A) Schematic of the experimental timeline in mice treated with MMP408. B) Serum creatinine (Cr) levels in each treatment group ( n = 5 per group). C) Representative histological images of kidney tissues stained with hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), and Masson's trichrome. Red arrowheads indicate inflammatory cell infiltration; yellow arrowheads indicate tubular injury. Scale bars: 50 µm. D) Western blot analysis of NGAL and α‐SMA protein expression in renal tissues ( n = 5 per group). E) qPCR analysis of Ngal and Fn1 mRNA expression in kidney tissues ( n = 5 per group). F) qPCR analysis of fibrosis‐related genes ( Acta2 , Fn1 , and Tgfb1 ) in NRK‐49F fibroblasts after 48‐hour transwell co‐culture with resident macrophages isolated from anti‐PD‐1‐treated mice, with or without MMP408 pretreatment ( n = 3 per group). G) Flow cytometric analysis of CD25 mean fluorescence intensity (MFI) on CD8⁺ T cells co‐cultured with resident macrophages in the presence or absence of CXCL9 neutralizing antibody ( n = 3 per group). H) qPCR analysis of Gzmb and Il1b expression in CD8⁺ T cells after 48‐hour co‐culture with resident macrophages with or without α‐CXCL9 treatment ( n = 3 per group). Data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA or unpaired t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Membranes were blocked with 5% non‐fat milk and incubated overnight at 4 °C with primary antibodies targeting MMP12 (Novus Biologicals, Centennial, CO, USA), α‐SMA (Proteintech, Wuhan, China), NGAL (R&D systems, USA), and β‐actin (Proteintech, Wuhan, China), followed by incubation with HRP‐conjugated secondary antibodies.

Techniques: Inhibition, Neutralization, Activation Assay, Staining, Western Blot, Expressing, Co-Culture Assay, Isolation, Fluorescence, Cell Culture

Journal: PLoS Medicine

Article Title: An Immune Basis for Lung Parenchymal Destruction in Chronic Obstructive Pulmonary Disease and Emphysema

doi: 10.1371/journal.pmed.0010008

Figure Lengend Snippet: (A) CD14 + , lymphocyte-depleted lung leukocytes were cultured with and without the indicated amounts of recombinant human IP-10 and IFN-γ, and supernatants were assessed for the presence of MMP12 by Western blotting. (B) Fold increase relative to unstimulated of MMP12 mRNA from lung macrophages stimulated without ( – ) and with ( + ) 500 ng/ml of IP-10 in the presence or absence of a function-blocking antibody to CXCR3 as determined by real-time PCR. (C and D) Lung tissue from a participant with emphysema (C) shows strong immune staining for MMP12 localized to macrophages (arrows), and (D) shows lung tissue from a control participant without emphysema and with undetectable MMP12. The insets show a high-power view of lung macrophages staining positive (C) and negative (D) for MMP12 (×60) *, p = 0.04.

Article Snippet: Paraffin-embedded, and fresh-frozen lung sections (5 μm) were immunostained using monoclonal antibodies against human MMP12 (R&D Systems) or non-immune antisera by an immunoperoxidase protocol (Vectastain Elite, Vector Labs, Burlingame, California, United States) and counterstained with hematoxylin as recommended by the manufacturer.

Techniques: Cell Culture, Recombinant, Western Blot, Blocking Assay, Real-time Polymerase Chain Reaction, Staining, Control