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