Journal: Cancer research

Article Title: Host-derived Matrix Metalloproteinase-13 Activity Promotes Multiple Myeloma-induced Osteolysis and Reduces Overall Survival

doi: 10.1158/0008-5472.CAN-20-2705

Figure Lengend Snippet: Stromal MMP-13 differentially regulates the cytokine secretion profile of MSCs. A, MMP-13 impacts MSC secretion of cytokines. WT and MMP-13−/− MSCs were incubated in serum-free α-MEM for 24 hours to generate conditioned media (CM) that was then subjected to cytokine array analysis. Cytokines with greatest changes and known roles in osteoclast biology were plotted and tabulated. B, MMP-13−/− MSCs have significantly higher levels of CXCL7 expression. RNA was isolated from WT and MMP-13−/− MSC for cDNA synthesis and quantitative PCR analysis. qPCR was performed for hits from cytokine array (ranging from * to ****). MMP-3 and CXCL7 are highlighted in A and B to note that overexpression of MMP-3, an upstream activator of CXCL7, didn’t sufficiently rescue CXCL7 levels in MMP-13−/− MSCs. C, CXCL7 protein is downregulated in MMP-13−/− MSC. CXCL7 western blot analysis was performed on MSC cell lysates and CM. D, CXCL7 protein levels were validated by ELISA in WT MSC treated with Compound 1, a highly selective MMP-13 inhibitor (MMP-13i). E, Human recombinant CXCL7 was incubated with human recombinant active MMP-13 overnight at 37°C and the impact on CXCL7 processing was assessed by western blot. F, CXCL7 was immunoprecipitated (IP) from WT and MMP-13−/− MSC CM. WT and MMP-13−/− MSC CM were incubated with anti-CXCL7 antibody and protein G sepharose beads overnight to precipitate CXCL7. Resultant CM was collected and corresponding beads washed for validation by western blot (top and bottom panel, respectively). G, CXCL7-depleted WT CM impairs osteoclastogenesis. Osteoclast differentiation assay was performed using WT bone marrow co-culture with WT, MMP-13−/−, and CXCL7-depleted CM supplemented with RANKL. Wells were fixed at day 4 and quantitated for number and size of osteoclasts. Resulting quantitation was statistically analyzed by One-way Anova with post hoc multiple comparison analysis (Tukey test; * and **). Asterisks denotes statistical significance (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001) while n.s. denotes non-significance.

Article Snippet: Transferred blots were blocked for 1 hour at 25°C (1XTBST containing 5% non-fat dairy milk) prior to overnight incubation in primary antibodies: α-mouse MMP-13 at 1:1000 (Triple Point Biologics, Cat#: RP1-MMP-13), and α-mouse CCL-2 (Thermofisher Scientific; Cat#: MA5–17040; RRID:AB_2538512), α-mouse CXCL7 (R&D Systems, Cat#: AF793; RRID:AB_355606), and α-mouse β-Actin at (Cell Signaling, Cat#: 3400S) all at 1:1000.

Techniques: Incubation, Expressing, Isolation, Real-time Polymerase Chain Reaction, Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Immunoprecipitation, Osteoclast differentiation Assay, Co-Culture Assay, Quantitation Assay

Journal: Cancer research

Article Title: Host-derived Matrix Metalloproteinase-13 Activity Promotes Multiple Myeloma-induced Osteolysis and Reduces Overall Survival

doi: 10.1158/0008-5472.CAN-20-2705

Figure Lengend Snippet: Selective pharmacological inhibition of MMP-13 impacts multiple myeloma viability and osteoclast formation in vitro, and significantly improves overall survival in vivo. A, Osteoclast differentiation assays were performed in the presence of three MMP-13 inhibitors (n=3/concentration for each compound). Inhibitor compounds were administered every other day in fresh osteoclast differentiation media. Plates were fixed at day 7, TRAcP stained and quantitated for osteoclast numbers. One-way Anova statistical test was performed to assess osteoclast inhibition at each concentration of compounds compared to control group (*). B, Compound 1 (MMP-13i) strongly inhibited multiple myeloma viability. Multiple myeloma cell lines (MM.1S, 5TGM1, OPM2 and U266) were cultured in concentration gradients of MMP-13i for 48 hours in 96-well plates (n=3/group). Cells were assessed by MTS and analyzed using a One-way Anova statistical test compared to the control group (*). C, MMP-13i treatment delayed multiple myeloma growth in immunocompetent syngeneic mouse model of multiple myeloma. luciferase-tagged 5TGM1 cells (n=106 cells/mouse) were inoculated into 5–6-week old syngeneic KaLwRiJ mice (n=30) by tail vein to establish skeletal lesions. Mice were randomly divided into Vehicle (10% DMSO 10% Tween80 in 1xPBS) and MMP-13i (20 mg/kg) group 7 days following tumor inoculation (n=15/group). Tumor growth was monitored by bioluminescence imaging and depicted as spider plot (BLI) twice a week until each mouse eventually reached endpoint hindlimb paralysis. D, Mice treated with MMP-13i demonstrated a significant increase in overall survival compared to vehicle control. Mice were euthanized upon reaching endpoints. Kaplan Meier curves were generated for each group (**; median survival: 35 vs 42 days for Vehicle and MMP-13i, respectively). E, Tibias harvested from control and MMP-13i MM-bearing mice (n=30) in addition to 7-week old control and MMP-13i treated tumor naïve groups (n=6/group) were subjected to μCT scanning and quantitation. MMP-13i treatment significantly increased bone volume in tumor-naive mice (Student’s t-test: *). Tumor-bearing mice treated with MMP-13i removed from study at end-points mice showed similar bone volumes as analyzed by Student’s t-test. Asterisks denotes statistical significance (*p<0.05 and **p<0.005) while n.s. denotes non-significance. F, Schematic of working model. 1. Multiple myeloma cells induce MMP-13 expression in MSCs. 2. MMP-13 can directly process CXCL7. 3. MMP-13 generated CXCL7 enhances osteoclast formation and activity. 4. Osteoclast mediated bone resorption generates further factors that promote myeloma cell survival and growth. 5. MMP-13 inhibition with highly selective inhibitors can block this myeloma and stromal derived MMP-13 to limit cancer associated bone disease and improve overall survival.

Article Snippet: Transferred blots were blocked for 1 hour at 25°C (1XTBST containing 5% non-fat dairy milk) prior to overnight incubation in primary antibodies: α-mouse MMP-13 at 1:1000 (Triple Point Biologics, Cat#: RP1-MMP-13), and α-mouse CCL-2 (Thermofisher Scientific; Cat#: MA5–17040; RRID:AB_2538512), α-mouse CXCL7 (R&D Systems, Cat#: AF793; RRID:AB_355606), and α-mouse β-Actin at (Cell Signaling, Cat#: 3400S) all at 1:1000.

Techniques: Inhibition, In Vitro, In Vivo, Concentration Assay, Staining, Cell Culture, Luciferase, Imaging, Generated, Quantitation Assay, Expressing, Activity Assay, Blocking Assay, Derivative Assay

Journal: Scientific reports

Article Title: Immuno-transcriptomic analysis based on machine learning identifies immunity signature genes of chronic rhinosinusitis with nasal polyps.

doi: 10.1038/s41598-025-02508-8

Figure Lengend Snippet: Fig. 7. Gene expression levels of candidate genes. Gene expression levels of CXCR1 (A), CCL13 (B), PPBP (C), CCR3 (D) and MMP9 (E) in CRSwNP and control samples. Gene expression levels of CXCR1 (F), CCL13 (G), PPBP (H), CCR3 (I) and MMP9 (J) in different inflammatory endotypes of CRSwNP. (*P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant).

Article Snippet: The membranes were closed in 5% skimmed milk for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CXCR1 (1:500; Servicebio, GB11625-100), PPBP (1:50; Proteintech, 13313-1-AP), GAPDH (1:3000; Servicebio, GB15004-100).

Techniques: Gene Expression, Control

Journal: Scientific reports

Article Title: Immuno-transcriptomic analysis based on machine learning identifies immunity signature genes of chronic rhinosinusitis with nasal polyps.

doi: 10.1038/s41598-025-02508-8

Figure Lengend Snippet: Fig. 8. Candidate gene protein expression levels in normal mucosal tissues and chronic rhinosinusitis with nasal polyps (CRSwNP) tissues were analysed using IHC staining. Unpaired t-tests were used to determine the significant differences for candidate gene protein expression between the normal mucosal(n = 5) and CRSwNP (n = 5) groups. (A) CXCR1. (B) CCL13. (C) PPBP. (D) CCR3. (E) MMP9. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: The membranes were closed in 5% skimmed milk for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CXCR1 (1:500; Servicebio, GB11625-100), PPBP (1:50; Proteintech, 13313-1-AP), GAPDH (1:3000; Servicebio, GB15004-100).

Techniques: Expressing, Immunohistochemistry

Journal: Scientific reports

Article Title: Immuno-transcriptomic analysis based on machine learning identifies immunity signature genes of chronic rhinosinusitis with nasal polyps.

doi: 10.1038/s41598-025-02508-8

Figure Lengend Snippet: Fig. 9. Effects of CXCR1 and PPBP on IL-8 secretion. (A) HNEpC cells were transfected with siRNA (targeting CXCR1), and CXCR1 expression in transfected cells was detected by RT-qPCR. (B) Detection of CXCR1 protein expression by western blot. (C) Detection of IL-8 in the supernatant of CXCR1 knockdown cells using an ELISA kit. (D) HNEpC cells were transfected with PPBP overexpression plasmid and PPBP expression was detected by RT-qPCR in the transfected cells. (E) PPBP protein expression was detected by western blot. (F) The level of IL-8 in the supernatant of PPBP overexpressing cells was detected using ELISA kit.

Article Snippet: The membranes were closed in 5% skimmed milk for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CXCR1 (1:500; Servicebio, GB11625-100), PPBP (1:50; Proteintech, 13313-1-AP), GAPDH (1:3000; Servicebio, GB15004-100).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Over Expression, Plasmid Preparation