Journal: The Journal of Experimental Medicine

Article Title: The tumor suppressor semaphorin 3B triggers a prometastatic program mediated by interleukin 8 and the tumor microenvironment

doi: 10.1084/jem.20072509

Figure Lengend Snippet: SEMA3B induces IL-8 expression in tumor cells, which accounts for increased monocyte attraction. (A) mRNA from transduced MDA-MB435 cells was used to probe a microarray representative of the human genome. Results are shown as a dot plot of individual genes, comparing the expression levels found in EV– and SEMA3B-transduced cells. A selected panel of 622 genes, including cytokines and factors mediating inflammatory response (identified by keywords as described in Materials and methods), is highlighted in red. IL-8 is the only gene in the panel significantly regulated more than twofold. ***, P < 0.001) (B) Real-time quantitative PCR mRNA expression analysis of SEMA3B- and EV-transduced tumor cells using Taqman probes for IL-8 and for the housekeeping gene β-actin (internal reference). Reported values indicate fold induction of IL-8 expression in SEMA3B-expressing cells relative to the respective EV control for each cell line, and values correspond to the mean ± SD of three individual experiments. *, P < 0.05. (C) The CM from MDA-MB435 EV control and SEMA3B-expressing cells was collected and concentrated 300 times. 10 μl of this preparation was analyzed by Western blotting with anti–IL-8 antibodies. The indicated amounts of purified IL-8 were loaded for internal reference. The concentration of IL-8 in the original CM of MDA-3B cells was thus estimated to be in the range of 5–7 ng/ml. (D) Western blot analysis of A549 cells expressing SEMA3B under a drug-inducible promoter; control cells (EV) expressed the doxycycline-dependent transactivator rTTA alone. Upon doxycycline treatment, SEMA3B expression in tumor cells (revealed by anti-Myc tag antibodies) and IL-8 levels in the CM (revealed with MAB208) were increased; purified IL8 was loaded as an internal reference. (E) The CM of SEMA3B tumor cells and EV controls were assayed in monocyte migration experiments, similar to those shown in . Where indicated, IL-8 neutralizing antibodies (MAB208) or an unrelated control antibody (i.e., anti-CD19; C Ab), or 100 ng/ml of purified IL-8 were added in the lower well. The number of migrated monocytes was scored as in , and the graph shows the mean of three independent experiments. IL-8 is responsible for the increased monocyte-attracting activity released by SEMA3B-expressing tumor cells. **, P < 0.01.

Article Snippet: For the treatment with IL-8 neutralizing antibodies, four groups of five mice were injected on both sides with either MDA-EV or MDA-3B cells, together with 100 μg/mouse of either IL-8 neutralizing monoclonal antibody (MAB208) or the control antibody CD19 (Antigenix America).

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, Control, Western Blot, Purification, Concentration Assay, Migration, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: The tumor suppressor semaphorin 3B triggers a prometastatic program mediated by interleukin 8 and the tumor microenvironment

doi: 10.1084/jem.20072509

Figure Lengend Snippet: SEMA3B knock down in tumor cells affects IL-8 production and monocyte recruitment. (A) RNA was collected from LoVo, A549, HeLa, and Sk-Mel-2 cells transduced with shRNA against SEMA3B (to suppress gene expression; shSEMA3B) and unrelated sequences (shCTR). Real-time PCR was performed using Taqman probes for SEMA3B (results shown at the bottom) and IL-8 (in the graph). Expression levels were normalized to controls for each cell line and are represented as fold changes. Data shown are the mean ± SEM. (B) Monocyte migration to CM derived from Sk-Mel-2 melanoma cells transduced with shRNAs. IL-8 neutralizing antibodies (MAB208) or unrelated control antibodies (anti-CD19) were included in the lower chamber. Knock down of SEMA3B expression in Sk-Mel-2 cells abolished IL-8–dependent monocyte-attracting activity in the CM. The number of migrated monocytes represents the mean ± SEM of two independent experiments. **, P < 0.01.

Article Snippet: For the treatment with IL-8 neutralizing antibodies, four groups of five mice were injected on both sides with either MDA-EV or MDA-3B cells, together with 100 μg/mouse of either IL-8 neutralizing monoclonal antibody (MAB208) or the control antibody CD19 (Antigenix America).

Techniques: Knockdown, Transduction, shRNA, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Migration, Derivative Assay, Control, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: The tumor suppressor semaphorin 3B triggers a prometastatic program mediated by interleukin 8 and the tumor microenvironment

doi: 10.1084/jem.20072509

Figure Lengend Snippet: Neutralizing IL-8 in SEMA3B-expressing tumors blocks macrophage recruitment and metastasis. (A) Nude mice were injected subcutaneously with either MDA-EV or MDA-3B tumor cells together with 100 μg of neutralizing IL-8 antibody (MAB208) or 100 μg of control antibody (anti-CD19). Antibodies were administered locally into tumors again at days 2, 5, 8, and 11 after tumor transplant. Data shown are the mean tumor volume ± SD from 10 tumors per each experimental group at the day of excision (day 15). (B) To reveal metastatic dissemination from the tumors, the lungs of mice described in A were analyzed by real-time RT-PCR using species-specific Taqman probes for mouse HPRT and human HPRT transcripts. Plotted values indicate the mean ratio between human and mouse RNA levels in the lungs of five mice per each experimental group ± SD. *, P < 0.05. (C) Tissue sections from SEMA3B and EV tumors, treated with control antibody or MAB208 (from the same experiment as in A), were stained with anti-F4/80 to detect monocytes/macrophages (red), whereas nuclei were revealed with DAPI (blue). Bar, 60 μm. Each data point represents the mean percentage of the macrophage-occupied area ± SD from eight tumors per each experimental group (10 fields were counted from at least three different sections for each tumor). **, P < 0.01; ***, P < 0.001. (D) Blood vessels and macrophages in sections of MDA-EV or MDA-3B tumors treated as described were revealed with anti–VE-cadherin (red) and anti-F4/80 (green); nuclei were marked with DAPI. Bar, 60 μm. (E) Blood vessels in sections of MDA-EV or MDA-3B tumors treated as described were revealed with anti-CD31 antibody (red), whereas nuclei were marked by DAPI (blue). Bar, 60 μm. The graph shows the mean vessel area ± SD from eight tumors for each experimental group (at least three sections and 10 fields were quantified per tumor). ***, P < 0.0005. (F) Sections from MDA-EV or MDA-3B tumors treated as described were subjected to TUNEL staining to reveal apoptotic cells. Bar, 60 μm. The graph shows the mean percentage of TUNEL-positive cells (±SEM) from five tumors for each experimental group in at least three sections and 10 fields. *, P < 0.05.

Article Snippet: For the treatment with IL-8 neutralizing antibodies, four groups of five mice were injected on both sides with either MDA-EV or MDA-3B cells, together with 100 μg/mouse of either IL-8 neutralizing monoclonal antibody (MAB208) or the control antibody CD19 (Antigenix America).

Techniques: Expressing, Injection, Control, Quantitative RT-PCR, Staining, TUNEL Assay

Journal: The Journal of Experimental Medicine

Article Title: The tumor suppressor semaphorin 3B triggers a prometastatic program mediated by interleukin 8 and the tumor microenvironment

doi: 10.1084/jem.20072509

Figure Lengend Snippet: p38–MAPK signaling elicited by SEMA3B mediates IL-8 induction. (A) Immunoblotting analysis to reveal phospho-p38 protein levels in MDA-MB435 tumor cells expressing SEMA3B (or EV controls), grown in the presence or absence of 10% FBS for 48 h. Results are displayed as fold induction of the phosphorylated/total p38 ratio in SEMA3B cells versus controls, as measured by densitometric analysis. (B) Serum-starved MDA-MB435 cells were treated for 30 min with CM collected from SEMA3B or EV control cells. The levels of phospho-p38 and phospho–ATF-2 were analyzed in protein lysates by immunoblotting. The same filter was furthermore decorated to reveal vinculin, providing loading controls. Plotted values indicate fold increase of the phosphorylated p38/vinculin or ATF2/vinculin ratios in SEMA3B-treated cells versus controls. (C) Immunoblotting analysis of A549 cells treated as in B with SEMA3B or EV control CM. Filters were probed to reveal phospho-p38, phospho-p42/44–MAPK, and vinculin levels (as loading control). The graph shows fold increase of the phosphorylated p38/vinculin or phospho-p42/44–MAPK/vinculin ratios in SEMA3B-treated cells versus controls. (D) MAB208 antibody was used to detect IL-8 expression by immunofluorescence (top) or by Western blotting (bottom) in SEMA3B-expressing and control cells grown in the absence of serum for 48 h, in the presence or absence of 10 μM of the selective p38–MAPK inhibitor SB202190. Micrographs were acquired with a bioimager (Pathway; BD Biosciences) and quantified by Autovision 1.5 (10 images per each data point). Results are displayed as mean ± SD. ***, P < 0.001. (E) CDKI p21 expression was detected by Western blotting in A549 cells, transduced with SEMA3B or EV control, upon serum starvation (48 h in 1% FCS). Cells treated with 20 nM staurosporin (STS) for 24 h provided a positive control for p21 activation. β-Actin levels provided a reference for protein loading. Plotted values indicate fold induction of the p21/β-actin ratio. (F) A549 cells were serum starved for 24 h and then treated for 30 min with CM from SEMA3B or EV control cells in the presence of 20 ng/ml anti-NP1 blocking antibody or the unrelated antibody anti–VSV-G. Cells treated with 20 μm cisplatin for 2 h provided a positive control for p38 activation. The levels of phospho-p38 and vinculin (as loading control) were analyzed in protein lysates by immunoblotting. (G) Consistent with a previous report, many tumor cells could not survive RNAi-mediated knock down of NP1 (reference ); however, we managed to stably express NP1-targeted shRNAs (shNP1) in HeLa cells previously transduced with SEMA3B or EV . Real-time PCR analysis indicated that NP1 expression levels were knocked down to 20% compared with cells transduced with unrelated sequences (shCTR; not depicted). Notably, SEMA3B autocrine stimulation was unable to up-regulate IL-8 levels in NP1-deficient cells, indicating the requirement for this receptor in the signaling pathway. The graph shows fold increase of expression relative to control cells. Data shown are the mean ± SEM.

Article Snippet: For the treatment with IL-8 neutralizing antibodies, four groups of five mice were injected on both sides with either MDA-EV or MDA-3B cells, together with 100 μg/mouse of either IL-8 neutralizing monoclonal antibody (MAB208) or the control antibody CD19 (Antigenix America).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Transduction, Positive Control, Activation Assay, Blocking Assay, Knockdown, Stable Transfection, Real-time Polymerase Chain Reaction